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ISSN: 2159-8967

Volume 3, Issue 2 2013


Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013


W.K. Kim

University of Florida, USA

University of Manitoba, Canada

Walid Q. Alali

M.B. Kirkham

University of Georgia, USA

Kansas State University, USA

Kenneth M. Bischoff

Todd Kostman


University of Wisconsin, Oshkosh, USA

Debabrata Biswas

Y.M. Kwon

University of Maryland, USA

University of Arkansas, USA

Claudia S. Dunkley

Maria Luz Sanz

University of Georgia, USA

MuriasInstituto de Quimica Organic General, Spain

Lawrence Goodridge

Melanie R. Mormile

Colorado State University, USA

Missouri University of Science and Tech., USA

Leluo Guan

Rama Nannapaneni

University of Alberta, Canada

Mississippi State University, USA

Joshua Gurtler

Jack A. Neal, Jr.


University of Houston, USA

Yong D. Hang

Benedict Okeke

Cornell University, USA

Auburn University at Montgomery, USA

Divya Jaroni

John Patterson

Oklahoma State University, USA

Purdue University, USA

Weihong Jiang Shanghai

Toni Poole

Institute for Biol. Sciences, P.R. China


Michael Johnson

Marcos Rostagno

University of Arkansas, USA


Timothy Kelly

Roni Shapira

East Carolina University, USA

Hebrew University of Jerusalem, Israel

William R. Kenealy

Kalidas Shetty

Mascoma Corporation, USA

North Dakota State University, USA

Hae-Yeong Kim Kyung Hee University, South Korea Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013


EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA Cesar Compadre University of Arkansas for Medical Sciences, USA

MANAGING and LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR Jessica C. Shabatura Fayetteville, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, USA

Philip G. Crandall University of Arkansas, USA

ABOUT THIS PUBLICATION Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701


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Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013


Consumers’ Interest in Locally Raised, Small-Scale Poultry in Georgia E. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, and S. C. Ricke

129 Isolation and Initial Characterization of Acetogenic Ruminal Bacteria Resistant to Acidic Conditions

P. Boccazzi and J. A. Patterson

145 Linoleic Acid Isomerase Expression in Escherichia coli BL21 (DE3) and Bacillus spp S. Saengkerdsub

REVIEW 103 Current and Near-Market Intervention Strategies for Reducing Shiga Toxin-Producing Escherichia coli (STEC) Shedding in Cattle.

T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, and D. J. Nisbet

121 Potential for Rapid Analysis of Bioavailable Amino Acids in Biofuel Feed Stocks D. E. Luján-Rhenals, and R. Morawicki

Introduction to Authors 162 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors. Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013

93 Copyright © 2013 Agriculture, Food and Analytical Bacteriology

Consumers’ Interest in Locally Raised, Small-Scale Poultry in Georgia E. J. Van Loo1,2, W. Q. Alali3, S. Welander4, C. A. O’Bryan1, P. G. Crandall1, S. C. Ricke1 Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR 2 Present address: Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 3 Center for Food Safety and Department of Food Science & Technology, University of Georgia, Griffin, GA 4 Georgia Organics, 200-A Ottley Drive, Atlanta, GA


ABSTRACT An online questionnaire was developed that targeted consumers with an interest in sustainable and local poultry production in Georgia. Approximately 97% of the respondents expressed an interest in supporting efforts to make sustainably raised poultry processed in Georgia available. Even for a high premium of $5.00/lb, some respondents would shift their current chicken purchases towards these locally raised chickens. Respondents reported some interest in attributes such as pasture raised, air chilled and Georgia grown for their poultry. Knowledge about the demand for local pastured poultry supports the need for infrastructure to support Mobile Processing Units for Georgia farmers interested in locally raised small-scale poultry production. Keywords: consumer, small-scale poultry production, pastured poultry

Agric. Food Anal. Bacteriol. 3: 94-102, 2013

Introduction The term pasture raised poultry refers to a production system in which chickens or other poultry are raised primarily on pasture, with the birds supplementing their feed grain by foraging for up to 20 percent of their dietary intake. Until the 1930s, when large concentrated animal feeding operations first developed, almost all chickens were raised on pasCorrespondence: Steven C. Ricke, Tel: +1-479-575-4678 Fax: +1-479-575-6936


ture. However, the concept of raising pastured poultry was never completely abandoned, and in 1993 Joel Salatin of Swoope, Virginia published Pastured Poultry Profit$, a book in which he outlined a model for modern pastured poultry production using small, mobile, floorless, enclosed chicken shelters or hoop houses. The modern pastured poultry movement has flourished because of the increased demand of consumers wanting to purchase pastured poultry products (Faulkner, 2011). Georgia produces more broilers than any other state, more than 1.3 billion birds in 2010 (USDA,

Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013

2011). However, the small-scale poultry industry in Georgia is severely limited in growth due to regulatory challenges imposed by the strict state rules, which do not allow an exemption from federal inspection for small farmers who process more than 1,000 birds/ year. An analysis of the Georgia consumers’ interest in these small-scale farmer poultry products was necessary to support the need for a new processing option for these farmers. The various processing options and the preferences of the farmers were previously studied (Van Loo et al., 2013). The purpose of this survey was to evaluate the consumer interest for locally raised pastured poultry in Georgia. A relatively high interest in these products could potentially justify the development of new processing options for the small-scale Georgia farmers.

meetings. The link to the survey was also posted on Georgia Organics’ website. A total of 508 Georgia consumers took the survey between September of 2008 and July of 2010. Table 1 contains the questions and choice of possible answers. Frequency tables, mean values and standard deviations were determined using JMP (release 9.0.0: SAS Institute, Inc.).The consumer study targeted consumers with an interest in sustainable and local foods produced in Georgia. The consumer survey consisted of questions about (i) current poultry consumption; (ii) consumer interest in locally pasture raised poultry as well as their purchasing behavior if this product would be available to them at different price levels; and (iii) consumers’ interest in different poultry characteristics/properties.

Materials and Methods

Results and Discussion

Notices of the pending survey were posted in the Georgia Organics print newsletter, and in an electronic newsletter, as well as by a targeted email to a list of interested poultry consumers based on connections Georgia Organics made at conferences and

Poultry Consumption A total of 508 consumers responded to the survey. Among those who reported purchasing any type of chicken in the previous 3 months, whole chicken and

Figure 1. Chicken purchasing habits of Georgia consumers (n = 508) during previous 3 months 500






0 Whole chickens

Parts with skin/bones Parts skinless with bones

Parts boneless/skinless

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Table 1. Questionnaire

1. Indicate to what degree you agree with the following statement: “I support efforts to make this new type of chicken available in Georgia.” 1 = not at all, 2 = yes, some interest, 3 = yes, absolutely 2. In the last three months, have you purchased chicken meat (any source, any type) in the following forms? • Whole chickens • Parts (with skin/bones) • Parts (skinless) with bones • Parts (skinless and boneless) • Other (please specify) 3. How many pounds of chicken (any source, any type) do you purchase per month? 4. How many whole chickens (any source, any type) do you purchase per month? Choices: 0, 1, 2, 3, 4, 5, 6, more than 6 (please specify) 5. If this new type of chicken were available as whole chickens at your favorite place to shop, please indicate the percentage of your current chicken purchasing you’d shift. For example, if you’d shift 40% of your purchasing to pasture raised Georgia poultry if it were available at $4.00/pound, select “40” from the drop-down menu on the $4.00 row. Price points $2.50, $3.00, $3.50, $4.00, $4.50, $5.00 6. Indicate your level of interest in the following, as it pertains to this new type of poultry. 1 = not at all interested, 5 = very interested. • • • • • • • • •


Boneless, Georgia grown, Air chilled Sustainably raised Skinless Pasture raised Certified organic Soy free Other (please specify)

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skinless boneless parts were the most popular types, 75% and 73% respectively (Figure 1). A smaller percentage, 63%, reported purchasing chicken parts with skin and bones in the past 3 months, and the least popular was skinless chicken parts with bones (32%). Other categories mentioned included ground chicken and livers/gizzards, with less than 1% of respondents each. The vast majority of the consumers reported monthly chicken purchases of between 1 and 10 pounds (Figure 2). For whole chickens in particular, almost 1/3 (32%) reported buying only one whole chicken per month (Figure 3).

Consumer’s interest and willingness to pay for sustainable locally raised and processed poultry in Georgia Consumers were asked about their interest in a new type of poultry that would be raised sustainably on Georgia pastures, meet or exceed all current sanitary and safety measures, be processed in Georgia, and be available for purchase at their favorite place to purchase chicken. Figure 4 illustrates that among these consumers there is a great interest in this type of poultry; 94% said that they were “absolutely” interested in being able to purchase this type of poultry. This can be explained by the nation-wide increase of consumers’ interest in local foods. Peer reviewed literature in agricultural economics substantiates a strong consumer preference for locally-produced foods (Zepeda and Li, 2006; Keeling-Bond et al., 2009; Carpio and Isengildina- Massa, 2009; Martinez et al., 2010). When asked, consumers list diverse reasons for their “buy local” preferences, including preference for fresher foods, minimal food miles, reduction of carbon footprint, and support for the local economy (Guptill and Wilkins, 2002; ERS, 2010). In a 2009 national study, respondents cited reasons for buying local food as: freshness (82%), support for the local economy (75%), and knowing the source of their food (58%) (Food Marketing Institute, 2009). The 2008 Farm Act defines the total distance a product can be transported and still be eligible for marketing as a “locally or regionally produced agricultural food

product” as less than 400 miles from its origin, or the State in which it is produced (USDA, 2008). As such the Georgia raised poultry sold in Georgia meets the definition for a local product. The price of meat is an extrinsic factor that can affect consumer’s purchasing decisions (Lange et al., 1999; Lockshin et al., 2006). The participating consumers in our study were willing to pay premium prices for these products. Consumers were asked the question “If this new type of chicken were available as whole chickens at your favorite place to shop, please indicate the percentage of your current chicken purchasing you would shift. For example, if you would shift 40% of your purchasing to pastured raised Georgia poultry if it were available at $4.00/pound, select “40” from the drop-down menu on the $4.00 row.” Depending on the price charged for whole pasture raised chicken, the consumers were willing to shift a different amount of their current whole chicken purchases (Figure 5). Van Loo et al. (2010) reported price as the main disincentive for organic chicken purchases. Similarly, our results indicate that price has a negative correlation to the demand of locally raised pastured poultry from Georgia. With a higher price point for the locally raised pastured poultry, the reported demand decreases. For the low price of $2.50/lb, 213 (42%) of the respondents were willing to shift 100% of their current whole chicken purchases towards locally raised chicken. At a higher price of $3.00/lb, 175 respondents were willing to shift 100% of their current chicken purchases towards this local product. However, even for a high premium of $5.00/lb, 75 (15%) of the respondents would shift 100% of their current chicken purchases towards this local product. Van Loo et al. (2011) indicated in previous research that consumers who are habitual buyers of sustainable meat products are also willing to pay a higher premium price for these products. Michel et al. (2011) reported that half of the participants were willing to pay a 30% premium for value-added chicken compared to conventional chicken products. Verbeke and Viaene (1999) conversely found that price was ranked fifth regarding perception of pork, beef and poultry attributes by consumers, behind quality, taste, free of hormones and healthy. Furnols et

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Figure 2. Pounds of chicken purchased during previous 3 months? (n = 508)

400 Number of consumers

350 300 250 200 150 100 50 0 0







Pounds purchased

Figure 3. Number of whole chickens purchased per month by survey respondents (n=508).

180 160

Number fo consumers

140 120 100 80 60 40 20 0 0





Number of chickens 98

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Figure 4. Consumer support for sustainably raised, Georgia pastured poultry. 1 = no interest, 2 = some interest, 3 = absolutely.

1 = Not at all 0%

2 = Some interest 5%

3 = Absolutely 95%

Figure 5. Percent of respondents willing to shift chicken purchases to pasture raised Georgia poultry at different price points per pound 100%



Number of respondents

10 6 23 17

40 22




56 36

36 71








94 72

45 42



58 175











Price point Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013


al. (2011) found that lamb meat price played only a minor role in determining consumer’s purchasing decisions, except that when sorted by demographics men considered that the price was the most important factor. No demographic information was collected in the survey reported here, so no such determination can be made, but we may conclude that price was not as big a factor for our respondents as other factors.

Interest and relative importance of different poultry characteristics Respondents rated the importance of/interest in 8 different meat quality criteria using the 5 point Likert scale, with 1 being not at all interested and 5 being very strongly interested (Table 2). When evaluating the average importance of the meat quality attributes, the most important chicken product attribute

was “sustainably raised” (3.70) followed by “Georgia grown” (3.63), “pasture raised” (3.45) and “certified organic“(3.13). These four product properties are characteristic for local pasture or organic raised poultry and appear to be more important than other characteristics. These other characteristics, related to general properties, not particularly characteristic for pasture raised, locally or organically raised poultry were found less important such as air chilled (2.84), boneless (2.59), skinless (2.47) and soy-free (2.42). These results indicate that the consumers who answered this portion of the survey were interested in sustainable locally raised and processed poultry products and suggest that there is a strong demand this product. Food selection and consumption can be affected by different intrinsic and extrinsic cues such as country of origin, price or type of feed such as grain versus grass fed (Verlegh and van Ittersum 2001; Furnols et al., 2011). For instance, Furnols et al. (2011) found that origin of lamb meat was one of

Table 2. The frequency distribution among the 5 levels of interest for different chicken product properties and average Likert scale value where 1 = not at all interested and 5 = very interested Chicken product







St. dev.

Sustainably raised








Georgia grown








Pasture raised








Certified organic









































*No respondents answered 5 100

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the most important factors in purchasing, with locally grown lamb being the most preferred. This is consistent with our study where “Georgia grown” is an attribute of high importance. The pasture raised attribute had a higher score than organic certified. This is similar with the study from Michel et al. (2011) where consumers also indicated a preference for free-range chicken over organic chicken and may be a consequence of the associated higher price of organic meat as compared to pasture or free-range poultry. However, we need to be careful drawing conclusions about the ranking of the importance of the attributes since varying totals for responding to these questions makes it difficult to make a definitive statement. For instance, “sustainably raised” has a Likert value of 3.70 but only 61 persons in the survey rated that attribute at any level. None of the respondents claimed to be strongly interested in any of the options. One possibility in our study is that other attributes might be more important to the respondents. In looking at comments, this has some validity as some of the respondents mentioned “humanely slaughtered” as an aspect they valued. Another possibility is that terms such as “sustainably raised”, “air chilled” or “soy free” were not defined and some respondents may not have been familiar with these terms.

Conclusions The polled consumers have a great interest in sustainable locally raised poultry products processed in Georgia and are willing to pay extra for these products compared to conventional poultry products. It is important to emphasize that the results are based on surveying consumers currently interested in sustainable and local foods and therefore cannot be generalized for all Georgian consumers. Therefore, we would suggest that future research not only focus on the consumers currently involved with Georgia Organics, but research involve a statistically representative group of all Georgia poultry consumers. The consumer awareness as well as their interest and

willingness to pay for local and sustainable poultry products will help decide the future for pastured poultry in Georgia and other regions.

Acknowledgements The preparation of this manuscript was partially funded by SARE grant LS11-245 and USDA-NIFSI grant #2008-51110-04339.

References Carpio, C. E., and O. Isengildina-Massa. 2009. Consumer willingness to pay for locally grown products: The case of South Carolina. Agribusiness 25:412–426. Faulkner, D. 2011. Pastured chicken. Farming magazine, Fall, 2011:28-29. Food Marketing Institute. 2009. U.S. Grocery shopper trends. Food Marketing Institute: Arlington, VA. Furnols, M. F., C. Realini, F. Montossi, C. Sanudo, M. M. Campo, M. A. Oliver, G. R. Nute, and L. Guerrero. 2011. Consumer’s purchasing intention for lamb meat affected by country of origin, feeding system and meat price: A conjoint study in Spain, France and United Kingdom. Food Qual. Pref. 22: 443-451. Guptill, A., and J.L. Wilkins. 2002. Buying into the food system: Trends in food retailing in the U.S. and implications for local foods. Agric. Human Values 19:39-51. Keeling-Bond, J., D. Thilmany, and C. Bond. 2009. What influences consumer choice of fresh produce purchase location. J. Agric. Appl. Econ. 41:61-74. Lange, C., F. Rousseau, and S. Issanchou. 1999. Expectation, liking and purchase behaviour under economical constraint. Food Qual. Pref. 10:31–39. Lockshin, L., W. Jarvis, F. d’Hauteville, and J-P. Perrouty. 2006. Using simulations from discrete choice experiments to measure consumer sensitivity to brand, region, price, and awards in wine choice. Food Qual. Pref. 17:166–178.

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Martinez, S., M. Hand, M. Da Pra, S. Pollack, K. Ralson, T. Smith, S. Vogel, S. Clark, L. Lohr, S. Low, and C. Newman. 2010. Local food systems: concepts, impacts, and issues. US Dep. Agric, Econ. Res. Serv., ERR Rep. 97. Available at http://www. Accessed February 2012. Michel, M. L., S. Anders, and W. V. Wismer. 2011. Consumer preferences and willingness to pay for value-added chicken product attributes. J. Food Sci.76: S469–S477. Salatin, J. 1996. Pastured Poultry Profit$. Chelsea Green Pub Co. 335 p. USDA. 2008. USDA 2008 Farm Bill. Main Outline. Accessed March 2011. http://www.5 MBILL2008. USDA. 2011. Poultry – Production and value. 2010 Summary. April 2011. Available at http://usda01. Accessed 02/22/2012. Van Loo, E. J., V. Caputo, R. M., Nayga, J. Meullenet, P. G. Crandall, and S. C. Ricke. 2010. The effect of organic poultry purchase frequency on consumer attitudes toward organic poultry meat. J. Food Sci. 75: S384–S397. Van Loo, E. J., V. Caputo, R. M. Nayga Jr., J-F. Meullenet, and S .C. Ricke. 2011. Consumers’ willingness to pay for organic chicken breast: Evidence from choice experiment. Food Qual. Pref. 22:603-613. Van Loo, E. J., W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, and S. C. Ricke. 2013. Independent poultry processing in Georgia: survey of producers’ perspective. Agric., Food, Anal. Bacteriol. 3:70-77. Verbeke, W., and J. Viaene. 1999. Beliefs, attitudes and behaviour towards fresh meat consumption in Belgium: Empirical evidence from a consumer survey. Food Qual. Pref. 10:437–445. Verlegh, P.W.J, and K. van Ittersum. 2001. The origin of spices: the impact of geographic product origin on consumer decision making, in Frewer L., E. Risvik, H. Schifferstein (Eds) Food, People and Society, Springer, New York, pp 267-279. Zepeda, L., and J. Li. 2006. Who buys local food? J. Food Dist. Res. 37:1-11. 102

Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013 Copyright © 2013 Agriculture, Food and Analytical Bacteriology

REVIEW Current and near-market intervention strategies for reducing Shiga Toxin-Producing Escherichia coli (STEC) shedding in cattle T. R. Callaway1, T. S. Edrington1, G. H. Loneragan2, M. A. Carr3, D. J. Nisbet1 Food and Feed Safety Research Unit, USDA/ARS, 2881 F&B Rd., College Station, TX 77845 2 Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409 3 Research and Technical Services, National Cattlemen’s Beef Association, Centennial, CO 80112 1

Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies neither approval of the product, nor exclusion of others that may be suitable.

