Bioresource Technology 131 (2013) 128–133
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High pressure thermal hydrolysis as pre-treatment to increase the methane yield during anaerobic digestion of microalgae Philip Keymer a, Ian Ruffell b, Steven Pratt a,⇑, Paul Lant a a b
School of Chemical Engineering, The University of Queensland, Brisbane, QLD 4072, Australia School of Chemical Engineering and Advanced Materials, University of Newcastle upon Tyne, Merz Court, Newcastle upon Tyne NE1 7RU, UK
h i g h l i g h t s " High pressure thermal hydrolysis (HPTH) pretreatment enhances algal digestibility. " HPTH increases methane yield from digestion of algae & lipid extracted algal residue. " HPTH has no affect on rate of methane production from algae. " Lipid extracted algal biomass has a higher rate of methane production than raw algae. " Digestion of algae solubilises nutrients which can be recycled for algal growth.
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Article history: Received 28 September 2012 Received in revised form 14 December 2012 Accepted 18 December 2012 Available online 25 December 2012 Keywords: High pressure thermal hydrolysis Anaerobic digestion Microalgae Methane Nutrients
a b s t r a c t Anaerobic digestion of algal biomass will be an essential component of algal biofuel production systems, yet the methane yield from digestion of algae is typically much lower than the theoretical potential. In this work, high pressure thermal hydrolysis (HPTH) is shown to enhance methane yield during algae digestion. HPTH pre-treatment was applied to both raw algae and algal residue resulting from lipid extraction. HPTH and even the lipid extraction process itself increased methane yield, by 81% and 33% respectively; in combination they increased yield by 110% over that of the raw algae (18 L CH4 g VS 1 substrate). HPTH had little effect on the rate of anaerobic digestion, however lipid extraction enhanced it by 33% over that for raw algae (0.21 day 1). Digestion resulted in solubilisation of nitrogen (and phosphorous to a lesser degree) in all cases, showing that there is potential for nutrient recycling for algal growth. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Anaerobic digestion of algal biomass will be an essential component of algal biofuel production systems (Sialve et al., 2009). The digestion product methane, a biofuel in its own right, can be generated from digestion of either algal biomass or the algae residue that is a by-product of lipid extraction for biodiesel production (Zamalloa et al., 2011). It has even been suggested that methane production from microalgae without lipid extraction is energetically more favourable than a system whereby the lipids are extracted prior to digestion, if the algal lipid content is less than 40% (Sialve et al., 2009). In practice, the methane yield from anaerobic digestion of algae is much lower than the theoretical methane potential based on the carbohydrate, lipid and protein composition of the algae (Becker, 2004). A number of factors may contribute to the lower than antic⇑ Corresponding author. Tel.: +61 7 33467843. E-mail address: s.pratt@uq.edu.au (S. Pratt). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.125
ipated methane yield, including the inhibitory effects of both culture substrate (sodium) and by-products (ammonia) as well as the physical properties of the algae themselves (rigid cell walls) (Appels et al., 2011; Ras et al., 2011). Biomass pre-treatment can be used to increase methane yields. A large number of pre-treatment options exist including ultrasonic, thermal, microwave, chemical, electrical and freeze/thaw methods (Komaki et al., 1998; Janczyk et al., 2007; Carrère et al., 2010; Carlsson et al., 2012). A powerful alternate pre-treatment technology that has already been commercialised for improving digestibility of waste activated sludge (WAS) from wastewater treatment plants, but not yet tested on algae, is high pressure thermal hydrolysis (HPTH) (Donoso-Bravo et al., 2011; Nielsen et al., 2011; Val del Río et al., 2011). The HPTH process is a two stage process whereby the substrate is first heated to 160 °C at a pressure of 6 bar. After these conditions have been held for 20–30 min the contents are then flashed to a lower pressure vessel, where the pressure change causes the cells to rupture and release the cell contents (Morgan-Sagastume