ABSTRACT Cattle can naturally contain foodborne pathogenic bacteria such as Shiga Toxin-Producing E. coli (STEC). These foodborne pathogenic bacteria are a threat to public health through contamination of foods and water supplies. In order to reduce human exposures and resultant illnesses, research has focused in recent years on the development of live animal intervention strategies that can be applied to reduce the burden of STEC entering the food chain. This review addresses the application of interventions that have been proposed or implemented to reduce STEC in live cattle. Recent years have seen increasing development of new interventions (e.g., vaccination, DFM, chlorate, phages) and into understanding what effect diet and the microbial population have on the microbial populations of the gut of cattle. This research has resulted in several novel interventions and potential dietary additions or changes that can reduce STEC in cattle, and many of them are in, or very near to entering, the marketplace. The live animal interventions must be designed in a coherent, complementary context as part of a multiple-hurdle scheme to reduce pathogens entry into the food supply. Keywords: Escherichia coli, shiga toxin, intervention, cattle, shedding, near-market, multiple hurdle Agric. Food Anal. Bacteriol. 3: 103-120, 2013

Introduction The beef industry has been significantly impacted by the emergence of Shiga toxin-producing EschCorrespondence: Todd Callaway, Tel: +1-979-260-9374 Fax: +1-979-260-9332.

erichia coli (STEC) bacteria which are naturally found in cattle (Karmali et al., 2010). STEC-caused illnesses are a zoonotic disease (Karesh et al., 2012) that costs the American economy more than $1 billion each year in direct and indirect costs from more than 175,000 human illnesses (Scallan et al., 2011; Scharff, 2010). While strategies focused on the prevention of transmission via carcasses have been largely suc-

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cessful, they are far from perfect (Arthur et al., 2007a; Barkocy-Gallagher et al., 2003). Thus it has been necessary to develop animal management controls as well as applicable intervention strategies for use in live cattle (Callaway et al., 2004b; LeJeune and Wetzel, 2007; Oliver et al., 2008; Sargeant et al., 2007). Because human STEC exposures are not limited only to food-based routes, but include animal contact, it is likely that reducing STEC in cattle can improve public health in rural communities, as well as in reducing foodborne illnesses (LeJeune and Kersting, 2010; Rotariu et al., 2012). As discussed previously (Callaway et al., 2013) the logic underlying focusing on reducing foodborne pathogenic bacteria in live cattle is straightforward: 1) reducing the amount of pathogens entering processing plants will reduce the burden on the plants and render the in-plant interventions more effective; 2) reducing horizontal pathogen spread from infected animals (especially in “supershedders”) in transport and lairage; 3) will reduce the pathogenic bacterial burden in the environment and wastewater streams; and 4) will reduce the direct risk to those in direct contact with animals via petting zoos, open farms, rodeos and to animal workers. This present review is intended to complement the accompanying STEC ecology and animal management-focused review (Callaway et al., 2013) and will stress the application of external intervention strategies focused on reducing STEC in live cattle. We will divide the interventions into two broad categories: 1) Probiotic approaches that utilize the competitive nature of the gastrointestinal microbiome, and 2) Anti-pathogen strategies that specifically target pathogens based on their physiology and ecological niches.

Probiotic approaches, harnessing microbial ecology In recent years, probiotic approaches (e.g., those that utilize live or dead cultures of microorganisms to alter the microbial population of the gut) have received increased interest as a method to reduce 104

foodborne pathogenic bacteria in cattle. Traditionally, probiotic products in the cattle industry have been used to enhance production efficiency of meat or milk (Callaway and Martin, 2006; Fuller, 1989; Tournut, 1989; Yoon and Stern, 1996). However recent years have an increase in the use of the probiotic types: direct fed microbials (DFM), competitive exclusion cultures (CE), and prebiotics to reduce E. coli O157:H7 populations in cattle (McAllister et al., 2011) and can be considered part of an “organic” approach to improving food safety (Siragusa and Ricke, 2012). In general it appears that probiotic products work to alter the microbial ecology of the gastrointestinal tract through a variety of mechanisms. As the DFM/ CE bacteria attach to the surface of the intestinal epithelium this physical binding can prevent opportunistic pathogens from attaching to the intestinal wall (Collins and Gibson, 1999; Kim et al., 2008). Volatile fatty acids produced by microbial fermentation can be toxic to some bacterial species (Ricke, 2003; Russell, 1992; Wolin, 1969), and other bacterial products (such as ethanol, traditional antibiotics, or colicins/ bacteriocins [described below]) are produced by some intestinal bacteria to eliminate competition within the same environmental niche (Jack et al., 1995). Collectively, these modes of action demonstrate the complexities involved with interrupting the cycle of transmission and colonization of cattle with E. coli O157:H7, and emphasize that a multiplehurdle using complementary interventions has the greatest chance of improving food safety at the live animal level.

Direct Fed Microbials Direct Fed Microbials are widely fed in beef and dairy cattle and are typically composed of yeast, fungal or bacterial cultures or end-products of fermentation, and the cultures may be live or dead. A DFM is fed to animals daily to improve the ruminal fermentation and production efficiency (Martin and Nisbet, 1992). Increasingly, companies claim some benefit to them in reducing E. coli O157:H7 shed-

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ding in cattle. Researchers compared several of the commercially-available growth enhancement probiotics and yeast products and found that feeding these probiotics provided no effect in regards to pathogen levels in cattle (Keen and Elder., 2000; Swyers et al., 2011). A probiotic culture comprised of Streptococcus bovis and Lactobacillus gallinarum from the rumen of cattle reduced E. coli O157 shedding when given to experimentally-infected calves, and this decrease was attributed to an increase in VFA concentration in the gut (Ohya et al., 2001). Probiotic products have been developed to specifically reduce E. coli O157:H7 shedding in cattle. A probiotic that contained S. faecium or a mixture of S. faecium, L. acidophilus, L. casei, L. fermentum and L. plantarum significantly reduced fecal shedding of E. coli O157:H7 in sheep, yet, a monoculture of Lactobacillus acidophilus was found to be ineffective (Lema et al., 2001). A DFM comprised of Bacillus subtilis did not affect the fecal prevalence or concentration of E. coli O157:H7 and did not impact average daily gain in feedlot cattle (Arthur et al., 2010a). Studies have also indicated that cultures of Lactobacillus acidilacti and Pediococus could directly inhibit E. coli O157:H7, likely through the production of organic acids and low pH (Rodriguez-Palacios et al., 2009). Other researchers demonstrated that a direct-fedmicrobial (DFM) L. acidophilus culture derived directly from the rumen of cattle reduced E. coli O157:H7 shedding by more than 50% when fed to feedlot cattle (Brashears and Galyean, 2002; Brashears et al., 2003a; Brashears et al., 2003b). In an independent evaluation, this DFM reduced fecal shedding of E. coli O157:H7 in cattle from 46% to 13% (Ransom et al., 2003). In a further refinement of this DFM, where the L. acidophilus cultures were combined with Propionibacterium freudenreichii (a propionate-producing commensal intestinal bacteria) a reduction in the prevalence of E. coli O157:H7 occurred in the feces from approximately 27% to 16% and reduced the prevalence on hides from 14% to 4% (Elam et al., 2003; Younts-Dahl et al., 2004). Further work with this DFM again showed that it reduced E. coli O157:H7 and Salmonella in feces and on hides (Stephens et

al., 2007b), and it further reduced concentrations of E. coli O157:H7 in the feces (Stephens et al., 2007a; Stephens et al., 2007b), which may be more of a critical impactor of carcass contamination than simple prevalence levels (Arthur et al., 2010b). Additional studies using only the L. acidophilus DFM found no impact of low dose DFM feeding on E. coli O157:H7 prevalence (Cull et al., 2012). It is important to note that in this study a low dose DFM product was utilized, and further research indicates that the effect on E. coli O157:H7 prevalence and concentrations is impacted by DFM dosage levels (Cull et al., 2012). This Lactobacillus-based DFM is currently marketed as Bovamine™ and Bovamine Defend™ based on dosing levels and both are widely used in the cattle industry because they have been reported to improve the growth efficiency of cattle, at least in a feedlot ration. There will likely not be a single DFM that can work effectively at reducing E. coli O157:H7 populations in cattle and improve production efficiency in all production systems (i.e., feedlots, cowcalf, stockers, and dairies). Therefore, alternative DFM cultures selected specifically for each production segment or situation need to be developed so that the food safety improvement can occur while economically balancing the cost of its inclusion in cattle rations thus “paying for” the enhancement of food safety.

Competitive exclusion Competitive exclusion (CE) is another probiotic approach that has been used to eliminate E. coli O157:H7 (as well as Salmonella) from cattle gastrointestinal tracts (Brashears and Galyean, 2002; Brashears et al., 2003a; Brashears et al., 2003b; Zhao et al., 2003). Competitive exclusion as a technology, involves the addition of a (non-pathogenic) bacterial culture (of one or more species) to the intestinal tract to reduce colonization or decrease populations of pathogenic bacteria (Fuller, 1989; Nurmi et al., 1992). An established gastrointestinal microbial population makes an animal more resistant to transient opportunistic infections (Fuller, 1989), because the species

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best adapted to occupy a particular niche within the intestinal tract succeeds, and pathogenic bacteria are generally viewed as opportunists. A CE culture should be derived from the animal of interest, thus CE cultures attempt to take advantage of co-evolution of host and microorganism. Depending on the stage of production of the animal (i.e., maturity of the gut), the goal of CE can be the exclusion of pathogens from the naïve gut of a neonatal animal, or the displacement of an already established pathogenic bacterial population (Nurmi et al., 1992). For example, many researchers have isolated commensal (non-pathogenic) E. coli strains that show tendencies to reduce E. coli O157:H7 populations, at least in vitro (Fox et al., 2009a; Reissbrodt et al., 2009; Zhao et al., 1998). Researchers used a defined population of multiple commensal E. coli strains that were isolated from cattle and found this generic E. coli CE culture could displace an established E. coli O157:H7 population from calves (Zhao et al., 1998). In a follow up study, calves that were colonized with the E. coli CE product shed less E. coli O111:NM and O26:H111 (both STEC strains isolated from cattle, but the CE product did not reduce E. coli O157:H7 (Zhao et al., 2003). Other researchers have isolated E. coli strains that display a “proximitydependent” killing of E. coli O157:H7 strains which could possibly be utilized in CE cultures or as a DFM (Sawant et al., 2011). While the mechanism of this killing has not been defined, it does not appear to be mediated by colicins or phages (Sawant et al., 2011).

Colicins and colicin-producing E. coli Colicins are antimicrobial proteins produced by certain E. coli strains that kill or inhibit the growth of other E. coli strains (Konisky, 1982; Lakey and Slatin, 2001; Smarda and Smajs, 1998), including E. coli O157:H7 (Jordi et al., 2001; Murinda et al., 1996; Schamberger and Diez-Gonzalez, 2002). The concept of using colicins as an intervention strategy to kill food borne pathogens is not new (Joerger, 2003; Murinda et al., 1996), but until recently has been lim106

ited by cost to use as treatment on finished meat products (Abercrombie et al., 2006; Liu et al., 2011; Patton et al., 2008) or vegetables (Nandiwada et al., 2004). Recently however, the costs of production and purification of colicins was lowered by recombination protein expression work (Stahl et al., 2004). Because of the increased availability of the colicins, scaled up studies could be conducted in a mouse model, where it was demonstrated that E. coli O157:H7 was prevented from colonization (Leatham et al., 2009). Recently, specific studies have examined the use of specific colicins against E. coli O157:H7 in vitro in gastrointestinal simulations (Callaway et al., 2004d) and against other E. coli in vivo (Cutler et al., 2007). In spite of the seemingly simple addition of a protein (colicin) to animal diets to control E. coli O157:H7, studies have indicated that the sensitivity of E. coli O157:H7 strains to any single colicin can be highly variable (Murinda et al., 1996; Murinda et al., 1998; Schamberger and Diez-Gonzalez, 2002). Because some E. coli O157:H7 strains are colicinogenic and produce specific concomitant immunity proteins (Murinda et al., 1998), these strains of E. coli O157:H7 can be resistant to certain added colicins or even a broad category of colicins (Alonso et al., 2000). Therefore, if colicins are to be used as a preharvest intervention strategy, there must be simultaneous administration of several categories of colicins. Furthermore, if colicins are to be a viable anti-E. coli O157:H7 intervention strategy, the proteins must be protected from gastric and intestinal degradation. As a way of getting colicins into the lower gut of cattle, researchers have proposed a specific form of DFM/CE of feeding colicin-producing E. coli in cattle rations (Schamberger and Diez-Gonzalez, 2002; Schamberger et al., 2004; Zhao et al., 1998). These strains have been shown to colonize the lower gut of cattle, but the reduction in concentration of E. coli O157 was approximately 2 log10 CFU/g, not a complete elimination (Nandiwada et al., 2004). The complex nature of ruminant animal gastrointestinal tract, and the long (12-18 month) life span of cattle going into a feedlot means that CE use in cattle as a “one shot” approach may not completely eliminate E. coli O157:H7 and other STEC shedding

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throughout the lifetime of the animal. So individual CE for various phases of production cycles or changes (e.g., entry to the feedlot) may need to be developed, or an early-established CE culture may be best supplemented over time by DFM and/or prebiotic feeding (synbiotics, described below).

Prebiotics Organic compounds that are unavailable to, or indigestible by the host animal, but are digestible by a specific segment of the microbial population are generally classified as “prebiotics” (Patterson and Burkholder, 2003; Schrezenmeir and De Vrese, 2001; Walker and Duffy, 1998). For example, fructo-oligosaccharides, are sugars that are not degraded by intestinal enzymes that can pass down to the cecum and colon to become “colonic food” for the host bacterial population and provide nutrients to the intestinal mucosa (Houdijk et al., 1998; Willard et al., 2000). Some prebiotics can provide a competitive advantage to specific members of the native microflora (e.g., Bifidobacteria, Butyrivibrio) that can help to exclude pathogenic bacteria from the intestine via direct competition for nutrients or for binding sites through the production of “blocking factors”, or antimicrobial compounds in a fashion similar to that of CE (Zopf and Roth, 1996). Other prebiotics (Celmanax) have been shown to have an anti-adhesive effect on E. coli O157:H7 in vitro using bovine cells, which should be investigated further (Baines et al., 2011). Coupling the use of CE and prebiotics is known as “synbiotics”, and could yield a synergistic effect in reduction of food-borne pathogenic bacterial populations in food animals prior to slaughter (Bomba et al., 2002). To date, prebiotics have not been widely implemented in cattle due to their expense, and the ability of ruminal microorganisms to degrade a wide variety of typical prebiotic substrates, however as costs change, their inclusion as part of a synbiotic directed anti-pathogen strategy may become feasible.

Anti-pathogen strategies, targeted treatment In spite of the potential of probiotic approaches, other pathogen-reduction strategies have been developed for use in the live animal that target pathogens directly. Many of these treatments utilize the host animal, natural members of the microbial ecosystem, or utilize an aspect of pathogen physiology to inhibit pathogen survival.

Antibiotics The use of antibiotics specifically to control E. coli O157:H7 shedding in cattle is controversial. Few researchers have delved into this area in cattle to date. Neomycin is an antibiotic that is approved for use in cattle to treat enteric infections and has been shown to reduce E. coli O157:H7 populations in the gut (Elder et al., 2002; Ransom et al., 2003) and on the hides of cattle (Ransom et al., 2003). Other researchers have found that in swine artificially infected with E. coli O157:H7, the feeding of chlortetracycline and tylosin decreased fecal shedding, while bacitracin did not impact E. coli O157:H7 populations (Cornick, 2010). It is hypothesized that the generalized disruption of the microbial ecosystem that is caused by antibiotic treatment indirectly affects the E. coli O157:H7 populations; the use of some antibiotics thus may provide E. coli O157:H7 a competitive advantage in the ruminant gastrointestinal tract. The use of antibiotics to reduce E. coli O157:H7 in cattle has not been recommended because of concerns relating to the development of antimicrobial resistance.

Bacteriophages Bacteria can be infected by naturally-occurring bacteriophages (bacterial viruses) that are found in many environments (Kutter and Sulakvelidze, 2005; Lederberg, 1996), including the intestinal tract of cattle (Callaway et al., 2006; Goodridge, 2008; Go-

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odridge, 2010). Phages can have very narrow target spectrums, and may only be active against a single bacterial species, or even strain because they target specific receptors on the surface of the bacterium (Lederberg, 1996). This specificity should allow phages to be used as an anti-pathogen treatment, a kind of “smart bomb” targeting on the species we wish to eliminate, without perturbing the overall microbial ecosystem (Johnson et al., 2008). Lytic phages “hijack” a targeted bacterium’s biosynthetic machinery to produce daughter phages; when intracellular nutrients are depleted, the host bacterium bursts, releasing phages to repeat the process in a fashion similar to a chain reaction. An exponential increase in the number of phages continues as long as target bacteria are present, allowing phages to persist in the environment rather than simply degrade over time as a chemical treatment. However, phage populations are self-limiting; if the targeted bacteria are removed from the environment, then phage populations diminish. One potential drawback to the use of phages is the rapid development of bacterial resistance to a single phage, thus much of the effort has been focused on the development of multi-phage cocktails (Tanji et al., 2005). Phages have been examined for use in two different approaches to reduce E. coli O157:H7, within the gut of cattle before slaughter, and as a hide or environmental decontaminant (Ricke et al., 2012). Commercial phage-based anti-E. coli O157:H7 are currently focused on the use of lytic phages in hide wash and surface cleansing products; FSIS has issued a letter of no objection to this use of phages. Phage products for use as a hide spray have been released into the marketplaces (Omnilytics and Elanco, Finalyse). Company-based research indicates a significant reduction in positive trim samples from cattle that were sprayed with this product. Processors are finding appropriate critical control points in which to include phage sprays on carcasses prior to de-hiding in relation to other hide spray intervention steps to reduce E. coli O157:H7 on the hides of cattle as they enter the food chain. Several phages isolated by European laboratories have shown promise as E. coli O157:H7 reduction agents sprayed on cattle hides, 108

but that they require an extended exposure time (1 h) to obtain maximal effect (Coffey et al., 2011). Interestingly, several phages have been isolated recently that are effective both against Salmonella spp. and E. coli O157:H7 (López-Cuevas et al., 2011; López-Cuevas et al., 2012; Park et al., 2012), which offers the hope of phage use as a broad-spectrum food safety improvement. Phages have been used successfully in several in vivo research studies examining the effect of phage on diseases that impact animal production efficiency or health (Huff et al., 2002; Smith and Huggins, 1982; 1983; 1987). Bacteriophage treatment reduced enterotoxigenic E. coli (ETEC)-induced diarrhea and splenic ETEC colonization in calves (Smith and Huggins, 1983; 1987). With the increasing focus on improving food safety throughout the food production continuum, bacteriophages have been used to control experimentally inoculated foodborne pathogenic bacteria, especially E. coli O157:H7 in cattle gastrointestinal tracts (Bach et al., 2003; Bach et al., 2009; Callaway et al., 2008; Kudva et al., 1999; Niu et al., 2008; Rozema et al., 2009). Several different phages have been isolated from feedlot cattle (Callaway et al., 2006; Niu et al., 2009; Niu et al., 2012; Oot et al., 2007) and other sources (Liu et al., 2012; McLaughlin et al., 2006) and have been used to reduce E. coli O157:H7 strains in experimentallyinfected animals as proofs of concept (Bach et al., 2009; Callaway et al., 2008; Rivas et al., 2010). In other studies, naturally phage-infected ruminants have been shown to be more resistant to E. coli O157:H7 colonization (Raya et al., 2006) and the presence of these endemic phages have often confused results of intervention studies (Kropinski et al., 2012). Commercialization studies for these on farm products have had mixed results (Stanford et al., 2010), but studies focusing on the development of appropriate, effective multi-phage cocktails are currently underway (Stanford and McAllister, personal communication). No matter what point in the beef production chain the phages are utilized in (e.g., hides or in the live animal), they must be carefully selected for: 1) action against multiple E. coli O157:H7 strains as well as other non-O157 STEC strains, 2) members

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of a cocktail must utilize different receptors to minimize resistance development, and 3) must be strictly lytic (i.e., does not transfer genetic material) because phage-mediated transfer is the mechanism by which STEC originally acquired their Shiga-toxin genes (Brabban et al., 2005; Law, 2000).

Vaccination Immunization has worked very effectively against pathogenic bacteria, including E. coli strains that cause edema disease in pigs and Salmonella in poultry (Gyles, 1998; Johansen et al., 2000). Unfortunately, because EHEC/STEC do not cause disease in cattle, the immunostimulation provided by these foodborne pathogens is not as potent, because it appears that natural exposure to E. coli O157:H7 does not confer protection to the host (Gyles, 1998). Thus vaccine production has specifically targeted aspects of the physiology of E. coli O157:H7 (Walle et al., 2012). Vaccination is widely accepted in the cattle industry, thus it is reasonable to predict that producers will implement this pathogen reduction technique if the vaccine is economically feasible, and can be incorporated into existing production systems. To date, two basic targeting strategies have been utilized to develop vaccines against E. coli O157:H7, and both have had their successes (Snedeker et al., 2012; Varela et al., 2013; Walle et al., 2012).

Siderophore Receptor and Porin (SRP) protein vaccines Siderophores are proteins excreted by bacteria in an effort to obtain iron from its environment, and E. coli O157:H7 utilizes secreted siderophores in the intestinal tract of cattle. The SRP vaccine targets this protein and disrupts iron transport into the bacterium, resulting in cell death. The EpitopixTM SRP vaccine has been conditionally approved for use in cattle in the U.S. and is undergoing additional safety and efficacy tests. Preliminary research results are promising when the vaccine is utilized in a 3 dose

treatment regimen (Thornton et al., 2009). Other researchers found that vaccination with the SRP reduced fecal concentrations of E. coli O157:H7 in cattle by 98%, but the vaccine did not affect cattle performance (Thomson et al., 2009). Vaccination of cattle with this SRP in another study reduced the prevalence of E. coli O157:H7 by nearly 50% (Fox et al., 2009b). A two-dose SRP vaccination reduced the prevalence and number of “high-shedding” cattle, with a reported efficacy of 53% and 77%, respectively (Cull et al., 2012). Vaccination of pregnant dams along with a second vaccination of calves was shown to reduce E. coli O157:H7 (from 25% to 15%, respectively) in feedlot cattle (Wileman et al., 2011).

Bacterial Extract Vaccines A vaccine produced from E. coli O157:H7 extracts (type III secreted proteins) has been produced as EconicheTM. This vaccine has been licensed in Canada and is pending a conditional license in the U.S. Preliminary experimental results indicated that this vaccine reduced E. coli O157:H7 shedding in feedlot cattle from 23% to less than 9% (Moxley et al., 2003; Potter et al., 2004; Van Donkersgoed et al., 2005). In an evaluation study, it was demonstrated that vaccination reduced fecal shedding from 46% to 14% (Ransom et al., 2003). Recent studies have shown an experimental three dose regimen reduced E. coli O157:H7 shedding by 65%, but that a 2 dose system was less effective (Moxley et al., 2009). However, in a follow up study, a two dose regimen was shown to reduce rectal colonization by E. coli O157:H7 in feedlot cattle (Smith et al., 2009b). The benefits of vaccinating cattle in reducing cattle hides positive for E. coli O157:H7 can be lost by comingling with non-vaccinated cattle during transport (Smith et al., 2009a). While the Econiche vaccine pioneered the use of bacterial extracts, other extract-type vaccines against multiple E. coli O157:H7 proteins (e.g., intimin and tir) have been produced that reduce fecal shedding in experimental-infection models (McNeilly et al., 2010); vaccines against a hemolysin pro-

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tein encoded in the locus of enterocyte effacement (LEE) island has also shown promise in reducing E. coli O157:H7 shedding in cattle (Sharma et al., 2011). Vaccines targeting EspA, EspB, shiga-toxin 2, and Intimin proteins have been used in pregnant cows, and it was shown that the antibodies were transferred to calves, but the effect of this vaccination on colonization was not determined (Rabinovitz et al., 2012). Further multi-protein vaccines have been developed that can reduce fecal shedding of E. coli O157:H7 in a sheep model (Yekta et al., 2011), including a Stx2B-Tir-Stx1B-Zot protein vaccine that successfully reduced E. coli O157:H7 shedding in a goat model (Zhang et al., 2012). Most excitingly, because the non-O157 STEC share the Type-III secretion system proteins, it appears that vaccines targeting these proteins (e.g., Tir, EspB, EspD, EspA, and NleA) can provide some degree of cross-protection from the non-O157 STEC (Asper et al., 2011). Bacterial ghosts (e.g., cellular membranes) have recently been used to produce an immune response that reduced E. coli O157:H7 populations in mice (Cai et al., 2010; Mayr et al., 2012) and calves (Vilte et al., 2012). A live-attenuated Salmonella strain that expresses the E. coli O157:H7 intimin protein has been demonstrated to induce immune responses in cattle (Khare et al., 2010). Others have devised chimeric multi-protein (eae, tir, intimin) vaccines (Amani et al., 2010) that can be produced in plants, potentially providing a source of an edible vaccine (Amani et al., 2011) that can be included in cattle rations rather than having to be injected via the stressful handling procedures currently required that add expense to the producers. However, for this approach to be utilized in ruminants, the proteins must be protected from the extensive proteolytic nature of the rumen microbial ecosystem, which will obviously add to the complexity and expense of vaccination via the edible vaccine approach.

Cattle Hide washing Currently, cattle hides are typically washed to remove visible contamination from hides. The hide 110

washes can contain antimicrobial compounds (e.g., organic acids [described in previous section], sodium hydroxide, trisodium phosphate [TSP], cetylpyridinium chloride [CPC] , hypobromous acid, or electrolyzed or ozonated water), which serves to reduce some of the bacterial contamination (including foodborne pathogens) entering the processing plant on the hide (Arthur et al., 2007b; Bosilevac et al., 2004; Bosilevac et al., 2005a; Bosilevac et al., 2005b; Schmidt et al., 2012). The most common hide/carcass rinse additive has been organic acids such as lactic or acetic acid (Berry and Cutter, 2000; Loretz et al., 2011). Hide washes significantly reduce the load of E. coli O157:H7 entering the plant on the hide, which has been linked to final carcass contamination levels (Arthur et al., 2007a; Arthur et al., 2010b), thus improving food safety; but they do not reduce the prevalence of E. coli O157:H7 entering the plant within the animal.

Sodium chlorate Addition of chlorate to E. coli cultures kills these bacteria because E. coli can respire under anaerobic conditions by reducing nitrate to nitrite via the dissimilatory nitrate reductase enzyme (Stouthamer, 1969). The intracellular bacterial enzyme nitrate reductase does not differentiate between nitrate and its analog, chlorate which is reduced to chlorite in the cytoplasm; chlorite accumulation kills bacteria (Stewart, 1988). Chlorate treatment in vitro quickly reduced populations of E. coli O157:H7 and Salmonella (Anderson et al., 2000a). Chlorate addition to animal rations reduced experimentally inoculated E. coli O157:H7 populations in swine and sheep intestinal tracts (Anderson et al., 2001; Edrington et al., 2003) as well as Salmonella in broiler intestinal contents (Byrd et al., 2003). Other studies indicated that soluble chlorate administered via drinking water significantly reduced E. coli O157:H7 ruminal, cecal and fecal populations in both cattle and sheep (Anderson et al., 2002; Callaway et al., 2002; Callaway et al., 2003). Hide contamination with E. coli O157:H7 plays a significant role in carcass/product contami-

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nation (Arthur et al., 2009; Arthur et al., 2010a; Arthur et al., 2010b), and chlorate treatment reduces both fecal and hide populations of E. coli (Anderson et al., 2005). In vitro and in vivo results have indicated that chlorate treatment does not adversely affect the ruminal or the cecal/colonic fermentation (Anderson et al., 2000b). Additional studies have demonstrated that chlorate alters neither the antibiotic resistance, nor toxin production by E. coli O157:H7 (Callaway et al., 2004a; Callaway et al., 2004c). The LD50 of sodium chlorate is from 1.2 to 4 g/kg BW; by way of comparison, the LD50 of sodium chloride is approximately 3 g/kg BW (Fiume, 1995). Therefore, it does not appear that chlorate poses a severe risk for use in animals due to inherent toxicity. Because of the dramatic impact chlorate has on food-borne pathogenic bacterial populations, it was suggested that chlorate could be supplemented in the last feeding before cattle are shipped to the slaughterhouse. The use of chlorate to reduce foodborne pathogenic bacteria in food animals is presently under review by the U. S. Food and Drug Administration, but has not been approved at this time.

What about potential unintended consequences? Before we attempt to completely eliminate STEC from the live animal, we must consider the law of unintended consequences, and its impact on food safety (Callaway et al., 2007). The poultry industry was hampered in the early part of the 20th century by fowl typhoid/cholera which impacted productivity and efficiency of production. This disease was caused by Salmonella Gallinarum and Pullorum, which do not cause illness in humans, but do cause illness solely in poultry (CDC, 2006). A concerted effort was made to rid the national poultry flock of these bacterial diseases, and this effort was successful at eliminating these diseases which were highly adapted to live only in their host (poultry). However, by removing a member of the microbial ecosystem from the intestinal meta-population, a niche in the ecosystem was opened (Kingsley and Bäumler, 2000).

This niche was occupied by another Salmonella that was not host-adapted and was transmitted from rodents to poultry, Salmonella Enteritidis (Kingsley and Bäumler, 2000). This foodborne pathogen has subsequently become widespread in the national poultry flocks and represents one of the most common serotypes isolated from human salmonellosis cases (CDC, 2006; Scallan et al., 2011). Therefore, in all our efforts to eliminate STEC from animals prior to slaughter, we must be aware that some other bacteria will undoubtedly fill the vacuum in the microbial ecosystem.

Conclusions Pre-harvest interventions to reduce E. coli O157:H7 and other STEC in cattle can reduce foodborne pathogen penetration into the food chain. However, implementation of these pre-harvest strategies does not eliminate the need for best practices in the processing plant and in the food preparation environment. Recent years have seen an increase in the research into developing new interventions (e.g., vaccination, DFM, chlorate, phages) and into understanding what effect the microbial population and host physiology has on STEC populations in the gut of cattle. This research has resulted in several novel interventions and potential dietary additions or changes that can reduce STEC in cattle, and many of them are in, or very near to entering, the marketplace. However, it must be noted that the live-animal interventions must be installed in a coherent, complementary fashion to reduce pathogens as part of an integrated multiple-hurdle approach that complements other post-harvest strategies to minimize pathogen contact and resultant human illnesses.

References Abercrombie, J. G., M. J. B. Paynter and S. S. Hayasaka. 2006. Ability of colicin V to control Escherichia coli O157:H7 in ground beef. J. Food Safety 26:103-114.

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are transferred to newborn calves. J. Dairy Sci. 95:3318-3326. Raya, R. R., P. Varey, R. A. Oot, M. R. Dyen, T. R. Callaway, T. S. Edrington, E. M. Kutter and A. D. Brabban. 2006. Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep. Appl. Environ. Microbiol. 72:64056410. Reissbrodt, R., W. P. Hammes, F. Dal Bello, R. Prager, A. Fruth, K. Hantke, A. Rakin, M. Starcic-Erjavec and P. H. Williams. 2009. Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli. FEMS Microbiol. Lett. 290:62-69. Ricke, S. C. 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult. Sci. 82:632-639. Ricke, S. C., P. Hererra and D. Biswas. 2012. Bacteriophages for potential food safety applications in organic meat production. In: S. C. Ricke, E. J. Van Loo, M. G. Johnson and C. A. O’Bryan Ed. Organic meat production and processing. John Wiley & Sons, Inc., New York. 407-424. Rivas, L., B. Coffey, O. McAuliffe, M. J. McDonnell, C. M. Burgess, A. Coffey, R. P. Ross and G. Duffy. 2010. In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7. Appl. Environ. Microbiol. 76:7210-7216. Rodriguez-Palacios, A., H. R. Staempfli, T. Duffield and J. S. Weese. 2009. Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5. J. Appl. Microbiol. 106:393-401. Rotariu, O., I. Ogden, L. MacRitchie, K. Forbes, A. Williams, P. Cross, C. Hunter, P. Teunis and N. Strachan. 2012. Combining risk assessment and epidemiological risk factors to elucidate the sources of human E. coli O157 infection. Epidemiol. Infect. 140:1414-1429. Rozema, E. A., T. P. Stephens, S. J. Bach, E. K. Okine, R. P. Johnson, K. I. M. Stanford and T. A. McAllister. 2009. Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in 118

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REVIEW Potential for Rapid Analysis of Bioavailable Amino Acids in Biofuel Feed Stocks D. E. Luján-Rhenals1,2 and Ruben Morawicki1 1


Food Science Department, 2650 Young Ave., University of Arkansas, Fayetteville, AR

Current address: Universidad de Córdoba, sede Berástegui. Km. 12 vía Cereté-Ciénaga de Oro, Córdoba, Colombia

ABSTRACT As the biofuels industry continues to grow and technologies to recover fermentable sugars from feedstocks improve, the leftover byproducts are becoming richer in proteins. These byproducts could potentially serve as sources of dietary protein for food animals. However, the uneven quality as well as the potentially large quantities would need the assessment of bioavailability of the amino acid content for the proper formulation of these for animal diets, which would require in vitro assays that best approximated animal bioavailability response. Among the in vitro methods, Escherichia coli – based biosensors show promise to fulfill this need. With the bulk of the work done on lysine and methionine, considerable research has been conducted on E. coli – based biosensors to optimize culture conditions and improve detection sensitivities. This review will discuss the current knowledge on E. coli – based biosensors and potential research directions for the future. Keywords: Escherichia coli, biosensors, amino acids, biofuels, feedstocks, rapid, bioassay Agric. Food Anal. Bacteriol. 3: 121-128, 2013

Introduction In recent years the emergence of cereal crops as biofuel feedstocks has grown to the extent that for some cereal grains, such as corn, bioethanol production has directly competed with its more traditional use as food and feed (Wisner and Baumel, 2004; Mayday, 2007). This competition has generated conCorrespondence: Ruben Morawicki, Tel: +1 -479-575-4923 Fax: +1-479-575-6936

siderable debate on the economics on further development of grain crops to generate ethanol (Wisner and Baumel, 2004; Johnson, 2007; Mayday, 2007; Buyx and Tait, 2011). Biofuel economic issues are reflected in not only the price and availability of the cereal grain substrates but also in the availability and efficiency of the fermentation (Somma et al., 2010). In addition, the efficiency of ethanol production by yeasts can be compromised by the presence of other undesirable microorganisms, which not only compete

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for substrates but potentially inhibit yeasts by producing organic acids with antimicrobial properties (Ricke, 2003; Muthaiyan and Ricke, 2010; Muthaiyan et al., 2011; Limayen and Ricke, 2012). Some of the inhibition issues can be resolved with further improvements on the genetics of ethanol-producing microorganisms (Ragauskas et al., 2006; Limayen and Ricke, 2012). Several opportunities exist for either genetically modifying microorganisms to utilize more diverse carbon substrates or isolating microorganisms with these capabilities (Limayen and Ricke, 2012). However, for ethanolic fermentations, achieving better carbon/sugar extraction efficiencies is only part of the solution. Despite the apparent competing interests between the biofuel industry and the livestock agricultural sector, the opportunities for a more synergistic relationship are possible. As the development of more sophisticated biofuel processes continues, it is anticipated that a more efficient extraction of carbon for biofuel generation will become more pronounced, which will leave carbon-poor-proteinenriched byproducts that are of limited use as biofuel substrates. However, these byproducts offer proteins that can serve as amino acid sources for meeting animal nutritional requirements. Besides the fact that these protein sources may have variable availability, there are other potential problems including non-optimal levels of essential amino acids and imbalances in amino acid profiles, which can be problematic when fed to animals as excess dietary protein. This can result in environmental nitrogen emission that can not only be an economic waste but also result in the contamination of surface and ground water sources from excess animal nitrogen emissions (Kim et al., 2006; Chalova et al., 2009a; Hunde et al., 2012). Some of these problems can be solved via supplementation of the deficient amino acids identified in the dietary formulation. However, the key for correct supplementation depends on both the identification of deficient amino acids and the quantification of their availability. The remainder of this review focuses on defining amino acid availability in general with particular emphasis on lysine, which is one of the most critical amino acids. To con122

clude, a discussion will cover the emergence of rapid methods for quantifying amino acids in general, particularly lysine.

Bioavailability of amino acids Bioavailability of a particular dietary amino acid is the fraction of the ingested amino acid that is absorbed in a chemical form suitable for metabolism or protein synthesis (Batterham, 1992; Johnson, 1992; Lewis and Bayley, 1995; Zebrowski and Buraczewski, 1998; Gabert et al., 2001). Traditionally, the gold standard to estimate amino acid availability has been the use of in vivo assays (Sauer and Ozimek, 1986; Applegate et al., 2004). Another traditional method uses a slope-ratio assay to estimate the bioavailability, in which the response—whole body protein deposition (Batterham, 1992) or amino acid oxidation (Moehn et al., 2005)—is correlated with the amino acid intake. Amino acid availability is determined by comparing the regression line of the test diet with a reference protein diet. The ratio of the slope of the test feed ingredient to the slope of the reference protein represents the relative bioavailability of the amino acid in question. Unfortunately, these animal-based bioavailability and digestibility methods are expensive, tedious, and time-consuming (2 to 4 weeks) and do not lend themselves well to high-throughput analyses that would be needed for large numbers of samples (Erickson et al., 2002). Additionally, they require special facilities and large amounts dietary materials. In addition, these in vivo methods are limited in the number of feed ingredients that can be compared simultaneously and increasing animal welfare concerns make it more difficult to conduct the trials (Erickson et al., 2002; Chalova et al., 2009a). There are no direct in vitro measures of amino acid bioavailability that duplicate exactly in vivo tests because of the complexity of the intestinal system and animal variability (Ravindran and Bryden., 1999; Erickson et al., 2002; Applegate et al., 2004; Chalova et al., 2009a, b, 2010). Although chemical separationbased methods, for instance high performance liq-

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uid chromatography (HPLC) or gas chromatography, are rapid and easily available, the determination of amino acid concentrations in feed ingredients does not reflect the actual amounts of amino acids absorbed under physiological conditions (Kivi, 2000; Ericksonet al., 2002; Froelich and Ricke, 2005). However, some of the in vitro methods that are available, such as amino acid enzyme-based digestibility and microbiological (biosensor) assays, can approximate values generated from animal studies (Erickson et al., 1999a; Chalova et al., 2007, Schasteen et al., 2007; Stein et al., 2007). Among them, microbiological assays for amino acid bioavailability are considered one of the most effective approaches in terms of time, cost and variability (Erickson et al., 2002; Froelich and Ricke, 2005; Chalova et al., 2009a,b, 2010).

Microbial-based amino acid biosensors Rapid tools with high specificity for food and fermentation analysis, such as new biosensor- based assays, are continually being developed to detect and quantify nutritional components, food additives, and contaminants. Biosensors consist of microorganisms—mainly bacteria due to rapid growth—or enzymes that can interact either physiologically or chemically with low concentrations of a compound of interest (Lei et al., 2006). Biosensors are very specific, sensitive, and flexible to use, and do not require large and expensive instrumentation as chemical analyses do (Chalova et al., 2009a, b). Common applications of bacterial-based biosensors include detection of antibiotics, ethanol, metals, phenolic compounds, sugars, urea and vitamins, as well as other compounds (Ricke and Zabala-Díaz, 2001; Lei et al., 2006; Chalova et al., 2009a, b). A microbial cell-based biosensor, also referred to as a whole cell sensor, consists of a viable bacterial cell that has been selected or genetically modified to quantify a particular metabolite. It is followed by a detection device that typically uses a colorimetric enzymatic response, a bioluminescence reaction, or fluorescence mediated by a green fluorescent pro-

tein (Lei et al., 2006; Chalova et al., 2009a). The ability of a microorganism to grow on a particular nutrient is the basis for detection and the extent of the growth provides data for quantification. Quantification can be done by following the optical density with a spectrophotometer, the luminescence in cells containing the lux gene, or the fluorescence in cells containing genes that synthesize green fluorescence proteins (GFP). The levels of growth are consistently proportional to the external concentration of the metabolite of interest (Erickson et al., 2000, 2002; Froelich and Ricke, 2005; Chalova et al., 2007; 2008b, 2009a, b, 2010; Bertels et al., 2012). More direct biosensor assays are possible by constructing gene fusions between promoter genes, which recognize a particular external metabolite, and a structural gene element responsible for synthesizing lux gene-based proteins or GFP (Lei et al., 2006; Zabala-Díaz et al., 2007; Chalova et al., 2008a, 2009c).