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et al., 2011). Macromolecular components are reduced to lower molecular weight derivatives or released as soluble monomeric substances (Wilson and Novak, 2009). When digesting secondary sludge from wastewater treatment plants, HPTH enables a 50– 100% increase in biogas production (Norli, 2006). This is sufficient to generate steam required for thermal treatment, which elevates sludge temperature thus negating the need to heat the digester. An energy balance suggests that while HPTH is an energy intensive process, the technology coupled with digestion can actually be energy positive (Kepp et al., 2000). Additional to methane production, anaerobic digestion is seen as a potential means for recycling nutrients, namely nitrogen and phosphorus, in algal production systems (Zamalloa et al., 2011). It has been proposed that these nutrients be recycled to support algal growth (Rösch et al., 2012). Although several studies have reported the nitrogen (primarily ammonia) release during the anaerobic digestion of algae, there is little information on the amount of nitrogen actually solubilised during algae digestion, as this is often masked by the nitrogen already present in the anaerobic digester inoculum (Samson and LeDuyt, 1986; Ehimen et al., 2011; Park and Li, 2012). There is even less data available for the solubilisation of phosphorous following anaerobic digestion of algae; recycling of phosphorous will be particularly important considering the ongoing depletion of mineral phosphorous reserves (Cordell et al., 2009). In this paper we aim to quantify the gain in methane yield and production rate that one may achieve by applying HPTH pre-treatment to algae; both raw algal biomass and algal residue from lipid extraction for biodiesel production are considered. This information will be useful for determining the potential energetic benefits of incorporating a HPTH stage in an algal biorefinery. The secondary objective of this study is to quantify the nutrient solubilisation during anaerobic digestion of algal biomass, as this is poorly reported for nitrogen and unreported for phosphorous. This information will assist in determining the potential for nutrient recycling in algal growth systems coupled with anaerobic digestion. 2. Methods Two treatment procedures were performed on the algal biomass prior to anaerobic digestion of the samples, namely high pressure thermal hydrolysis (HPTH) and lipid extraction. Fig. 1 shows the work-flow followed for the generation of the four samples used for the anaerobic digestion tests. An untreated raw algal sample was used to determine the baseline methane yield of the algae. The raw algae was either hydrolysed or subjected to a procedure for lipid extraction to provide the HPTH material (referred to in this work as high pressure thermally hydrolysed (HPTH) algae) and algae residue (referred to in this work as lipid extracted (LE) material). High pressure thermally hydrolysed lipid extracted (HPTH LE) material was produced by applying HPTH to a portion of the algae residue from lipid extraction. Methane production tests and analytical tests were performed in triplicate and the results were reported as means with standard errors. 2.1. Growth and culture conditions The algae used in this study was a mixed culture enriched with Scenedesmus microalgae collected from a local freshwater pond at The University of Queensland. It was cultivated in a conical 370L (300 L working volume) opaque, open-top bioreactor. The bioreactor was located outdoors, was aerated at a rate of 7 L min 1, and supplemented with 11 g of soluble garden fertiliser (Aquasol, Australia, 23% total nitrogen and 4% phosphorous by mass) every four days. The bioreactor was operated in a semi-batch fashion with an
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average retention time of 17 days. The algae was grown to a dry weight of approximately 1 g L 1 with a COD of 1180 mg gVS 1, nitrogen content of 140 mg gVS 1 a phosphorous content of 46 mg gVS 1 and a lipid fraction of 9% by mass. The typical composition of Scenedesmus microalgae is 50–56% protein, 10–17% carbohydrate and 12–14% lipid (Becker, 2004), the carbohydrate primarily contain glucose monomers ( 60%) (Brown et al., 1997). The algae was concentrated to a paste by centrifugation in a Beckman Coulter, Allegra™ X-12 at 3270g for 5 min in 500 ml batches. 2.2. Lipid extraction Prior to lipid extraction, the algal paste was dried overnight in a 55 °C oven and ground to a fine powder in a ceramic mortar and pestle. Lipids were extracted in hexane using a standard Soxhelt apparatus for 6 h (Wang and Weller, 2006). After the extraction the residual algal biomass was incubated at 55 °C for 48 h. The elevated temperature ensured the removal of residual solvent by volatisation. 