E. coli-based Amino Acid Biosensors Microbial methods for quantification of amino acid bioavailability are generally considered user friendly, relatively precise, specific, and economical (Shockman, 1963; Erickson et al., 2002). They include different assay microorganisms, which are based on their nutritive requirements for the respective amino acid (Shockman, 1963). E. coli has been one of the most highly investigated microorganisms for amino acid bioavailability quantification because this bacterium offers several advantages over other microorganisms as originally outlined by Payne and Tuffnell (1980). These advantages include: (1) it has one of the lowest doubling times (one of the fastest growth rates) among bacteria; (2) it is relatively easy to growth with minimal nutritional supplementation of the media; (3) the genetics are extremely well established and universally recognized; and (4) it can be easily manipulated to produce desired phenotypic responses for each respective nutrient to be assayed, such as amino acids. Additionally, E. coli is naturally found in

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the majority of animals and human intestine with a similar absorption of amino acids and peptides, attributes that make E. coli very functional as a biosensor microorganism for these substances (Ingraham et al., 1983; Chalova et al., 2009b). Several studies have been conducted over the years to develop specific E. coli-based assays for amino acids (Payne and Tuffnell, 1980; Hitchins et al., 1989; Erickson et al., 2002; Froelich and Ricke, 2005, Chalova et al., 2009a, b, 2010; Bertels et al., 2012). The basis for most of these E. coli sensors is that cell growth correlates with amino acid concentrations. However, since all the 20 essential amino acids can be synthesized by the wild type E. coli in a medium containing only a carbon source and inorganic salts (Neidhardt et al., 1990), this wildtype strain cannot be used directly for amino acid quantification. Instead, multiple mutants of E. coli have been created by genetic manipulation and studied for the purpose of quantifying amino acid bioavailability (Payne and Tuffnell, 1980; Hitchins et al., 1989; Erickson et al., 2002; Froelich and Ricke, 2005; Bertels et al., 2012). As a result, the genetically modified E. coli becomes an auxotroph, for a particular amino acid, and is consequently incapable of synthesizing that amino acid. Thus, the cell growth of the auxotroph is a direct function of the concentration of the amino acid evaluated (Gavin, 1957; Erickson et al., 2002), and its quantity can be determined by the extent of cell growth.

Microbial Biosensor for Methionine Availability Assays Extensive characterization and modification of microbial biosensors have been conducted for the essential amino acid methionine (Schwab, 1996; Webel and Baker, 1999; Boisen et al. 2000; Froelich and Ricke, 2005; Chalova et al., 2009a, b). Froelich et al. (2002a) determined that the growth kinetics of E. coli methionine mutant was not influenced by the presence of antibiotics or antifungal agents, which could potentially be used to eliminate interfereing background microflora during E. coli growth assays. 124

This was similar to the previous result observed by Erickson et al. (1999b) for an E. coli lysine auxotroph. This auxotroph was demonstrated to work as an ODbased assay for estimating crystalline methionine in poultry feeds (Zabala Díaz et al., 2004). It was found that it produced similar growth kinetics with either methionine or a commercial source of a nutritional methionine analogue supplement (Froelich et al., 2002b). Further improvements included adaptation to a microtiter-based assay and construction of luminescent and GFP-based strains (Zabala Díaz et al., 2003; Froelich et al., 2002c, 2005; Bertels et al., 2012). By deleting genes involved in amino acid biosynthesis, Bertels et al. (2012) developed E. coli biosensors able to quantify eleven amino acids—arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine—at a sensitive level comparable to HPLC analysis.

Microbial Biosensor for Lysine Bioavailability Assays Lysine is nutritionally one of the most important amino acids and is often the first limiting amino acid for humans and monogastric animals (Hurrell and Carpenter, 1981; Jørgensen et al., 1997; Chalova et al., 2009a). A variety of microorganisms have been used over the years as lysine biosensors, but E. coli has been one of the most extensively examined microbial-based assays for quantification of lysine bioavailability (Tuffnell and Payne, 1985; Anantharaman et al., 1983; Hitchins et al., 1989; Erickson et al., 2002). Early on, a high correlation (>0.9) between the microbiological and chemical methods in the quantification of available lysine was achieved (Anantharaman et al., 1983); and therefore E. coli estimates appear to be an accurate predictor of lysine bioavailability in a variety of protein sources (Tuffnell and Payne, 1985; Hitchins et al., 1989; Erickson et al. 1999a). Several studies were focused on improvements on growth response by modifications of the growth protocol, including agitation to reduce the time of incubation, increasing the number of cells in

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the inocula, determining the level of growth interference by the presence of Maillard-lysine products, and developing cryopreservation procedures (Li et al., 1999, 2000; Li and Ricke 2002a,b, 2004). Initially genetic refinement focused on constructing more precise lysine auxotrophic E. coli mutants by insertion mutagenesis; but since then more stable deletion mutants have been generated (Li and Ricke, 2003a,b,c, Bertels et al., 2012). Increasing detection sensitivity was originally based on bioluminescent emitting lysine auxotrophs but due to the requirement for multiple reagents later work focused on using fluorescent dyes and eventually generation of GFP expressing lysine auxotrophs (Erickson et al., 2000; Zabala Díaz and Ricke, 2003; Zabala Díaz et al., 2007; Chalova et al., 2004,2006, 2007, 2008a; Bertels et al., 2012).

CONCLUSIONS Protein-rich byproducts from the biofuel industry have the potential to be valuable sources of dietary protein for food animal feed. However, the uneven quality of these byproducts as well as their large quantities generated would require a systematic evaluation of the amino acids bioavailability almost on batch-to-batch basis. Unfortunately, animalbased bioavailability assays would not be able to accommodate this need, thus requiring the development of rapid in vitro assays. The construction of E. coli whole-cell-biosensors offer an opportunity to satisfy this need; but, further refinement, such as development of solid phase or bead anchored systems, are still needed to make these biosensors more user friendly and have a broader application spectrum. Given the advancements made in other biological detection systems, such as those for foodborne pathogens, the adaptation of these systems to E. coli could be a fairly straight forward process.

Acknowledgements This review was partially supported by the Arkan-

sas Soybean Promotion Board. Deivis Enrique LujánRhenals was supported in part by Colciencias of Colombia and the Universidad de Córdoba (Colombia).

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Chalova, V.I., C.L. Woodward, and S.C. Ricke. 2009c. CadBA induction of an Escherichia coli lysine auxotroph transformed with a plasmid carrying cad-gfp transcriptional fusion. Antonie van Leeuwenhoek J. Gen. Mol. Microbiol. 95:305-310. Chalova, V.I., C.L. Woodward, and S.C. Ricke. 2008a. Construction of a cad-gfpmut3 plasmid-construct in Escherichia coli for gene induction-based quantification of lysine in acid hydrolysates of feedstuffs. Lett. Appl. Microbiol. 46:107-112. Chalova, V., I.B. Zabala-Díaz, C.L. Woodward, and S.C. Ricke. 2008b. Development of a whole cell sensor - green fluorescent sensor method for lysine quantitification. World J. Microbiol. Biotechnol. 24:353-359. Chalova, V.I., Kim, W.K., Woodward, C.L., Ricke, S.C. 2007. Quantification of total and bioavailable lysine in feed protein sources by a whole-cell green fluorescent protein growth based Escherichia coli biosensor. Appl. Microbiol. Biotechnol. 76: 91-99. Chalova, V., C.L. Woodward, and S.C. Ricke. 2006. Assessment of an Escherichia coli lysine auxotroph containing green fluorescent protein for quantifying bioavailable lysine in animal protein samples under nonsterile and autofluorescence background. Lett. Appl. Microbiol. 42:625-270. Chalova, V., C.L. Woodward, and S.C. Ricke. 2004. Fluorescent DNA binding dye-based approach for measuring the growth of an Escherichia coli lysine auxotroph and quantifying lysine. J. Rapid Meth. Automation Microbiol. 11:313-324. Erickson, A.M.. X. Li, I.B. Zabala-Diaz, and S.C. Ricke. 2002. Potential for measurement of lysine bioavailability in poultry feeds by microbiological assaysA Review. J. Rapid Methods Automation Microbiol.10:1-8. Erickson, A.M., Zabala-Diaz, I.B., Kwon, Y.M., Ricke, S.C. 2000. A bioluminescent Escherichia coli auxotroph for use in an in vitro lysine availability assay, J. Microbiol Methods 40:207-212. Erickson, A.M., X. Li, C.L. Woodward and S.C. Ricke. 1999a. Optimisation of enzyme treatment for the degradation of feed proteins for an Escherichia coli auxotroph lysine availability assay. J. Sci. Food Agric. 79:1929-1935. 126

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reactions, Progress in Food & Nutrition Science 5:159-176. Ingraham, J.L., O. Maaloe, F. C. Neidhardt. 1983. Growth of the bacterial cell; Sinauer Associates, Inc. Sunderland, MA, USA. Johnson, J. 2007. Ethanol—is it worth it? Chem. Eng. News 85:19–21. Johnson, R.J. 1992. Principles, problems and application of amino acid digestibility in poultry. World’s Poult. Sci. J. 48:232-246. Jørgensen H, V.M. Gabert, and B.O. Eggum. 1997, The nutritional value of high-lysine barley determined in rats, young pigs and growing pigs, J. Sci. Food Agric. 73: 287-295. Kim, W.K., C.A. Froelich, Jr., P.H. Patterson, and S.C. Ricke. 2006. The potential to reduce poultry nitrogen emissions with dietary methionine or methionine analogues supplementation. World’s J. Poult. Sci. 62: 338-353. Kivi, J.T. 2000. Amino acids. In: L. M. L. Nollet. Ed. Food analysis by HPLC. Marcel Dekker Inc., New York, NY, USA; p. 55. Lei, Y., W. Chen, and A. Mulchandani. 2006. Microbial biosensors. Anal. Chim. Acta 568:200-210. Lewis, A. J., and H. S. Bayley. 1995. Amino acid bioavailability. p 35–65. In: C. B. Ammerman, D. H. Baker, and A. J. Lewis. Eds. Bioavailability of nutrients for animals, amino acids, minerals, and vitamins. Academic Press, New York, NY. Li, X. and S.C. Ricke. 2003a. A suicide vector for constructing a lysA insertion mutation in Escherichia coli. J. Rapid Meth. Automation Microbiol. 10:281290. Li, X. and S.C. Ricke. 2003b. Characterization of Escherichia coli lysA insertion targeted mutant using phenotype arrays. Bioresource Technol. 89:249253. Li, X. and S.C. Ricke. 2003c.Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay. Letts. Appl. Microbiol. 36:458-462. Li, X. and S.C. Ricke. 2004. Comparison of cryoprotectants for Escherichia coli lysine bioavailability assay culture. J. Food Proc. and Pres. 28:39-50.

Li, X. and S.C. Ricke. 2002a. Influence of soluble lysine Maillard reaction products on Escherichia coli amino acid lysine auxotroph growth based-assay. J. Food Sci. 67:2126-2128. Li, X. and S.C. Ricke. 2002b. Specificity of Escherichia coli amino acid lysine auxotroph growth kinetics and lysine assay response to various soluble Maillard reaction products. J. Food Proc. Pres. 26:279294. Li, X., A.M. Erickson, and S.C. Ricke. 2000. Agitation during incubation reduces the time required for a lysine microbiological growth assay using an Escherichia coli auxotrophic mutant. J. Rapid Meth. Automation Microbiol. 8:83-94. Li, X., A.M. Erickson, and S.C. Ricke. 1999. Comparison of minimal media and inoculum concentration to decrease the lysine growth assay response time of an Escherichia coli lysine auxotroph mutant. J. Rapid Methods Automation Microbiol. 7:279-290. Limayen, A. and S.C. Ricke. 2012. Lignocellulosic biomass for bioethanol production: Current perspectives, potential issues and future prospects. Prog. Energy Comb. Sci. 38:449-467. Mayday, J. 2007. Food, feed, or fuel: ethanol boom reverberates throughout the food system. Meat & Poultry 53:10–12. Moehn, S., R. F. P. Bertolo, P. B. Pencharz, and R. O. Ball. 2005. Development of the indicator AA oxidation technique to determine the availability of AA from dietary protein in pigs. J. Nutr. 135:2866– 2870. Muthaiyan, A. and S.C. Ricke. 2010. Current perspectives on detection of microbial contamination in bioethanol fermentors. Bioresource Technol. 101:5033-5042. Muthaiyan, A., A. Limayen, and S.C. Ricke. 2011. Antimicrobial strategies for limiting bacterial contaminants in fuel bioethanol fermentations. Prog. Energy Comb. Sci. 37:351-370. Neidhardt, F.C., Ingraham, J.L.; Schaechter, M. 1990. Growth of cells and populations. In: F. C. Neidhardt, J. L. Ingraham, M. Schaechter. Eds. Physiology of the bacterial cells: a molecular approach; Sinauer Associates, Sunderland, UK; p. 197. Payne, J.W., and J. M. Tuffnell. 1980. Assays for ami-

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no acids, peptides and proteins. In: J. W. Payne. Ed. Microorganisms and nitrogen sources; John Wiley & Sons: New York, NY, USA; p. 727. Ragauskas A.J., C.K. Williams, B. H. Davison, G. Britovsek, J. Cairney, C. A. Eckert, W.J. Frederick, Jr., J.P. Hallett, D.J. Leak, C.L. Liotta, J.R. Mielenz, R. Murphy, R. Templer and T. Tschaplinski, 2006, The path forward for biofuels and biomaterials, Science 311:484-489. Ravindran, V. and W. L. Bryden. 1999. Amino acid availability in poultry-in vitro and in vivo measurements. Aust. J. Agric. Res. 50:889-908. Ricke, S.C. 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sci. 82:632-639. Ricke, S.C., and I. B. Zabala-Díaz. 2001. A potential need for development of rapid microbiological assays to quantify folate concentrations in foods. Recent Developments in Nutrition 4 (Research Signpost, Trivandrum, India):1-5. Sauer, W. C., and L. Ozimek. 1986. Digestibility of AA in swine: Results and their practical applications. A review. Livest. Prod. Sci. 15:367–388. Schasteen, C.S., J. Wu, M.G. Schulz, and C.M. Parsons. 2007. Correlation of an immobilized digestive enzyme assay with poultry true amino acid digestibility for soybean meal. Poult. Sci. 86:343–348. Schwab, C.G. 1996. Rumen-protected amino acids for dairy cattle: Progress toward determining lysine and methionine requirements. Anim. Feed Sci.Technol. 59:87–101. Shockman, D. 1963. Amino acids. In Analytical microbiology; F. Kavanagh, Ed.; Academic Press: New York, NY, USA; p. 567. Somma, D. H. Lobkowicz, and J.P. Deason. 2010. Growing America’s fuel: An analysis of corn and cellulosic ethanol in the United States. Clean Techn. Environ. Policy 12:373-380. Stein, H.H., Sève, B. Fuller, M. F., Moughan, P. J. , and De Lange, C. F. M. 2007. Amino acid bioavailability and digestibility in pig feed ingredients: terminology and applications. J. Anim. Sci. 85:172-180. Tuffnell, J.M., and Payne, J.W. 1985. A colorimetric enzyme assay using Escherichia coli to determine nutritionally available lysine in biological materials. 128

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Isolation and Initial Characterization of Acetogenic Ruminal Bacteria Resistant to Acidic Conditions P. Boccazzi1,2 and J. A. Patterson1 1

Department of Animal Sciences, Purdue University. West Lafayette, IN 47907 2 Current address: Pharyx Inc., 801 Albany st, Ste 112C. Boston, Ma 02140

ABSTRACT Methanogenesis is a predominant fermentation reaction in the gut ecosystem of ruminants. A functional replacement of methanogenesis with acetogenesis in the rumen could potentially decrease energy losses and increase the efficiency of ruminant production. Hydrogen limited continuous cultures, at pH 6.0, were used to isolate over 40 potentially acetogenic bacteria from ruminal contents of a fistulated dairy cow. The dairy cow was at mid-lactation, consuming a 56% hay and 44% corn silage-concentrate diet. Eight bacterial isolates had the ability to grow on CO2 and H2 as their sole carbon and energy source producing acetate as the main end product. Keywords: acetogen, ruminal buffer, acetate production, H2 utilization

Agric. Food Anal. Bacteriol. 3: 129-144, 2013

Introduction During rumen fermentation, complex carbohydrates (e.g., cellulose) are degraded to monomeric carbohydrates (e.g., glucose and other soluble carbohydrates) which are primarily fermented to pyruvate via the Embden-Meyerhof-Parnas pathway (Pinder et al., 2012; Weimer et al., 2009). Pyruvate Correspondence: J. A. Patterson, Tel: +1 -765-494-4826 Fax: +1-765-494-9347

is subsequently metabolized to volatile fatty acids (VFA; acetate, propionate, and butyrate), CO2, H2, microbial cells and intermediate endproducts which can serve as crossfeeding substrates for other ruminal microorganisms such as Selenomonas ruminantium (Ricke et al., 1996). While fermentation acids provide 60 to 80% of the daily metabolizable energy intake of ruminants (Annison and Armstrong, 1970), microbial cells provide an important source of amino acids, vitamins, and cofactors (Hungate, 1966). Interspecies H2 transfer is a syntrophic interac-

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tion between H2-producing and H2-consuming organisms, that plays an important role in regulating fermentation environments (McInerney et al., 2011). Hydrogen produced by fermentative microorganisms is consumed by H2 utilizing microorganisms (methanogens, sulfidogens, and acetogens). The decrease in H2 concentration, due to interspecies H2 transfer, influences VFA fermentation patterns of many ruminal microorganisms. When H2 concentrations are high, pyruvate is utilized as a reducing equivalent acceptor and more reduced fermentation products (e.g., propionate, lactate, and ethanol) are produced. When H2 concentrations are low, there is an increase in acetate and ATP production that could be converted into an increase in overall microbial cell yields. Since energy lost as methane has been estimated to be 2.4 to 7.4% of the gross energy intake (Branine and Johnson 1990) or 10 to 15% of the apparent digestible energy of the diet of ruminants (Blaxter and Clapperton, 1965), there has been an interest to specifically inhibit methanogenesis to enhance animal productivity. Direct inhibition of methanogenesis, however, also results in loss of energy in the form of H2, and reduced microbial proteins (Chalupa, 1980).

Acetogenesis has been demonstrated to be the predominant fate of H2 in some humans, swine, xylophagus termites, cockroaches and rats (Breznak and Blum, 1991; Ljoie et al., 1988). Replacing methanogenesis with acetogenesis in the rumen may have potential in decreasing energy losses in ruminants. Blautia producta (Peptostreptococcus productus, Bryant et al., 1958), Eubacterium limosum (SharakGenthner, 1981), and Acetitomaculum ruminis (Greening and Leedle, 1989) are chemolithoautotrophic acetogenic bacteria that have been isolated from the bovine rumen. However they are not considered the primary H2 consuming organisms in this environment, since their numbers are consistently lower than methanogens. Factors dictating whether acetogenesis or methanogenesis will predominate in anaerobic environments are not well understood. Breznak and Kane (1990) suggested several possible factors that may influence the competitiveness of acetogens with methanogens. One factor is that methanogenesis has a higher energy yield than acetogenesis (Breznak and Blum, 1991). Another important factor is that methanogens have a higher affinity for H2 than acetogens. The

Maintaining the beneficial effects of interspecies H2 transfer while minimizing loss of energy as methane could enhance energy provided to ruminants by 22% (Schaefer, D., personal communication; Thauer, et al., 1977). However, an alternative electron sink is required to trap electrons into a form utilizable by the animal if methanogens are to be directly inhibited. Historically, the major method used to manipulate rumen fermentation and influence ruminant animal production has been the use of ionophore antibiotics such as monensin and lasalocid (Raun et al., 1976; Richardson et al., 1976; Berger et al., 1981; Ricke et al., 1984; Newbold et al., 2013). These compounds improve the efficiency of animal production by decreasing methane production and increasing ruminal propionate concentration by 15%. Methane production decreases primarily because monensin inhibits H2-producing microorganisms, therefore decreasing the amount of H2 available for methanogenesis.

normal rumen H2 concentration is between 10-5 and 10-6 atm (Robinson et al., 1981). Ruminal methanogens have an affinity for H2 between 1 and 4x10-6 atm (Greening et al., 1989). Different acetogenic isolates have been shown to have affinities for H2 between 10-4 and 10-5 atm (Greening et al., 1989; LeVan et al., 1998). In general, methanogens have been found to have H2 thresholds 10 to 40 fold lower than acetogens (Greening et al., 1989; Breznak and Blum, 1991). However, in our laboratory, acetogens with H2 thresholds only 2 to 4 fold higher than those of methnogens were isolated from ruminal contents (Boccazzi and Patterson, 2011). Finally, tolerance of bacteria to lower pH levels can also be a key factor in determining competitiveness in gastrointestinal environments including the rumen (Ricke, 2003; Russell, 1992). The objective of this study was to isolate chemolithoautotrophic acetogenic bacteria from ruminal contents of a dairy cow at low pH (5.5 to 6.0) and under H2-limiting conditions in order to select


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for bacterial strains with low H2 thresholds resistant to acidic environments.