2.3. High pressure thermal hydrolysis The high pressure thermal hydrolysis (HPTH) system consisted of two 1 Gallon pressure vessels (Parr Instrument Company, USA). The first vessel was the heated pressure vessel, whilst the second vessel was used as the receiving flash vessel (Fig. 2). The reactor was filled with a thickened solution of algae or lipid extracted algae (14 g L 1) and was heated to 170 °C at 800 kPa. The reactor contents were agitated (200 rpm) by an impeller, ensuring uniform heating of the sample. The reactor was held at this elevated temperature and pressure for 30 min, after which the contents were systematically vented (10 s open and 50 s closed) to the flash tank to complete the HPTH process. 2.4. Biochemical methane potential The biochemical methane potential (BMP) assay provides a measure of the anaerobic digestibility of a given substrate, and was used to determine the methane yield of the algal biomass (Batstone et al., 2009). Algal biomass was batch digested in 270 mL sealed glass serum bottles (230 mL working volume). Batches were inoculated at an inoculate to substrate ratio of 2 (volatile solids basis) with anaerobic digester sludge. The anaerobic digester sludge was sourced from a local wastewater treatment plant (Luggage Point Advanced Water Treatment Plant, Myrtleton, Queensland, Australia). The digester sludge had been conditioned to high ammonia levels making it optimal for the digestion of nitrogen rich algal biomass. The digester sludge was degassed and kept under anaerobic conditions at 38 °C for no longer than seven days prior to commencing the experiments. The remainder of the batches consisted of concentrated algal biomass and deionised water to ensure an equal headspace was maintained for all serum bottles. Anaerobic conditions were established by purging the headspace of each serum bottle with high purity nitrogen immediately followed by sealing with a gas-tight butyl-rubber plug secured by an aluminium crimp. The batches were incubated at 38 °C and agitated every 2–3 days. Periodically (daily for 8 days, then every 2nd day for 4 days, then every 3rd day for 3 days and then every 5th day for 20 days), headspace gas samples were extracted with a precision gas-tight syringe (SGE International Pty Ltd., Australia). The serum bottles were maintained under pressure. The pressure in each serum bottle was kept below 3 bar by removing gas as required. The total volume of the removed gas was measured with a bench-top manometer, and the composition (CH4, CO2 and H2) was measured by GC. The tests were run for 35 days by which stage the methane
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Fig. 1. Sequence of treatments followed to generate samples used in this study, rectangles represent treatments performed and free-standing text represent samples.
2.5. Analytical methods
Fig. 2. HPTH system consisting of two 1 Gallon pressure vessels connected by a transfer line for thermal hydrolysis.
The composition of gas produced during digestion was determined with a Perkin–Elmer loop injection GC. The Perkin–Elmer GC-TCD (AutoSystem GC, Perkin–Elmer, Waltham, MA, USA) is fitted with a 2.44 m stainless steel column (Haysep at 80/100 mesh) and a GC Plus Data Station (model 1022, Perkin–Elmer, Waltham, MA, USA). High purity nitrogen was used as the carrier gas at a flow pressure of 55 kPa. The injection port temperature was set at 75 °C, the oven temperature at 40 °C and the detector at 100 °C (American Public Health Association, 2005). The GC was calibrated using external gas standards from British Oxygen Company (Sydney, NSW, Australia). Total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (sCOD) were measured according to Standard Methods (American Public Health Association, 2005) using SpectroquantÒ photometric cell tests (114555, Merk, Germany), a Thermoreactor TR 300 (Merck, Germany) and an SQ 118 Photometer (Merck, Germany). TSS and VSS were measured according to standard methods (American Public Health Association, 2005). NHþ 4 —N, 3 NO —N, NO —N, PO —P, total Kjeldahl phosphorous (TKP) and 3 2 4 total Kjeldahl nitrogen (TKN) were measured with a Lachat QuikChem 8000 Flow Injection Analyser (Lachat Instrument, Milwaukee, USA).
3. Results and discussion production had dropped dramatically. Samples of the untreated substrates, inoculum and the completed batches were analysed 3 for NHþ 4 —N, NO3 —N, NO2 —N, PO4 —P, TKP, TKN, total chemical oxygen demand (TCOD), soluble chemical oxygen demand (sCOD), total solids (TS) and volatile solids (VS).
3.1. Solubilisation of organics during pre-treatments The ratios of sCOD to TCOD for the samples, for each pre-treatment, prior to digestion are shown in Fig. 3. Both HPTH and HPTH
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Table 1 Parameter estimation for degradability (fd) and first order production rate coefficient (khyd) showing standard errors of predictions. Sample
fd (L CH4 gVS subs 1)
fd (gCOD CH4 gCOD subs 1)
khyd (day 1)
v2
Raw algae HPTH algae LE material HPTH LE material
0.18 ± 0.01 0.33 ± 0.01 0.24 ± 0.01 0.38 ± 0.01
0.29 ± 0.01 0.58 ± 0.01 0.77 ± 0.02 0.83 ± 0.01
0.21 ± 0.01 0.22 ± 0.01 0.28 ± 0.03 0.29 ± 0.01
0.0037 0.015 0.026 0.013
or yield, fd [L CH4 gVS 1] and the apparent hydrolysis rate, khyd [day 1]. These parameters were determined by fitting the data to the following first order equation (Batstone et al., 2009):
Y CH4 ¼ V CH4 =VS ¼ fd ð1 expð khyd tÞÞ: Fig. 3. Soluble COD fraction of raw and treated algal biomass.
LE samples showed an approximately ten fold increase in the soluble COD fraction over that of their non-hydrolysed counterparts. This is expected as the high pressure thermal hydrolysis process is particularly effective at solubilising organic matter (Wilson and Novak, 2009; Donoso-Bravo et al., 2011). Wilson and Novak (2009) showed that the increase in soluble COD was a consequence of an increase in the soluble fraction of all components. For waste activated sludge, HPTH boosts the soluble protein by a factor of at least five and the soluble polysaccharides by a factor of at least 10. The algal residue remaining after lipid extraction showed a 2.5fold increase in soluble COD fraction as compared to the raw algae. This too was expected as the residual biomass from the extraction is expected to have an increased soluble fraction due to the disruption of the cells during the process. The combination of both the lipid extraction and HPTH had a cumulative effect as practically all the COD was solubilised. One may expect that this solubilisation of cell contents would have an affect on the subsequent methane yield and nutrient release following anaerobic digestion.
3.2. Methane production A first order kinetic model was employed for analysis of the BMP data, to allow for the estimation of the apparent degradability
ð1Þ
Where YCH4 is the specific methane yield at a given time [L CH4 gVS 1], VCH4 is the volume of methane produced [L CH4], VS is the mass of volatile solids present in the BMP vessel [gVS] and t is the time since inoculation of the BMP [days]. The model was implemented in Aquasim 2.1d. The objective function used was the sum of squared errors (v2). Average data from triplicate experiments were used, and uncertainty was assessed through parameter uncertainty analysis. Results of the BMP tests with model simulations are shown in Fig. 4. The error bars indicate the standard errors from triplicate tests. Model lines shown in this figure are based on the best fit of fd and khyd with standard errors which are shown in Table 1. The HPTH treatment consistently increased the methane yield compared to that obtained from the raw algae (0.15 L CH4 gVS 1 or 81% increase over raw algae) and the algal residue from lipid extraction (0.14 L CH4 gVS 1 or 58% increase over LE material). Lipid extraction itself had an effect on methane yield and gave an increase of 33% over that of the raw algae. When combined with HPTH the effect was cumulative and methane yield increased 110% over that of the raw algae. This increase in methane yield follows a similar trend to that observed for the solubilisation of COD. The methane yields observed in this study are similar to those reported in other studies, ranging between 0.10 and 0.40 L CH4 gVS 1 (Nallathambi Gunaseelan, 1997; Ras et al., 2011). The raw algal methane yield was fairly low when compared to other studies, however this was expected as the algal biomass was not enriched
Fig. 4. Results of biochemical methane potential tests for different algal samples (blank corrected production amounts). Error bars indicate standard error in triplicate tests and lines show model predicted trends.