Materials and Methods Source of Organisms Acetobacterium woodii (ATCC 29683) was obtained from the American Type Culture Collection (Rockville, MD). Acetogenic bacterial strains A10 and 3H (previously referred to as H3HH) were isolated and characterized previously in our laboratory (Boccazzi and Patterson, 2011; Pinder and Patterson, 2011, 2012, 2013; Jiang et al., 2012). This research was conducted prior to IACUC protocols being required for farm animal research. However animals were treated in accordance with currently approved IACUC protocols.

closed with butyl rubber stoppers and aluminum seals. The bottles were first flushed for 30 sec with an appropriate gas mixture by inserting both a gassing and a release needle through the serum stoppers and then they were pressurized to 200 kPa by removing the release needle. Oxygen traces were removed from gas mixtures by passing the gas through a reduced copper column. Pressurized bottles, unless otherwise specified, were incubated on their side on a rotatory shaker (New Brunswick Scientific Co. Inc., Model M52) operating at 200 rpm. For growth on solid medium, plates were incubated in an anaerobic growth vessel (made by the Agricultural and Biological Systems Department. Purdue University, IN) able to withstand high gas pressures. Prior to incubation the container was flushed for 2 min and then pressurized to 16 psi with gas mixtures specified in the text for each experiment.

Isolation of Acetogenic Bacteria Media and Growth Conditions Growth and H2 threshold experiments were conducted with a basal rumen fluid based acetogen medium or with Mac-20 medium containing casein hydrolysate and no rumen fluid (Table 1). Both media were prepared as described in Table 1 with the anaerobic techniques of Hungate (1966) as modified by Bryant (1972) and Balch and Wolfe (1976). The prepared medium was dispensed anaerobically into 60 mL or 120 mL serum bottles (West Company, Phoenixville, PA) or 20 mL serum tubes (Bellco Inc., Vineland, NJ) in an anaerobic glove box (Coy Laboratories, Ann Arbor, MI) containing a H2:CO2 (5:95) gas phase. Serum tubes and bottles were sealed with butyl rubber serum stoppers and aluminum seals (Bellco Inc., Vineland, NJ). All stock solutions utilized to formulate media were prepared anaerobically by boiling and cooling distilled water under CO2 and sterilized either by autoclaving or by injecting the solution through a 0.2 μm filter (Nalgene, Nalge Company, Rochster, NY). For chemolithoautotrophic growth in broth medium, bacterial cultures were grown in serum bottles

Determination of buffer capacity: MES versus citrate plus phosphate A batch culture experiment was performed to determine the best buffer system to use for isolating acetogenic bacteria at a pH range between 5.5 to 6.0. The experiment was conducted with the acetogen A10 (Boccazzi and Patterson, 2011). The four duplicate treatments were control plus 2-(N-morpholino)ethanesulfonic acid (MES), control plus citrate, A10 plus MES and A10 plus citrate. Serum bottles (60 mL) were anaerobically filled with 0.35 g alfalfa, 6 ml acetogen medium, 4 mL ruminal contents, 4 mL of A10 culture, or 4 mL of acetogen medium (control). MES was added to give a final concentration of 40 mM, citrate and KH2PO4 were added to a final concentration of 20 and 40 mM, respectively and 2-bromoethanesulfonic acid (BES) was added, to inhibit methanogens, to a final concentration of 5 mM. The alfalfa was dried at 60°C and ground through a 1 mm screen. Ruminal contents were collected anaerobically from a Holstein Friesian dairy cow prior to morning feeding and set on ice during transport-

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Table 1. Media compositiona Acetogen Medium

MAC-19 Mediumb

(amounts per liter)

(amounts per liter)

Rumen Fluid

50.0 mL


Mineral 1c

40.0 mL

40.0 mL

Mineral 2d

40.0 mL

40.0 mL

Additional Trace Min. Sol.e

10.0 mL

10.0 mL

Wolfe’s Trace Min. Sol.f

10.0 mL

10.0 mL

Vitamin Solutiong

10.0 mL

10.0 mL


4.0000 g

4.0000 g

Yeast Extract

0.5400 g

2.0000 g

Casein Hydrolysate


1.0000 g



1.0000 g


0.5400 g

0.5400 g


0.5000 g

0.5000 g

Resazurin solution

0.0010 g

0.0010 g

Hemin solution

0.0001 g

0.0001 g

All components, except Na2CO3 and Cysteine.HCl, were added to distilled water and brought to a volume of 1,000 mL. The resulting solution was mixed thoroughly and the pH adjusted to 7.0 with 1 M NaOH then gently heated and brought to a boil. Boiling was continued for 1 min., Na2CO3 added and cooled rapidly to 25°C under 100% CO2. Finally, cysteine.HCl was added, mixed thoroughly and autoclaved anaerobically for 12 min at 121°C and 15 psi. a


Modification of AC-19 medium by Breznak et al. (1988)


Mineral 1 (g/liter): 6.00 K2HPO4


Mineral 2 (g/liter): 12.00 NaCl, 6.00 K2HPO4, 6.00(NH4)2SO4, 2.45 MgSO4.7H20, 1.60 CaCl2.2H2O


Additional Trace Mineral Solution (g/Liter): 0.10 NiCl2.6H2O, 0.01 H2SeO3

Wolfe’s Trace Mineral Solution (g/liter): 3.00 Mg SO4.7H20, 1.00 NaCl, 0.50 MnSO4.H20, 0.10 CoCl2.6H20, 0.10 FeSO4.7H20, 0.10 CaCl2.2H20, 0.18 CoSO4.6H20, 0.19 ZnSO4.7H20, 0.02 AlK(SO4)2.12H20, 0.01 CuSO4.5H20, 0.01Na2MoO4.2H20 f

Vitamin Solution (g/liter): 0.10 pyridoxine.HCl, 0.056 ascorbic acid, 0.05 choline chloride, 0.05 thiamine.HCl, 0.05 D,L-6,8-thioctic acid, 0.05b riboflavin, 0.05 D-calcium panthotenic acid, 0.05 p-amino benzoic acid, 0.05 niacinamide, 0.05 nicotinic acid, 0.05 pyridoxal.HCl, 0.05 pyridoxamine, 0.05 myo-inositol, 0.02 biotin, 0.02 folic acid, 0.001 cynocobalamin.



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Figure 1. Continuous culture system utilized to isolate acetogenic bacteria, at low pH, from ruminal contents and to study the possibility to functionally replace methanogenesis with reductive acetogenesis in the rumen.

ing to the lab. Serum bottles were incubated on a rotatory shaker at 37°C. Strain A10 was grown in serum bottles (120 mL) filled with 50 mL of acetogen medium plus 10 mM glucose (0.5% inoculum) under 200 kPa of a H2:CO2:N2 (1:24:75:) gas mixture for 72 h. Measurements were final pH, headspace gas volume, and H2 and CH4 concentrations at 0 and 72 h of incubation.

Isolation of acetogenic bacteria from bovine ruminal contents using a continuous culture at pH 6.0 The continuous culture system used to isolate acetogenic bacteria is shown in Figure 1. The continuous culture system included two 20 L reservoirs

filled with 16 L of sterile medium, four 500 mL growth vessels (450 mL working volume), a Plexiglas water bath, a water heater/circulator (Vankel Heater/Circulator Bench SaverTM- Series VK 650A, Edison, NJ), a four channel peristaltic pump (Gilson Medical Electronics Inc., Middleton, WI), a magnet system operated by an electrical motor was used to turn stirrers in growth vessels and four 3.8 L plastic containers were used to collect culture effluent. The isolation medium was the acetogen medium (Table 1) modified by the addition of 40 mM MES (final concentration), 2.5 % (v/v), instead of 5% of clarified ruminal contents (Greening and Leedle, 1989) and 5 mM BES (final concentration). The pH of the medium was adjusted to 6.0 with 1 M HCl. Monensin was also added to two of the four growth vessels to a final concentration of 5 μM. Monensin and BES

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Figure 2. Continuous culture system utilized for the isolation of acetogenic bacteria from bovine ruminal contents.

R 1= reservoir 1 with acetogen medium and 5 mM BES R 2= reservoir 2 with acetogen medium, 5 mM BES and 5 ÎźM monensin GV 1 and GV 3= growth vessels 1 and 3 at dilution rate = 0.28 h-1 GV 2 and GV 4= growth vessels 2 and 4 at dilution rate = 0.06 h-1

were added to the medium reservoirs as anaerobic sterile stock solutions. The medium in each reservoir was continuously stirred and gassed with a 100% CO2 gas. Two different dilution rates (D) were used to simulate ruminal dilution rates of animals on a high forage diet (D=0.06 h-1) or a high concentrate diet (D=0.28 h-1). The four isolation treatments utilized are summarized in Figure 2. Ruminal contents utilized as inoculum were collected prior to the morning feeding from a Holstein Friesian dairy cow producing a daily average of 55 134

lb of milk and eating a 56:44 concentrate:forage diet. Ruminal contents were collected anaerobically from 3 sites in the rumen and immersed in ice during transporting to the lab. In the lab, ruminal contents were blended for 1 min and filtered through a double layer of cheesecloth under CO2. Each growth vessel, already containing 200 ml of reduced isolation medium, received 250 mL of ruminal contents as inoculum. Each growth vessel was continuously stirred and gassed with limited H2:CO2 (80:20) through stainless steel needles. After 8 turnovers, 1 mL of fermentation fluid was

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Table 2. Anaerobic dilution solution (ADS) compositiona Component

(amounts per liter)

Mineral 1b

75.0 mL

Mineral 2c

75.0 mL


0.5000 g

Resazurin solution

0.0010 g

All components, except Cysteine.HCl, were added to distilled water and brought to a volume of 1,000 mL. These components were mixed thoroughly and the pH adjusted to 7.0 with 1 M NaOH followed by gently heating and brought to a boil. Boiling was continued for 1 min., Cysteine.HCl was added under 100% CO2, mixed thoroughly and autoclaved anaerobically for 12 min at 121°C and 15 psi. Leedle and Hespell, 1980. a

b c

Mineral 1 (g/liter): 6.00 K2HPO4 Mineral 2 (g/liter): 12.00 NaCl, 6.00 K2HPO4, 6.00(NH4)2SO4, 2.45 MgSO4.7H20, 1.60 CaCl2.2H2O

withdrawn with a sterile syringe, from each growth vessel and then serially diluted to 10-8 with anaerobic dilution solution (ADS, Table 2). From each dilution tube, 250 μL were plated on solid isolation medium in triplicate 60 mm petri plates. The solid medium was the same as the isolation medium plus 2% of washed agar (Leedle and Hespell, 1980). Plates were incubated in an anaerobic growth vessel, pressurized to 16 psi with a H2:CO2 (80:20) gas mixture, at 37°C for 5 days. Approximately ten single colonies for each treatment were anaerobically transferred into serum bottles (120 mL) containing 10 mL acetogen medium plus 5 mM BES (final concentration). The bottles were pressurized to 200 kPa with a H2:CO2 (80:20) gas mixture and incubated on a rotatory shaker for 5 days at 37°C.

Initial screening of newly isolated potential acetogenic bacteria Potential acetogen isolates G1.4b, G1.5a, G1.5c, G1.5d, G1.5e, G2.4a, G3.2a, G4.4a, acetogenic bacteria Acetobacterium woodii, Sporomusa termitida,

and strains A10 and 3H were grown in duplicate serum bottles (60 or 120 mL) containing 10 mL of acetogen medium (Table 1), and pressurized to 200 kPa with a H2:CO2 (80:20) or N2:CO2 (80:20) gas mixtures. The inoculum was 10% of a third transfer of each bacterium grown for 48 h in acetogen medium under 200 kPa of H2:CO2 (80:20) at 37°C. After 72 h incubation, 3 mL of culture from each bottle was transferred to 5 mL glass tubes and growth was determined by optical density (660 nm). Potential acetogenic isolates were identified by difference in growth under H2 and N2. Hydrogen threshold concentrations were measured in a separate experiment. To increase cell mass, cultures (2% inoculum) were grown initially for 12 h at 37°C, in duplicate serum bottles (60 mL), in acetogen medium (5ml) containing 2.5 mM glucose under 200 kPa of a H2:CO2 (80:20) gas mixture. Bottles were then brought inside the glove box where 5 ml of fresh acetogen medium, without glucose, was added to each bottle. Serum bottles were then resealed with sterile butyl rubber stoppers and aluminum seals, pressurized to 200 kPa with a H2:CO2:N2 (1:24:75) gas mixture and incubated at 37°C on a rotatory shaker for 7 days. At the end of this period,

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Figure 3. Comparison of buffering capacity between citrate (20 mM) and MES (40 mM) in ruminal contents in presence or absence (C) of the acetogen strain A10. Methanogens were inhibited by 5 mM BES.

headspace gas volume and gas composition in each bottle was measured as described previously (Boccazzi and Patterson, 2011). Potential acetogenic isolates G1.5a, G1.5c, G1.5d, G1.5e, G2.4a, G3.2a, and acetogenic bacteria A.woodii and strain 3H were further screened for acetate production. Bacterial cultures were grown in single serum bottles (60 mL) containing 10 mL acetogen medium, modified by the addition of 0.75 g/L instead of 0.5 g/L (w/v) of yeast extract, under 200 kPa of a H2:CO2 (80:20) or a N2:CO2 (80:20) gas mixture. Serum bottles were incubated at 37°C on a rotatory shaker for 3 days. Growth was measured by optical density (660 nm) and acetate concentrations were measured enzymatically (Boeringer Mannheim, Indianapolis, IN). Growth and acetate production values of the N2:CO2 incubations were subtracted from the values of the H2:CO2 incubations of the same strain. 136

Growth curves of newly isolated acetogenic bacteria G1.4b, G2.4a and G3.2a were obtained by growing the bacteria in duplicate serum bottles (275 mL, Bellco Inc., Vineland, NJ) modified by the addition of a side arm for optical density measurements. Bacteria were grown in 20 mL acetogen medium, modified by the addition of 0.75 g/L (w/v) of yeast extract, plus or minus 2.5 mM of glucose under 200 kPa of a H2:CO2 (80:20) gas mixture. Serum bottles were incubated vertically in a water bath (Precision Scientific Company, Model 50, Chicago, IL) shaking at 80 rpm at 37°C. Growth was measured by optical density (660 nm) at 0, 1, 2, 3, 4, 5.5, 7.5, 9.0, 11, 20, 22, 24, 27, 32, and 48 h after inoculation.

Analytical Methods Bacterial growth: optical density was measured

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Figure 4. The effect of citrate (20 mM) and MES (40 mM) on initial H2 concentration (0 h) and on H2 uptake by ruminal contents in the presence or absence of the acetogen strain A10. Methanogens were inhibited by 5 mM BES.

at 660nm using a Spectronic 70 spectrophotometer (Bausch and Lomb, Rochster, NY). VFA analysis: volatile fatty acid concentrations were measured by gas-liquid chromatography (GLC; Holdeman et al., 1977). At sampling time, samples were acidified by adding 20% (v/v) of meta-phosphoric acid (25% w/v) and then frozen. Samples to be analyzed were thawed, centrifuged at 15,000 rpm for 5 min, and the supernatant was analyzed. A 3 foot long column, packed with SP1220 (Supelco, Bellefonte, PA, USA), was used in a Hewlett Packard 5890 GLC equipped with a flame ionization detector. Oven temperature was 130°C (isothermal), injector temperature was 170°C, detector temperature was 180°C, the carrier gas was N2 flowing at a rate of 30 mL per minute.

For the measurements of H2 and methane concentrations, gas samples were analyzed using a Varian 3700 Gas Chromatograph equipped with a thermal conductivity detector, and a 6 feet silica gel column (Supelco). Temperatures of the injector, oven, and detector were room temperature, 130°C, and 120°C respectively. The carrier gas was N2 flowing at a rate of 30 mL per minute. The volume of gas injected for standards and samples was 0.5 mL. The GC was standardized with 5 different concentrations of H2 (400 to 25,000 ppm) and CH4 (900 to 32,000 ppm). A regression line was obtained from the output values of the standard concentrations. The regression line was then utilized to calculate H2 and CH4 concentrations in experimental samples. All gas mixtures were purchased from Airco (Indianapolis, IN).

Gas Analysis

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Table 3. Growtha and H2 utilizationb of potential and known acetogenic bacteria grown in acetogen medium under 200 kPa of a H2:CO2 (80:20) or N2:CO2 (80:20) gas mixture Strain



ppm H 2


>0.1 – <0.2
















>0.1 – <0.2





>0.1 – <0.2





>0.1 – <0.2














>0.2 - < 0.4

>0.1 – <0.2



Acetobacterium woodii


>0.1 – <0.2



Sporomusa termitida


>0.2 - < 0.4



strain 3H


>0.1 – <0.2




Growth was measured after 72 h of incubation at 37°C by optical density (OD 660 nm): Ranges of OD values are given to indicate relative amounts of growth. b H2 utilization was determined in a different experiment where bacteria were incubated in acetogen medium under 200 Kpa of a H2:CO2:N2 (1:24:74) gas mixture c Uninoculated acetogen medium d Not determined a


Isolation of Acetogenic Bacteria A batch culture experiment with ruminal contents was performed to determine which buffer system to use for the isolation of acetogenic bacteria from ruminal contents at pH 6.0. After 72 h of incubation, both control and strain A10 treatments that were buffered with citrate had higher pH values than the same treatments buffered with MES (Figure 3). Hydrogen concentrations were also lower for the citrate buffered treatments than for the MES buffered treatments (Figure 4). 138

The isolation of acetogenic bacteria from bovine ruminal contents at pH 6.0 was performed with a continuous culture system. The experiment resulted in the isolation of 40 potential acetogenic bacteria. Eight new isolates and 3 known acetogens grew at least 3 times to a higher yield under a H2:CO2 than a N2:CO2 atmosphere, indicating capability of chemolithoautotrophic growth (Table 3). Strains G1.4b, G1.5a, G1.5c, G1.5d, and G1.5e were isolated from growth vessel (GV) 1 that received acetogen medium plus BES with a dilution rate (D)= 0.28 h-1. Strain G2.4a was isolated from GV 2 that received acetogen medium plus BES with a D= 0.06 h-1. Strain G3.2a was isolated from GV 3 that received acetogen medium plus BES and monensin with a D= 0.28 h-1.

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Figure 5. Growth (OD 660 nm) and acetate production of six new potential acetogen isolates and of the acetogens Acetobacterium woodii and 3H. Bacteria were grown in single serum bottles for 72 h in acetogen medium under 200 kPa of a H2:CO2:N2 (1:24:75) gas mixture. Values for the same bacteria growing in acetogen medium under 200 kPa of a N2:CO2 gas mixture were subtracted from the data.

Strain G4.4a was isolated from GV 4 that received acetogen medium plus BES and monensin with a D= 0.06  h-1. In a subsequent batch culture study, with 8 of the new acetogen isolates, strain G1.5e had the lowest H2 threshold at 635 ppm, while the other isolates had H2 thresholds ranging from 800 to 1062 ppm (Table 3).

Initial screening of newly isolated acetogenic bacteria Growth and acetate production of 6 new and two known acetogenic bacteria was determined in a batch culture experiment. Isolate G3.2a had the

highest yield of acetate per OD unit (Yacetate= mM acetate per OD= 1) of 206.2 mM. Strains G1.5e, G1.5a, G1.5d, G2.4a and G1.5c had a Yacetate of 137.4, 114.9, 95.1, 88.2 and 67.6 mM, respectively (Figure 5). The two known acetogens A. woodii and strain 3H had a Yacetate of 77.7 and 146.6 mM, respectively (Figure 5). The acetogenic strains G1.4b, G2.4a, and G3.2a were considered to be the most promising of the new acetogen isolates. Strains G1.4b, G2.4a, and G3.2a had growth rates (µ) of 0.094, 0.029 and 0.025 h-1, respectively, growing on H2:CO2 alone and of 0.86, 0.37 and 0.35 h-1, respectively, growing on glucose plus H2:CO2 (Figures 6 through 8).

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Figure 6. Growth of acetogen isolate G1.4b in acetogen medium under 200 kPa of a H2:CO2 (80:20) gas mixture plus (circles) or minus (squares) 2.5 mM glucose.

Figure 7. Growth of acetogen isolate G2.4a in acetogen medium under 200 kPa of a H2:CO2 (80:20) gas mixture plus (triangles) or minus (squares) 2.5 mM glucose.


Agric. Food Anal. Bacteriol. â&#x20AC;˘ â&#x20AC;˘ Vol. 3, Issue 2 - 2013

Figure 8. Growth of acetogen isolate G3.2a in acetogen medium under 200 kPa of a H2:CO2 (80:20) gas mixture plus (triangles) or minus (circles) 2.5 mM glucose.

discussion A preliminary batch culture experiment demonstrated that citrate plus phosphate was a better buffer system than MES for the isolation of acetogenic bacteria from ruminal contents. However, when the citrate plus phosphate was used as buffer in continuous culture, citrate utilizing bacteria were selected (data not reported). Therefore, MES was utilized as the buffer system for isolation of acetogenic bacteria in continuous culture. In many beef operations, monensin is used to manipulate rumen fermentations; therefore, monensin was included in the medium used to isolate acetogens. A preliminary study was performed to determine the minimum inhibitory concentration (MIC) of monensin on acetogenic bacteria that had been previously isolated by our lab. Both gram positive acetogens, strain A10 and strain 3H, were insenstive to the highest concentration of monensin tested

(60  μM). This concentration of monensin is higher than the concentration (3 to 5 μM) normally found in the rumen (Russell and Strobel, 1989). Our results, thus indicated that monensin may not inhibit some acetogens. The second experiment on the isolation of acetogenic bacteria resulted in the isolation of ten potential acetogenic strains from each of the four growth vessels utilized. Post-isolation studies were conducted to determine the ability of these strains to grow on a H2:CO2 gas mixture and to produce acetate. A total of eight isolates were identified as acetogenic bacteria. After further studies comparing growth and acetate production on H2:CO2 versus N2:CO2 gas mixtures, three of the eight isolates were selected for further study because of their greater H2 utilization and acetate production. The three isolates chosen were strains G1.4b, from growth vessel one; G2.4a, from growth vessel 2; G3.2a, from growth vessel 3. Of these three isolates, G3.2a had higher growth and produced more acetate than the other two.

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BES was used to inhibit methanogenesis, since the results of batch culture experiments indicated that BES was a more effective inhibitor than 9,10-anthraquinone, or lumazine. Also, due to the results obtained in batch culture experiments, the inoculum of acetogenic bacteria at each turnover was set to give a final concentration of 5x108 CFU/mL in the first experiment, and 3x108 CFU/mL in the second experiment. While in the first experiment, acetogens were grown on glucose plus H2:CO2, in the second experiment the acetogens were grown only on H2:CO2. This change in growth conditions was made so that acetogenic cultures were growing totally chemolithoautotrophicly. The dilution rate was set to 0.084 h-1 in the first experiment and 0.06 h-1 in the second. The change in the second experiment was made, because in the first experiment the control culture did not produce CH4 as expected. This could have been an indication that methanogens were not able to reproduce fast enough, and were washed out of the system when no acetogen was added. The amount of CH4 produced by the other four growth vessels that received BES was minimal or zero. In these treatments, the H2 concentration was lower in the control culture that did not receive acetogens than in the cultures that received acetogens. This was also unexpected, since in batch culture experiments cultures that received the acetogen strain A10 had lower H2 concentrations than did the control cultures. The treatment that received a mixture of the acetogens strains A10 and G3.2a had a higher H2 concentration than the control and A10 treatments, but not significantly different. The growth vessel that received the acetogen strain G3.2a had the highest H2 concentration. The pH averages over seven turnovers for each growth vessel ranged from 6.26 to 6.61, with no significant difference among the six growth vessels. Acetate production was not significantly different among the six treatments. However, the control which received BES and no acetogen had the lowest concentration of acetate, and propionate and highest concentration of butyrate. In conclusion, a total of 8 isolates were identified as acetogenic bacteria. Among these strain G3.2a, isolated with a dilution rate of 0.28 h-1 in the presence 142

of monensin had the highest acetate production per unit of OD growing with H2/CO2. In batch culture studies acetogen bacteria G1.5a, G2.4a, G3.2a, A10 and 3H could effectively reduce H2 concentrations in ruminal contents in the presence of BES as the methanogenesis inhibitor.

Acknowledgements We would like to thank the College of Agriculture and Department of Animal Sciences for financial support for this project.

References Annison, E.F., and D.G. Armstrong. 1970. Volatile fatty acids metabolism and energy supply. In: A. T. Phillipson (eds). Physiology of digestion and metabolism in the ruminant. Oriel Press, Newcastle, England, pp 422-437. Balch, W.E. and R.S. Wolfe. 1976. A new approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol. 32: 781-791. Berger, L.L., S.C. Ricke and G.C. Fahey, Jr. 1981. Comparison of two forms and two levels of lasalocid with monensin of feedlot cattle performance. J. Anim. Sci. 53:1440-1445. Blaxter, K.L. and J.L. Clapperton. 1965. Prediction of the amount of methane produced by ruminants. Br. J. Nutr. 19: 511-522. Boccazzi, P. and J.A. Patterson. 2011. Using hydrogen limited anaerobic continuous culture to isolate low hydrogen threshold ruminal acetogenic bacteria. Agric. Food Anal Bacteriol. 1: 33-44. Branine, M.E. and D.E. Johnso. 1990. Level of intake effects on ruminant methane loss across a wide range of diets. J. Anim. Sci. 68 (Suppl.1):509. Breznak, J.A., J.M. Switzer, and H.-J. Seitz. 1988. Sporomusa termitida sp.nov., an H2/CO2-utilizing acetogen isolated from termites. Arch. Microbiol.

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150:282-288. Breznak, J.A. and M.D. Kane. 1990. Microbial H2/CO2 acetogenesis in animal guts: nature and nutritional significance. FEMS Microbiol. Rev. 87:309-314. Breznak, J.A. and J.S. Blum. 1991. Mixotrophy in the termite gut acetogen, Sporomusa termitida. Arch. Microbiol. 156:105-110. Bryant, M.P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Amer. J. Clin. Nutr. 25: 1324-1328. Chalupa, W. 1980. Chemical control of rumen microbial metabolism. In: Y. Ruckebusch, P. Thivend. (eds). Digestive Physiology and Metabolism in Ruminants. MTP Press, Lancaster, England.Greening, R.C., and J.A.Z. Leedle. 1989. Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen. Arch. Microbiol. 151:399-406. Hungate, R.E. 1966. The rumen and its microbes. Academic Press, New York, NY. Jiang, W.H., J.A. Patterson, and L.R. Steenson. 1995. Isolation and characterization of a temperate bacteriophage from a ruminal acetogen. Current Microbiology. 32:336-339. Jiang, W., R.S., Pinder, and J.A. Patterson. 2012. Influence on growth conditions and sugar substrate on sugar phosphorylation activity in acetogenic bacteria. Agric. Food Anal. Bacteriol. 2:94-102. Lajoie, S.F., S. Bank, T.L. Miller, and M.J. Wolin. 1988. Acetate production from hydrogen and [14] carbon dioxide by the microflora of human feces. Appl. Environ. Microbiol. 54: 2723-2737. Leedle, J.A.Z. and R.B. Hespell. 1980. Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations. Appl. Environ. Microbiol. 39:709-719. LeVan, T.D., J.A. Robinson, J. Ralph, R.C. Greening, W.J. Smolenski, J.A.Z. Leedle, and D.M. Schaefer. 1998. Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis. Appl. Microbiol. Biotechnol. 64: 3429-3436. McInerney, M.J., J.R. Sieber, and R.P. Gunsalus. 2011. Microbial syntrophy: Ecosytem-level biochemical

cooperation. Microbe 6:479-485. Newbold, C.J., R.J. Wallace, and N.D. Walker-Bax. 2013. Potentiation by metal ions of the efficacy of the ionophores, monensin and tetronasin, towards four species of ruminal bacteria. FEMS Microbiol Lett 338:161-167. Pinder, R.S. and J.A. Patterson. 2011. Isolation and initial characterization of plasmids in an acetogenic ruminal isolate. Agric. Food Anal. Bacteriol. 1: 186-192. Pinder, R.S. and J.A. Patterson. 2012. Glucose and hydrogen utilization by an acetogenic bacterium isolated from ruminal contents. Agric. Food Anal. Bacteriol. 2:253-274. Pinder, R.S. and J.A. Patterson. 2013. Growth of acetogenic bacteria in response to varying pH or carbohydrate concentration. Agric. Food Anal. Bacteriol. 3:6-16. Pinder, R.S., J.A. Patterson, C.A. O’Bryan, P.G. Crandall, and S.C. Ricke. 2012. Dietary fiber content influences soluble carbohydrate levels in ruminal fluids. J. Environ. Health Sci. Part B 47:710717. Raun, A.P., C.O. Cooley,, E.L. Potter, R.P. Rathmacher, and L.F. Richardson. 1976. Effect of monensin on feed efficiency of feedlot cattle. J. Anim. Sci. 43: 670-677. Richardson, L.F., A.P. Raun, E.L. Potter, C.O. Cooley, and R.P. Rathmacher. 1976. Effect of monensin on rumen fermentation in vitro and in vivo. J. Anim. Sci. 43:657-664. Ricke, S.C. 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sci. 82:632-639. Ricke, S.C., S.A.Martin, and D.J. Nisbet, D.J. 1996. Ecology, metabolism, and genetics of ruminal selenomonads. Crit Rev Microbiol. 22: 7-56. Ricke, S.C., L.L. Berger, P.J. van der Aar and G.C. Fahey, Jr. 1984. Effects of lasalocid and monensin on nutrient digestion, metabolism and rumen characteristics of sheep. J. Anim. Sci. 58:194-202. Robinson, J.A., J.M. Strayer, and J.M. Tiedje. 1981. Method for measuring dissolved hydrogen in anaerobic ecosystems: application to the rumen. Appl. Environ. Microbiol. 41:545-548.

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Russell, J. B. 1992. Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. J. Appl. Microbiol. 73:363-370. Russell, J.B. and H.J. Strobel. 1989. Effect on ionophores on ruminal fermentation. Appl. Environ. Microbiol. 55:1-6. Sharak-Genthner, B.R., C.L. Davies, and M.P. Bryant. 1981. Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol and H2-CO2-utilizing species. Appl. Environ. Microbiol. 42:12-19. Thauer, R.K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic bacteria. Bacteriol. Rev. 41:100-180. Weimer, P.J., J.B. Russell, and R.E. Muck. 2009. Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass. Bioresour. Technol. 100:5323-5331.


Agric. Food Anal. Bacteriol. â&#x20AC;˘ â&#x20AC;˘ Vol. 3, Issue 2 - 2013 Copyright © 2013 Agriculture, Food and Analytical Bacteriology

Linoleic Acid Isomerase Expression in Escherichia coli BL21 (DE3) and Bacillus spp S. Saengkerdsub 1

Center for Poultry Excellence, University of Arkansas, Fayetteville, AR 72701

ABSTRACT Linoleic acid isomerase (LAI) is responsible for converting linoleic acid conjugated linoleic acids (LAs), which are believed to lower cancer risk and enhance immunity. In this study, the Bifidobacterium LAI gene was cloned into Escherichia coli BL21 (DE3) using pET24a(+) as the expression vector while Propionibacterium acnes LAI gene fused with pSSBm97 derivatives was expressed in Bacillus species. The protein expressed by Bifidobacterium LAI was found in E. coli, but no activity was detectable. By changing cysteine residues to alanine, P. acnes LAI activity was present in B. megaterium YYBm1 but activity was not improved. Prepropeptide B. subtilis amyE fused with P. acnes LAI at N-terminus resulted in unstable proteins. By transferring plasmids carrying prepropeptide Staphylococcus hyicus lipase and prepropeptide B. subtilis amyE fused with P. acnes LAI into B. licheniformis NRRLB-14212, LAI was not found due to possible proteolytic degradation. Keywords: Bacillus, Bifidobacterium, conjugated linoleic acid






Agric. Food Anal. Bacteriol. 3: 145-158, 2013

Introduction Conjugated linoleic acids (CLA) are a family of isomers of linoleic acid (LA) found mainly in the meat and dairy products derived from ruminants (Banni, 2002). Therefore, CLA is found in foods such as beef and lamb, as well as dairy foods derived from these ruminant sources (Chin et al., 1992; Griinari et Correspondence: Suwat Saengkerdsub,

al., 2000; Ma et al., 1999). As the name implies, the double bonds of CLAs are conjugated, with only one single bond between them. The three-dimensional stereo-isomeric configuration of CLA may be in combinations of cis and/or trans configurations. The predominant geometric isomer in foods is the cis9, trans-11-CLA isomer, also known as rumenic acid (Fritsche et al., 1999; Kramer et al., 1998, Ma et al., 1999), followed by trans-7,cis-9-CLA, cis-11,trans-13CLA, cis-8, trans-10-CLA, and trans-10, cis-12-CLA (Fritsche et al., 1999).

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Numerous health benefits have been attributed to CLAs. Several studies have demonstrated that CLA changes body composition, especially by reducing the accumulation of adipose tissue in mice, rats, pigs, and humans, (Dugan et al., 1999, Park et al., 1997, Sisk et al 2001; Smedman and Vessby, 2001). The role of CLA as an aid in the management of type 2 diabetes in humans was examined by Belury (2002), who found that those receiving a CLA supplement (6.0 g CLA/day) had significantly decreased fasting blood glucose, plasma leptin, body mass index, and weight (Belury, 2002). Dietary CLA has also been shown to inhibit numerous cancer models in experimental animals, particularly skin tumor initiation and neoplasias in the forestomach (Ha et al., 1987, 1990), as well as mammary and colon tumorigenesis (Belury et al., 1996; Ip et al., 1991; Liew et al., 1995). There is also evidence that CLA reduces atherosclerotic plaque formation in experimental animals, including rabbits (Lee et al., 1994) and hamsters (Nicolosi et al., 1996). However, concerns have emerged that the use of CLA supplements by the morbidly obese may tend to cause or to aggravate insulin resistance, which may increase their risk of developing diabetes (Risérus et al., 2002). CLA is currently marketed as a dietary supplement, and these commercially available supplements contain equal mixtures of two CLA isomers: the cis-9, trans-11 isomer as well as the trans-10, cis-12 isomer. It is the trans-10, cis-12 isomer that is linked to this and other adverse side effects (Poirier et al., 2006). The CLA dietary supplements are produced by alkaline isomerization of linoleic acid (LA) or vegetable oils containing triglyceride esters of LA (Peng et al., 2007); however, chemical synthesis produces a mixture of CLA (Reaney et al., 1999; Sehat et al., 1998) and the processes required to separate the respective single isomers are expensive (Berdeaux et al., 1998; Chen et al., 1999; Hass et al., 1999). In contrast to chemical processes, biological processes originating from microorganisms can provide production of a single isomer of CLA (Deng et al., 2007). The LA C12 isomerase has been detected in a variety of bacteria (Coakley et al., 2003; Peng et al., 2007; Rosson et al., 2001; Verhulst et al., 1985). 146

Biotransformation of LA using microbial cells and enzyme extracts has been explored for the production of cis-9, trans-11 CLA (Ando et al., 2004; Rainio et al., 2001). Propionibacterium acnes was reported to contain an LA C9 isomerase for converting LA to trans-10, cis-12 CLA (Deng et al., 2007). There is an interest in developing commercial processes for the production of single isomers of CLA by biotransformation of LA using microbial cells and enzymes (Ando et al., 2004; Kim et al., 2000; Rainio et al., 2001). However, the evaluation of these strains suggested that growth and linoleic acid isomerase (LAI) production levels by these anaerobes are insufficient to support economic commercial production of single CLA isomers (Peng et al., 2007). A better alternative would be to clone the LAI gene and generate new production strains using recombinant technology. The aim of this study was to clone the linoleate isomerase gene from Bifidobacterium species and Propionibacterium acnes into E. coli BL21 (DE3) and Bacillus species.

Materials and Methods Bacterial strains The bacterial strains used in this study are described in Table 1. All Bifidobacterium strains were grown in anaerobic jars (BD Diagnostics, Franklin Lake, NJ) with anaerobic generator (GasPak envelope, BD Diagnostics, Franklin Lake, NJ) in de Man Rogosa Sharp (MRS) broth (EMD Chemicals, Gibbstown, NJ) supplemented with 0.05% (w/v) Lcysteine (98% pure; Sigma, St. Louis, MO) and incubated at 37°C overnight.

DNA preparation Genomic DNA was prepared from Bifidobacterium strains by using a QIAamp DNA Stool Mini Kit. Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA).

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Table 1. Bacterial strains used in this study

Bacterial strain

E. coli BL21 (DE3)



This strain lacks the lon and opmT proteases and contains a copy of RNA the T7 RNA polymerase gene under the control of the lacUV5 promoter. These modifications enable stable expression of proteins using T7 promoter driven constructs

Novagen, Darmstadt, Germany

Wild type

Center for Food Safety, University of Arkansas

Bifidobacteria B. longum ATCC15700

B. breve ATCC15700 Wild type

ATCC, Manassas, VA

B. adolescentis ATCC15703

Wild type

ATCC, Manassas, VA

B. infantis ATCC25962

Wild type

ATCC, Manassas, VA

B. subtilis

Wild type

Center for Food Safety, University of Arkansas

B. licheniformis NRRLB-14212

Wild type

Center for Food Safety, University of Arkansas

B. megaterium YYBm1

This strain is deficient in the major extracellular protease NprM and xylose metabolism XylA.

Stammnen et al. (2010)

Bacillus spp

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Table 2. Oligonucleotides used in this study Primer name





Forward primer containing an NdeI site for cloning LA from B. breve ATCC 15700



Reverse primer containing an XhoI site for cloning LA from B. breve ATCC 15700

Bifido R1His


Reverse primer containing an XhoI site for cloning LA from B. breve ATCC 15700 with a C-terminal His6-tag



Forward primer containing an NdeI site for cloning LA from B. longum ATCC 15707 or B. infantis ATCC 25962



Reverse primer containing an XhoI site for cloning LA from B. longum ATCC 15707 or B. infantis ATCC 25962



Reverse primer containing an XhoI site for cloning LA from B. longum ATCC 15707 or B. infantis ATCC 25962 with a C-terminal His6-tag



Forward primer containing an NdeI site for cloning LA from B. adolescentis ATCC 15703



Reverse primer containing an XhoI site for cloning LA from B. adolescentis ATCC 15703



Reverse primer containing an XhoI site for cloning LA from B. adolescentis ATCC 15703 with a C-terminal His6-tag

Cloning of LA gene into pET24a(+) The linoleic acid isomerase (LAI) gene from B. breve (accession number AX647943) and two finished genome sequences, B. longum NC004307 (accession number AE014295) and B. adolescentis ATCC15703 (accession number AP009256), were aligned by using T-coffee (Notredame et al., 2000). Primers (Table 2) were designed according to the potential linoleic acid (LA) sequences. The PCR conditions (30 cycles) were: initial denaturation 95°C, 120 148

sec; denaturation, 95°C, 30 sec, annealing, 45°C, 30 sec, extension 72°C, 120 sec, final extension, 72°C, 7 min. PCR conditions were identical for all primer sets, except the annealing temperature (40°C) for primers Bifido1F and Bifido1RHis. The 1990-bp PCR products were confirmed by agarose gel electrophoresis. PCR products were digested with XhoI and NdeI and were ligated to vector pET24a(+). Recombinant plasmids pET2 to pETH5 (Table 3) were transformed into E. coli BL21 (DE3) by electroporation. Individual colonies from LB agar plates containing 50 µg/mL of kanamycin were selected.

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Table 3. Plasmids used in this study plasmid



pCR® 4-TOPO®

Cloning vector for PCR products, Ampr, Kanr



Expression vector with an N-terminal His6-tag, Kanr



pET24a(+) with NdeI-XhoI LA gene from B.longum ATCC 15707 This work


pET24a(+) with NdeI-XhoI LA gene with His6-tag from B.longum This work ATCC 15707


pET24a(+) with NdeI-XhoI LA gene from B. breve ATCC 15700

This work


pET24a(+) with NdeI-XhoI LA gene with His6-tag from B. breve ATCC 15700

This work


pET24a(+) with NdeI-XhoI LA gene from B. adolescentis ATCC 15703

This work


pET24a(+) with NdeI-XhoI LA gene with His6-tag from B. adolescentis ATCC 15703

This work


pET24a(+) with NdeI-XhoI LA gene from B. infantis ATCC 25962 This work


pET24a(+) with NdeI-XhoI LA gene with His6-tag from B. infantis This work ATCC 25962


P. acnes linoleate isomerase inserted into SpeI and EagI sites of pPPlip; PxylA-(-35+ rbs+)-prepeptidelipA-propeptidelipA- LAI P. acnes

This work


pLPPL derivative, C46A

This work


pLPPL derivative, C154A

This work


pLPPL derivative, C286A

This work


pLPPL derivative, C344A

This work


pLPPL derivative, C412A

This work


pLPPL derivative, C46A, C154A, C286A, C344A, C412A

This work


Prepropeptide B. subtilis amyE inserted into BsrGI and SpeI sites of pLPPL; PxylA-(-35+ rbs+)-prepeptideamyE-propeptideamyELAI P. acnes

This work


pE0 derivative, C46A

This work


p E0 derivative, C154A

This work


p E0 derivative, C286A

This work


pE0 derivative, C344A

This work


pE0 derivative, C412A

This work


pE0 derivative, C46A, C154A, C286A, C344A, C412A

This work

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For Propionibacterium acnes LAI, the new DNA sequence was designed by JCat software (http:// (Grote et al., 2005) and was synthesized by Integrated DNA Technologies, Coralville, IA. The P. acnes LAI was used as the template and primers LF to A5F were used to change from 5 cysteine positions to alanine (Table 4). The PCR products were flanked by SpeI-EagI restriction sites, were digested with these enzymes, and subsequently inserted into pLPPL after SpeI-EagI digestion, creating the plasmids pA1, pA2, pA3, pA4, pA5, and pA6. For prepropeptide B. subtilis amyE fused with P. acnes LAI, PCR product was amplified by using primers amyEF and amyER (amyEF: 5’tat atg taca ATG TTT GCA AAA CGA TTC AAA ACC TC -3’; AmyER: 5’ tat aag atc tac tagt CTC ATT CGA TTT GTT CGC CGT-3’). The PCR products were flanked by BsrGISpeI restriction sites, were digested with these enzymes, and subsequently inserted into pLPPL, pA1, pA2, pA3, pA4, pA5, pA6 after BsrGI-SpeI digestion, creating the plasmids pE0, pE1, pE2, pE3, pE4, pE5, pE6. Protoplast B. megaterium YYBm1 cells were transformed with the appropriate expression plasmids using a polyethylene glycol-mediated procedure described by Christie et al. (2008) while plasmids were transferred into B. licheniformis NRRLB-14212 by electroporation as described in Xue et al. (1999).

Darmstadt, Germany) and the protein samples were analyzed by SDS-PAGE. All Bacillus plasmid strains were grown in baffled shake flasks at 30°C in LB medium at 200 rpm. Recombinant expression of genes under transcriptional control of the xylose-inducible promoter was induced by the addition of 0.5% (w/v) xylose when OD578 reached 0.4. The secreted proteins were separated from cells by centrifugation at 10,000 x g at 4°C for 10 min. After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane and detected with 6X his tag antibody (Abcam, Cambridge, MA) and horseradish peroxidase–anti-rabbit immunoglobulin G conjugates.

Determination of linoleate isomerase activity Determination of linoleate isomerase activity was carried out as described by Peng et al. (2007). Briefly, appropriate dilutions were made in 0.1M Tris, pH 7.5 (total volume of 2 mL) in glass tubes (15×100 mm) with screw caps. Linoleic acid was added to 140µM and tubes were shaken for 1 h at 200 rpm at room temperature. Changes in LA and CLA concentrations were determined by GC analysis. reactions were extracted with 1ml of hexane and analyzed on a HP 8452A diode array spectrophotometer. The absorbance spectrum was between 200 and 400 nm.

Enzymatic activity measurements For E. coli BL21 (DE3), 50 mL of Luria-Bertani (LB) broth containing 50 µg/mL kanamycin (Sigma, St. Louis, MO) was inoculated (1:100 v/v) with a freshly grown overnight culture of strains hosting an isomerase expression plasmid. After growing at 37°C for 3 hr, cultures were induced with 1 mM isopropylthioβ-D-galactoside (IPTG) for 2 hr at 26°C. Cultures were harvested by centrifugation at 10,000 x g for 10 min at 4°C. Cells were suspended in BugBuster (Novagen, Darmstadt, Germany). The subcellular localization of heterologous protein production was separated following the protocol described in pET System Manual 11th edition (Novagen, 150

Preparation of fatty acid methyl esters The preparation of fatty acid methyl esters (FAME) was described in Lewis et al. (2000). Briefly Cells were weighed into clean, 10 mL screw capped tubes and a fresh solution of transesterification reaction mix (methanol:hydrochloric acid:chloroform (10:1:1 v/v/v, 3 mL)) was added. Cells were suspended by vortex mixing and immediately placed at 90°C for 60 min for transesterification. Tubes were removed from the heat and cooled; water (1 mL) was added and the FAME extracted with a hexane/chloroform mix (4:1, v/v). Samples were diluted with chloroform contain-

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Table 4. Oligonucleotides used for LAI engineering in this study


Sequence 5’ to 3’




tat aag atc t CGG CCG TTA GTG ATG GTG





















ing a known concentration of tridecanoic acid as an internal standard. Chromatography was performed with a Shimadzu GC2010 chromatography system (Shimadzu Scientific Instruments, Columbia, MA, USA) equipped with a flame ionization detector. Helium was used as carrier and make-up gas. The injection volume was 1 μL which was used with a split ratio of 1:50. The injection port and detector temperatures were 240 and 250°C, respectively. The col-

umn temperature program was as follows: temperature was held at 30°C for 2 min, increased to 180°C at 20°C/min, held at 180°C for 2 min, increased to 207°C at 4 °C/min, held at 207°C for 3 min, increased to 220°C at 2°C/min, held at 220°C for 2 min, and then increased to 240°C at 2°C/min before finally being held at 240°C for 2 min.

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Figure 1. Total cell protein fraction isolated from E. coli clones containing LAI gene originated from Bifidobacterium strains. 2: LAI gene originated from B. longum ATCC1570; 3: LAI gene originated from B. breve ATCC15700; 4: LA gene originated from B. adolescentis ATCC15703; 5: LAI gene originated from B. infantis ATCC25962; H: LAI gene fused with 6X His tag

Figure 2. Soluble cytoplasmic protein fraction isolated from E. coli clones containing LAI gene originated from Bifidobacterium strains. 2: LAI gene originated from B. longum ATCC1570; 3: LAI gene originated from B. breve ATCC15700; 4: LAI gene originated from B. adolescentis ATCC15703; 5: LAI gene originated from B. infantis ATCC25962.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) After Sephadex LH-20 cleanup, the extract was mixed with a 1 M solution of dihydroxybenzoic acid (DHB) in 90% methanol in a 1:1 ratio, and 1 µL of the mixture was spotted onto a ground stainless steel MALDI target for MALDI analysis using the dry droplet method. A Bruker Reflex III MALDI-TOF-MS (Billerica, MA) equipped with a N2 laser (337 nm) was used in the MALDI analysis, and all the data were obtained in positive ion reflectron TOF mode.


Results and Discussion

LA expression in E. coli BL21 (DE3) as the host In this study, E. coli BL21 (DE3) was chosen as the host. Among expression systems, E. coli is considered to be the first choice since numerous vectors, readily available engineered strains, and minimal technical requirements are already in place. In addition, this system is rapid due to short doubling times of approximately 20 minutes per generation (Brondyk, 2009). In this system it was suggested that the target protein would be synthesized to an

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Figure 3. Soluble cytoplasmic protein fraction isolated from E. coli clones containing LAI gene originated from Bifidobacterium strains. 2: LAI gene originated from B. longum ATCC1570; 3: LAI gene originated from B. breve ATCC15700; 4: LAI gene originated from B. adolescentis ATCC15703; 5: LAI gene originated from B. infantis ATCC25962; H: LAI gene fused with 6X His tag.

Figure 4. Soluble (S) and insoluble (I) cytoplasmic protein fraction from E. coli clones containing LAI gene originated from Bifidobacterium strains after adding IPTG 4 hours at 25°C incubation. 2: LAI gene originated from B. longum ATCC1570; 3: LAI gene originated from B. breve ATCC15700; 4: LAI gene originated from B. adolescentis ATCC15703; 5: LAI gene originated from B. infantis ATCC25962; H: LAI gene fused with 6X His tag.

equivalent of more than 50% of the total cell protein within a few hours after induction (pET system manual 11th edition, 2006). By using SDS-PAGE, our results showed that the expression of LAI in E. coli was tightly controlled by IPTG (Figure 1). The soluble cytoplasm proteins in E. coli carrying plasmids were collected and detected by SDS-PAGE (Figure 2 and 3). The results demonstrated that these proteins were unstable in the cytoplasm and some strains, particularly strains H3 and H5, did not produce soluble cytoplasm proteins. The soluble and insoluble cytoplasm proteins were collected to identify the localization of proteins (Figure 4). The results demonstrated that the 6X His tag adversely affected the solubility of LAI, particularly LAI originating from B.

longum ATCC15707 (clones 2 versus H2, Figure 4). Since expressed proteins were sequestered in inclusion bodies, the lower temperature incubation might have enhanced enzymatic folding. At an incubation temperature of 21.5°C, the results from SDS-PAGE did not show improved folding of the enzyme (Figure 5). Deng et al. (2007) reported that the folding of P. acnes LAI expressed in E. coli was interfered with by the C-terminal 6X His tag. The activities of LAI from these clones were undetectable due to possible improper folding of the enzyme or very low enzyme activities. Deng et al. (2007) reported that the activity of P. acnes LAI expressed in E. coli BL21 (DE3) was only 1 nmol/min/mL. The primary method for minimizing inclusion body formation and maximizing the

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Figure 5. Soluble (S) and insoluble (I) cytoplasmic protein fraction isolated from E. coli clones containing LAI gene originated from Bifidobacterium strains after adding IPTG 4 hours at 21.5°C incubation. 2: LAI gene originated from B. longum ATCC1570; 3: LAI gene originated from B. breve ATCC15700; 4: LAI gene originated from B. adolescentis ATCC15703; 5: LAI gene originated from B. infantis ATCC25962; H: LAI gene fused with 6X His tag.

formation of soluble, properly folded proteins in the cytoplasm is lowering the incubation temperature to 15 to 30°C during the expression period because the reduced temperature reduces the rate of transcription, translation, refolding and thus increases proper folding (Brondyk, 2009). In this study, the incubation temperature was reduced to 21.5°C; however, this method did not enhance enzymatic activity. In addition, protein accumulated in inclusion bodies as observed in this study is one of the disadvantages of protein expression in E. coli (Terpe, 2006).

LA expression in B. megaterium YYBm1 and B. licheniformis NRRLB-14212 as the host Because of these known limitations of E. coli, other hosts, such as Bacillus spp, have gained interest (Schmidt, 2004; Wong, 1995;). Bacillus megaterium is known for its high protein secretion potential, and strains have the advantage of highly stable, freely replicating plasmids and the lack of alkaline proteases (Vary, 1994). The plasmidless B. megaterium strain MS941 was generated from the wild-type strain DSM319 by directed gene deletion, then the xylA gene for xylose metabolism was inactivated leading 154

to the strain YYBm1, which does not metabolize the inducer of gene activation (Stammen et al., 2010). B. licheniformis was chosen due to its ability to secrete large quantities of extracellular enzymes (Schallmey et al., 2004). B. megaterium YYBm1 carrying pLPPL secreted P. acnes LAI; however, no activity was detectable. Based on P. acnes LAI amino acid sequence, there are 5 cysteine residues (Figure 6). All five cysteine residues were changed to alanine by using primers LF to A5F (Table 4) with P. acnes LAI as the template but no activity in B. megaterium YYBm1 carrying pA1 to pA6 was detectable. Liu and Escher (1999) reported that the bioluminescence activity of the secreted Renilla luciferase could be improved after selective removal of sulfhydryl groups by substitution of cysteine residues. Since wild type Renilla luciferase protein contains an odd number of cysteine residues in its amino acid sequence, they proposed that a free cysteine residue and/or unfavorable disulfide bond in secreted Renilla luciferase could affect its bioluminescence activity and alanine, an amino acid considered to be one of the most neutral, was used for this purpose. Based on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), propeptide S. hyicus lipase was still attached to

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Figure 6. P. acnes LAI amino acid sequence. The underlines show the positions of cysteine


the P. acnes LAI and might impede enzymatic activity. In the next step, propeptide B. subtilis amplase (amyE) was chosen since this propeptide enhanced the secreted human interferon-α in B. subtilis as the host. In addition, the propeptide B. subtilis amyE is only 8 amino acids in length, as compared to 207 amino acids in length for propeptide S. hyicus lipase. The plasmids pE0 to pE6 were transferred into B. megaterium YYBm1. Based on Western blot detected with 6X his tag antibody, no secreted proteins were found in these strains. The results demonstrated that the protein fused with propeptide B. subtilis amyE was unstable in B. megaterium YYBm1 as the host (data not shown), compared to human interferon-α in B. subtilis. Since attempts with B. megaterium YYBm1 were unsuccessful, B. licheniformis NRRLB-14212 was examined as a possible expression host. The plasmids pLPPL and pE0 were transferred into B. licheniformis NRRLB-14212 by electroporation. The plasmid pE0 constructed in E. coli could not be successfully trans-

ferred into the expression strain B. licheniformis, indicating a lethal effect. This result agrees with Brockmeier et al. (2006) that 25 of 173 prepeptides could not be transferred into B. subtilis TEB1030. The secreted protein in B. licheniformis NRRLB-14212 carrying pLPPL could not be detected by Western blot (data not shown). Possibly, B. licheniformis NRRLB-14212, a wild type strain, has extracellular proteases or propeptide S. hyicus lipase was unable to protect P. acnes LAI from proteolytic degradation.

CONCLUSIONs Bifidobacterium LAI expressed in E. coli BL21 (DE3) did not function due to insoluble formation in inclusion bodies. Amino acid modification of P. acnes LAI expressed in B. megatreium YYBm1 did not improve the activity. Also, the propeptide of B. subtilis amyE and both propeptides could not protect P. acnes LAI from proteolytic degradation in B. mega-

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terium YYBm1 and B. licheniformis NRRLB-14212 as the hosts, respectively.

Acknowledgements Author Saengkerdsub was supported by an Arkansas Biosciences Institute Grant. I would like to thank Simon Stammen, Rebekka Biedendieck, and Dieter Jahn of Institute of Microbiology, Technische Universitat Braunschweig for providing plasmids and B. megterium YYBm1. I appreciate Robert Preston Story Jr. at the Center for Food Safety in the Department of Food Science, the University of Arkansas for providing Bidifobacterium longum ATCC15707, Bacillus subtilis, Bacillus licheniformis NRRL B-14212.

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od for expressing a recombinant protein. Methods Enzymol. 463:131-147. Chen, C.-A., W. Lu, and C. Sih. 1999. Synthesis of 9Z,11E-octadecadienoic and 10E,12Z-octadecadienoic acids, the major components of conjugated linoleic acid. Lipids 34:879-884. Chin, S. F., W. Liu, J. M. Storkson, Y. L. Ha, and M. W. Pariza. 1992. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Compost. Anal. 5:185-197. Christie, G., M. Lazarevska, and C. R. Lowe. 2008. Functional consequences of amino acid substitutions to GerVB, a component of the Bacillus megaterium spore germinant receptor. J. Bacteriol. 190:2014-2022. Coakley, M., R. P. Ross, M. Nordgren, G. Fitzgerald, R. Devery, and C. Stanton. 2003. Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species. J. Appl. Microbiol. 94:138-145. Deng, M. D., A. D. Grund, K. J. Schneider, K. M. Langley, S. L. Wassink, S. S. Peng, and R. A. Rosson. 2007. Linoleic acid isomerase from Propionibacterium acnes: purification, characterization, molecular cloning, and heterologous expression. Appl. Biochem. Biotechnol. 143:199-211. Dugan, M. E. R., J. L. Aalhus, L. E. Jeremiah, J. K. G. Kramer, and A. L. Schaefer. 1999. The effects of feeding conjugated linoleic acid on subsequent pork quality. . Can. J. Anim. Sci. 79:45-51. Fritsche, J., R. Rickert, and H. Steinhart. 1999. Formation, contents, and estimation of daily intake of conjugated linoleic acid isomers and trans-fatty acids in foods., In: M. P. Yurawecz, M. M. Mossobo, J. K. G. Kramer, M. W. Pariza, and G. J. Nelson. Eds. Advances in Conjugated Linoleic Acid Research. AOCS, Champaign, IL. p. 378-396. Griinari, J. M., B. A. Cori, S. H. Lacy, P. Y. Chouinard, K. V. V. Nurmela, and D. E. Bauman. 2000. Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by delta(9)-desaturase. J. Nutr. 130:2285-2291. Grote, A., K. Hiller, M. Scheer, R. Münch , B. Nörtemann, D. C. Hempel, and D. Jahn. 2005. JCat: a novel tool to adapt codon usage of a target gene

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to its potential expression host. Nucleic Acids Res.33:W526-W531. Ha, Y. L., N. K. Grimm, and M. W. Pariza. 1987. Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 8:18811887. Ha, Y. L., J. Storkson, and M. W. Pariza. 1990. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives oflinoleic acid. Cancer Res. 50:1097-1101. Haas, M., J. Kramer, G. McNeill, K. Scott, T. Foglia, N. Sehat, J. Fritsche, M. Mossoba, and M. Yurawecz. 1999. Lipase-catalyzed fractionation of conjugated linoleic acid isomers. Lipids 34:979-987. Ip, C., S. F. Chin, J. A. Scimeca, and M. W. Pariza. 1991. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res. 51:6118-6124. Kim, Y. J., R. H. Liu, D. R. Bond, and J. B. Russell. 2000. Effect of linoleic acid concentration on conjugated linoleic acid production by Butyrivibrio fibrisolvens A38. Appl. Environ. Microbiol. 66:5226-5230. Kramer, J. K., P. W. Parodi, R. G. Jensen, M. M. Mossoba, M. P. Yurawecz, and R. O. Adlof. 1998. Rumenic acid: a proposed common name for the major conjugated linoleic acid isomer found in natural products. Lipids. 33:835. Lee, K. N., D. Kritchevsky, and M. W. Pariza. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis. 108:19-25. Lewis, T., P. D. Nichols, and T. A. McMeekin. 2000. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods. 43:107-116. Liew, C., H. A. Schut, S. F. Chin, M. W. Pariza, and R. H. Dashwood. 1995. Protection of conjugated linoleic acids against 2-amino-3- methylimidazo[4,5f]quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms. Carcinogenesis. 16:3037-3043. Liu, J., and A. Escher. 1999. Improved assay sensitivity of an engineered secreted Renilla luciferase. Gene. 237:153-159. Ma, D. W., A. A. Wierzbicki, C. J. Field, and M. T. Clandinin. 1999. Conjugated linoleic acid in ca-

nadian dairy and beef products. J. Agric. Food Chem. 47:1956-1960. Nicolosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis inhypercholesterolemic hamsters. Artery. 22:266-277. Notredame, C., D. G. Higgins, and J. Heringa. 2000. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302:205-217. Pariza, M. W. 2004. Perspective on the safety and effectiveness of conjugated linoleic acid. Am. J. Clin. Nutr. 79:1132S-1136S. Pariza, M. W., Y. Park, and M. E. Cook. 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40:283-298. Park, Y., K. J. Albright, W. Liu, J. M. Storkson, M. E. Cook, and M. W. Pariza. 1997. Effect of conjugated linoleic acid on body composition in mice. Lipids. 32:853-858. Peng, S. S., M.-D. Deng, A. D. Grund, and R. A. Rosson. 2007. Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes. Enzyme Microb. Technol. 40:831-839. Poirier, H., J. S. Shapiro, R. J. Kim, and M. A. Lazar. 2006. Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of whiteadipose tissue. Diabetes. 55:16341641. Rainio, A., M. Vahvaselkä, T. Suomalainen, and S. Laakso. 2001. Reduction of linoleic acid inhibition in production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. shermanii. Can. J. Microbiol. 47:735-740. Reaney, M. J. T., L. Ya-Dong, and N. D. Westcott. 1999. Commercial production of conjugated linoleic acid. Adv. Conjugat. Linoleic Acid Res. 1:3954. Risérus, U., S. Basu, S. Jovinge, G. N. Fredrikson, J. Arnlöv, and B. Vessby. 2002. Supplementation with conjugated linoleic acid causes isomer-dependent oxidative stress and elevated C-reactive protein: a potential link to fatty acid-induced insulin resis-

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tance. Circulation. 106:1925-1929. Rosson, R. A., M.-D. Deng, A. D. Grund, and S. S. Peng. 2001. Linoleate isomerase. PCT Patent # WO0100846-A. Schallmey, M., A. Singh, and O. P. Ward. 2004. Developments in the use of Bacillus species for industrial production. Can. J. Microbiol. 50:1 - 17. Schmidt, F. R. 2004. Recombinant expression systems in the pharmaceutical industry. Appl. Microbiol. Biotechnol. 65:363-372. Sehat, N., M. P. Yurawecz, J. A. Roach, M. M. Mossoba, J. K. Kramer, and Y. Ku. 1998. Silver-ion highperformance liquid chromatographic separation and identification of conjugated linoleic acid isomers. Lipids 33:217-221. Sisk, M. B., D. B. Hausman, R. J. Martin, and M. J. Azain. 2001. Dietary conjugated linoleic acid reduces adiposity in lean but not obese Zucker rats. J. Nutr. 131:1668-1674. Smedman, A., and B. Vessby. 2001. Conjugated linoleic acid supplementation in humans-metabolic effects. Lipids. 36:773-781. Stammen, S., B. K. Müller, C. Korneli, R. Biedendieck, M. Gamer, E. Franco-Lara, and D. Jahn. 2010. High-yield intra- and extracellular protein production using Bacillus megaterium. Appl. Environ. Microbiol. 76:4037-4046. Terpe, K. 2006. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72:211-222. Vary, P. S. 1994. Prime time for Bacillus rnegateriurn. Microbiology. 140:1001-1013. Verhulst, A., G. Semjen, U. Meerts, G. Janssen, G. Parmentier, S. Asselberghs, H. Vanhespen, and H. Evssen. 1985. Biohydrogenation of linoleic acid by Clostridium sporogenes, Clostridium bifermentans, Clostridium sordellii and Bacteroides sp. FEMS Microbiol. Ecol. 31:255-259. Wong, S. L. 1995. Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr. Opin. Biotechnol. 6:517-522. Xue, G. P., J. S. Johnson, and B. P. Dalrymple. 1999. High osmolarity improves the electro-transforma158

tion efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Methods. 34:183-191.

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Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013



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Greenhouse Gas Emissions from Livestock and Poultry C. S. Dunkley and K. D. Dunkley


Can Salmonella Reside in the Human Oral Cavity? S. A. Sirsat


Shiga Toxin-Producing Escherichia coli (STEC) Ecology in Cattle and Management Based Options for Reducing Fecal Shedding T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, D. J. Nisbet


Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration R. S. Pinder, and J. A. Patterson


Independent Poultry Processing in Georgia: Survey of Producers’ Perspective E. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, S. C. Ricke

Introduction to Authors 79

Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors. Agric. Food Anal. Bacteriol. • • Vol. 3, Issue 2 - 2013




Authors must submit their papers electronically ( According to instructions provided online at our site: www.afabjournal. com. Authors who are unable to submit electronically should contact the editorial office for assistance by email at

We invite you to consider submitting your research and review manuscripts to AFAB. The journal serves as a peer reviewed scientific forum for to the latest advancements in bacteriology research on Agricultural and Food Systems which includes the following fields:

• • • • • • • • • • • • • • • • • • •


Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control Food microbiology Food quality Food Safety

• • • • • • • • • • • • • • • • • • •

Foodborne pathogens Gastrointestinal microbiology Microbial education Microbial genetics Microbial physiology Modeling and microbial kinetics Natural products Phytoceuticals Quantitative microbiology Plant microbiology Plant pathogens Prebiotics Probiotics Rumen microbiology Rapid methods Toxins Veterinary microbiology Waste microbiology Water microbiology

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With an open access publication model of this journal, all interested readers around the world can freely access articles online. AFAB publishes original papers including, but not limited to the types of manuscripts described in the following sections. Papers that have been, or are scheduled to be, published elsewhere should not be submitted and will not be reviewed. Opinions or views expressed in papers published by AFAB are those of the author(s) and do not necessarily represent the opinion of the AFAB or the editorial board.


peer review, the journal editorial office has the capability to shorten the review timing to one week or less. Any type of manuscript whether it be a full length manuscript, brief communication or review paper can be submitted as a rapid communication. There will be additional costs for processing and page charges will be double the normal rate. Authors who choose this option must select Rapid Communications as the paper type when submitting the paper and the editors will judge whether a rapid review is possible and let the author know immediately.

Brief Communications

Full-Length Research Manuscripts AFAB accepts full-length research articles containing four (4) figures and/or tables or more. AFAB emphasizes the importance of sound scientific experimentation on any of the topics listed in the focus areas followed by clear concise writing that describes the research in its entirety. The results of experiments published in AFAB must be replicated, with appropriate statistical assessment of experimental variation and assignment of significant difference. Major headings to include are: Abstract, Introduction, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowledgements (optional), Appendix for abbreviations (optional), and References. Manuscripts clearly lacking in language will be returned to author without review, with a suggestion that English editing be sought before the paper is reconsidered. AFAB offers a fee based language service upon request. Please contact for more information about our fees and services.

Rapid Communications

Brief communications are short research notes giving the results of complete experiments but are considered less comprehensive than full-length articles with three (3) figures and/or tables or less. Manuscripts should be prepared with the same subheadings as full length research papers. The running head above the title of the paper is “Brief Communications.”

Unsolicited Review Papers Review papers are welcome on any topic listed in the focus section and have no page limits. Reviews are assessed the same pages charges as all other manuscripts. All AFAB guidelines for style and form apply. Major headings to include are: Abstract, Introduction, Main discussion topics and appropriate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter than 20 pages of double spaced text and references will be considered mini-reviews with the subheading above the title on the first page. The running head above the title of the paper is either “Review” or “Mini-review”.

Solicited Review Papers

Under normal circumstances, AFAB aims for receipt-to-decision times of approximately one month or less. Accepted papers will have priority for publication in the next available issue of AFAB. However, if an author chooses or requires a much more rapid

Solicited reviews will have no page limits. The editor-in-chief will send invitations to the authors and then review these contributions when they are submitted. Nominations or suggestions for potential timely reviews are welcomed by the editors or edito-

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rial board members and should be sent to submit@ There will be no page charges for solicited review papers but the solicitation must originate from the editor-in-chief or one of the editors. Requests from authors will automatically be classified as unsolicited review papers. The running head above the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews AFAB welcomes opportunities to publish papers from symposia, scientific conference, and/or meetings in their entirety. Conference organizers need simply to contact AFAB at and a rapid decision is guaranteed. If in agreement, the conference organizers must guarantee delivery of a set number of peer reviewed manuscripts within a specified time and submitted in the same format as that described for unsolicited review papers. Conference papers must be prepared in accordance with the guidelines for review articles and are subject to peer review. The conference chair must decide whether or not they wish to serve as Special Issue Editor and conduct the editorial review process. If the conference chair/organizer chooses to serve as special issue editor, this will involve review of the papers and, if necessary, returning them to the authors for revision. The conference organizer then submits the revised manuscripts to the journal editorial office for further editorial examination. Final revisions by the author and recommendations for acceptance or rejection by the chair must be completed by a mutually agreed upon date between the editor and the conference organizer. Manuscripts not meeting this deadline will not be included in the published symposium proceedings but if submitted later can still be considered as unsolicited review papers. Although offprints and costs of pages are the same as for all other papers, the symposium chair may be asked to guarantee an agreed upon number of hard copies to be purchased by conference attendees. If the decision is not to publish the symposium as a special issue, the individual authors retain the right to submit their papers for consideration for the journal as ordinary unsolicited review manuscripts. 164

Book Reviews AFAB publishes reviews of books considered to be of interest to the readers. The editor-in-chief ordinarily solicits reviews. Book reviews shall be prepared in accordance to the style and form requirements of the journal, and they are subject to editorial revision. No page charges will be assessed solicited reviews while unsolicited book reviews will be assigned the regular page charge rate.

Opinions and Current Viewpoints The purpose of this section will be to discuss, critique, or expand on scientific points made in articles recently published in AFAB. Drafts must be received within 6 months of an article’s publication. Opinions and current perspectives do not have page limits. They shall have a title followed by the body of the text and references. Author name(s) and affiliation(s) shall be placed between the end of the text and list of references. If this document pertains to a particular manuscript then the author(s) of the original paper(s) will be provided a copy of the letter and offered the opportunity to submit for consideration a reply within 30 days. Responses will have the same page restrictions and format as the original opinion and current viewpoint, and the titles shall end with “Opinions.” They will be published together. Letters and replies shall follow appropriate AFAB format and may be edited by the editor-in-chief and a technical editor. If multiple letters on the same topic are received, a representative set of opinions concerning a specific article will be published. A disclaimer will be added by the editorial staff that the opinion expressed in this viewpoint is the authors alone and does not necessarily represent the opinion of AFAB or the editorial board.

COPYRIGHT AGREEMENT The copyright form is published in AFAB as space permits and is available online (

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AFAB grants to the author the right of re-publication in any book of which he or she is the author or editor, subject only to giving proper credit to the original journal publication of the article by AFAB. AFAB retains the copyright to all materials accepted for publication in the journal. If an author desires to reprint a table or figure published from a non-AFAB source, written evidence of copyright permission from an authority representing that source must be obtained by the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS Authors will be requested to provide the names and complete addresses including emails of five (5) potential reviewers who have expertise in the research area and no conflict of interest with any of the authors. Except for manuscripts designated as Rapid Communication each reviewer has two (2) weeks to review the manuscript, and submit comments electronically to the editorial office. Authors have three (3) weeks to complete the revision, which shall be returned to the editorial office within six (6) weeks after which the authors risk having their manuscript removed from AFAB files if they fail to ask the editorial office for an extension by email. Deleted manuscripts will be reconsidered, but they will have to be processed as new manuscripts with an additional processing fee assessed upon submission. Once reviewed, the author will be notified of the outcome and advised accordingly. Editors handle all initial correspondence with authors during the review process. The editor-in chief will notify the author of the final decision to accept or reject. Rejected manuscripts can be resubmitted only with an invitation from the editor or editor-in chief. Revised versions of previously rejected manuscripts are treated as new submissions.

PRODUCTION OF PROOFS Accepted manuscripts are forwarded to the editorial office for technical editing and layout. The manuscript is then formatted, figures are reproduced, and author proofs are prepared as PDFs. Author proofs of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and checked against the typed manuscript, because the responsibility for proofreading is with the author(s). Corrections must be returned by e-mail. Changes sent by e-mail to the technical editor must indicate page, column, and line numbers for each correction to be made on the proof. Corrections can also be marked using “track changes” in Microsoft Word or using e-annotation tools for electronic proof correction in Adobe Acrobat to indicate necessary changes. Author alterations to proofs exceeding 5% of the original proof content will be charged to the author. All correspondence of proofs must be agreed to by the editorial office and the author within 48 hours or proof will be published as is and AFAB will assume no responsibility for errors that result in the final publication.

PUBLICATION CHARGES AFAB has two publication charge options: conventional page charges and rapid communication. The current charge for conventional publication is $25 per printed page in the journal. There is no additional charge for the publication of pages containing color images, micrographs or pictures. For authors who wish to have their papers processed as a rapid communication, authors will pay the rapid communication fee when proofs are returned to the editorial office in addition to twice the conventional page charges. Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one week and $100 per journal page.

HARD COPY OFFPRINTS If you are wishing to obtain a physical hard copy of the AFAB journal, offprints are available in any quantity at an additional charge: $100/page for black-white and $150/page for color prints. You may order your offprints at any time after publication on our website. Scientific conference organizers may be expected to agree to a set number of offprints as a part of their agreement with AFAB.

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MANUSCRIPT CONTENT REQUIREMENTS Preparing the Manuscript File Manuscripts must be written in grammatically correct English. AFAB offers a fee based language service upon request ( Manuscripts should be typed double-spaced, with lines and pages numbered consecutively. All documents must be submitted in Microsoft Word (.doc or .docx, PC or Mac). All special characters (e.g., Greek, math, symbols) should be inserted using the symbols palette available in this font. Tables and figures should be placed in separate sections at the end of the manuscript (not placed in the text). Failure to follow these instructions will cause delays of the processing and review of the manuscript.

Title Page At the very top of the title page, include a title of not more than 100 characters. Format the title with the first letter of each word capitalized. No abbreviations should be used. Under the title, the authors names are listed. Use the author’s initials for both first and middle names with a period (full-stop) between initials (e.g., W. A. Afab). Underneath the authors, a list affiliations must be listed. Please use numerical superscripts after the author’s names to designate affiliation. If an authors address has changed since the research was completed, this new information must be designated as “Current address:”. The corresponding author should be indicated with an asterisk e.g., * Corresponding author. The title page shall include the name and full address of the corresponding author. Telephone and e-mail address must also be provided for the corresponding author, and emailaddresses must be provided for all authors.

at the beginning of the manuscript. In vivo, in vitro and bacterial names must be italicized (obligatory). Authors must avoid single sentence paragraphs and merge such paragraphs appropriately. Authors must not begin sentences with “Figure or Table shows…” as these are inanimate objects and cannot “show” anything. When number are reported in text or in tables, always put a zero in front of decimal numbers: “0.10” instead of “.10”.

Manuscript Sections Abstract The abstract provides an abridged version of the manuscript. Please submit your abstract on a separate page after the title page. The abstract should provide a justification of your work, objectives, methods, results, discussion and implications of study or review findings . Your abstract must consist of complete sentences without references to other work or footnotes and must not exceed 250 words. On the same page as your abstract, please provide at least ten (10) keywords to be used for linking and indexing. Ideally, these keywords should include significant words from the title.

Introduction The introduction should clearly present the foundation of the manuscript topic and what makes the research or the review unique. The introduction should validate why this topic is important based on previously published literature, and the relevance of the current research. Overall goals and project objectives must be clearly stated in the final sentence of the last paragraphs of the introduction.

Materials and Methods Editing Author-derived abbreviations should be defined at first use in the abstract and again in the body of the manuscript. If abbreviations are extensive authors may need to provide a list of abbreviations 166

Information on equipment and chemicals used must include the full company name, city, and state (country if outside the United States or Province if in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent replication of experiments and quantification of variation among replicates is required by AFAB. Reviewers and/or editors may request additional statistical analysis depending on the nature of the data and it will be the responsibility of the authors to respond appropriately. Statistical methods commonly used in the bacteriology do not need to be described in detail, but an adequate description and/or appropriate references should be provided. The statistical model and experimental unit must be designated when appropriate. The experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, the average of replicate tubes per single study per treatment is the experimental unit; therefore, individual studies must be replicated. Repeated time analyses of the same sample usually do not constitute independent experimental units. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis of time effects, assess as a rate of change over time. Standard deviation refers to the variability in the biological response being measured and is presented as standard deviation or standard error according to the definitions described in statistical references or textbooks.

Results Results represent the presentation of data in words and all data should be described in same fashion. No discussion of literature is included in the results section.

Discussion The discussion section involves comparing the current data outcomes with previously published work in this area without repeating the text in the results section. Critical and in-depth dialogue is encouraged.

Results and Discussion Results and discussion can be under combined or separate headings.

Conclusions State conclusions (not a summary) briefly in one paragraph.

Acknowledgments Acknowledgments of individuals should include institution, city, and state; city and country if not U.S.; and City or Province if in Canada. Copies being reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented as follows: Adams and Harry (1992) or Smith and Jones (1990, 1992). If more than two authors of one article, the first author’s name is followed by the abbreviation et al. in italics. If the sentence structure requires that the authors’ names be included in parentheses, the proper format is (Adams and Harry, 1982; Harry, 1988a,b; Harry et al., 1993). Citations to a group of references should be listed first alphabetically then chronologically. Work that has not been submitted or accepted for publication shall be listed in the text as: “G.C. Jay (institution, city, and state, personal communication).” The author’s own unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications and unsubmitted unpublished data must not be included in the References section. Two or more publications by the same authors in the same year must be made distinct with lowercase letters after the year (2010a,b). Likewise when multiple author citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section.

Book Chapter: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication.

b) Citing References In Reference Section In the References section, references are listed in alphabetical order by authors’ last names, and then chronologically. List only those references cited in the text. Manuscripts submitted for publication, accepted for publication or in press can be given in the reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section. Journal manuscript: Author(s). Year. Article title. Journal title [abbreviated]. Volume number:inclusive pages.

Inclusive pages of chapter.

Examples: O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010. Assessing consumer concerns and perceptions of food safety risks and practices: Methodologies and outcomes. In: S. C. Ricke and F. T. Jones. Eds. Perspectives on Food Safety Issues of Food Animal Derived Foods. Univ. Arkansas Press, Fayetteville, AR. p 273-288. Dissertation and thesis: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX.

Examples: Chase, G., and L. Erlandsen. 1976. Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum. J. Bacteriol. 127:572-583.

Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van Doesburg, and A. J. M. Stams. 2009. A typical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermioacetica strain. Arch. Microbiol. 191:123-131.

Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p.

Book: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number of pages.

Examples: Hungate, R. E. 1966. The rumen and its microbes Academic Press, Inc., New York, NY. 533 p. 168

Web sites, patents: Examples: Davis, C. 2010. Salmonella. /article. htm. Accessed July, 2010. Afab, F. 2010, Development of a novel process. U.S. Patent #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently and effectively. NABC report 19, Agricultural Biofuels: Tech., Sustainability, and Profitability. p.27 Musgrove, M. T., and M. E. Berrang. 2008. Presence of aerobic microorganisms, Enterobacteriaceae and Salmonella in the shell egg processing environment. IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10) Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p. 86 (Abstr.)

Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the experimental approach sufficiently to interpret the data. Consequently, all statistical analyses should be very carefully presented along with variation estimates and what constitutes an independent replication and the number of replicates used to calculate the averages presented in the table or figure. Each table and figure must be on a separate page in the submitted paper. In addition, you will need to submit all data for charts, tables and figures in native format when possible (e.g., Microsoft Excel, Powerpoint). Photographs should be submitted as high-resolution (600 dpi) .jpg or tif. files. All figures should be clearly presented with well defined axis and units of measurement. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

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AFAB Volume 3 Issue 2