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Fig. 5. 95% Confidence envelopes for parameters (fd and khyd) predicted for each of the samples digested.
sult observed for solubilisation of COD and indicates that ammonification of protein takes place during TH, as concluded by Wilson and Novak (2009). Conversely lipid extraction alone did not give rise to a significant change in pre-digestion soluble nitrogen fraction, which indicates that lipid extraction only acted to liberate soluble organics rather than instigate protein degradation. Anaerobic digestion resulted in a significant increase in the soluble fraction of nitrogen in all four materials (40–53% soluble N fraction increase), with HPTH samples having higher final fractions than their untreated counterparts (raw algae and LE material). These findings are consistent with previous observations of other studies where algal digestion results in a significant increase in soluble nitrogen, due to the high protein content of the algae (Ras et al., 2011). The trends observed for the pre-digestion soluble fractions of phosphorous, as shown in Fig. 6b, suggests that neither HPTH or lipid extraction lead to a significant change in pre-digestion phosphorous solubility, however when both processes are combined
(a) for lipids and the algae had a high culture age (retention time of 17 days). Both these factors have been known to decrease the methane yields in WAS treatment (Venkata Mohan et al., 2008). Fig. 5 shows 95% confidence envelopes of the parameters estimated for the four samples allowing one to visualise the effect of the treatments on both parameters of interest. These envelopes were calculated by the minimisation an objective function based on the residual of the sum of squares as described by Batstone et al. (2003). From Fig. 5 it is apparent that HPTH had little effect on the rate of methane production (khyd), however it did significantly increase the overall degradability (fd) of both raw and lipid extracted algae. This lack of change in khyd is somewhat contrary to what has been previously observed for WAS where the rate was shown to increase (Kim et al., 2003). The lipid extraction increased both the degradability and rate of digestion of the algae, as seen by comparing the confidence envelopes of the raw algae and the LE material. In this case the extraction led to a 33% increase in hydrolysis rate. A similar effect (32% increase in hydrolysis rate) was observed when comparing the confidence envelopes of the HPTH algae and the HPTH LE material. This supports the previous observations where the effect of the combined treatments were cumulative. It is apparent that the extraction process enhanced the rate at which the biomass was degraded whilst the HPTH did not. Organic solvents are known to disrupt cell walls and denature proteins and it is possible that during the lipid extraction process the hexane chemically degraded parts of the algal biomass, making it easier to degrade rapidly whereas the HPTH may have only served to physically disrupt the biomass and solubilise its contents (Isken and de Bont, 1998). One should note that the confidence envelope of the LE material, shown in Fig. 5, was larger than of the other treatments. This was due to difficulty in sampling the algal residue following lipid extraction as the resulting biomass formed a waxy solid which did not dissolve well in the aqueous phase.
(b)
3.3. Nutrient solubilisation of digested algae Fig. 6a shows, the soluble nitrogen fraction (combined NHþ 4 —N, 3 NO 3 —N and NO2 —N per TKN) and phosphorous fraction (PO4 —P per TKP) before and after anaerobic digestion. It can be seen that HPTH increased the pre-digestion soluble fraction of nitrogen in both raw (6-fold N fraction increase) and lipid extracted algae (8fold N fraction increase), which is consistent with the previous re-
Fig. 6. Soluble fractions of nitrogen and phosphorous for the various samples preceding and following anaerobic digestion.
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(HPTH LE material) the soluble P fraction increased by 27% over that of the raw algae. After digestion the soluble fraction of phosphorus increased for all samples with the exception of the HPTH LE material which showed no significant change. It can be seen that the nitrogen release during anaerobic digestion is correlated with the methane yield, as these are both governed by the biological degradation of the algae. On the other hand, net phosphate release is not closely related to methane yield and this is most likely due to the action of other chemical processes which govern phosphate solubility in addition to biological degradation (Yuan et al., in press). One such chemical process is the dependence of phosphate solubility on pH, and it is suspected that adjusting the post digestion pH would have led to further phosphate release (Münch and Barr, 2001). These results show that there is potential to recycle both nitrogen (43–66% recovery as soluble N) and to a lesser degree phosphorous (20–39% recovery as soluble P) from digested algae. But even with the enhanced nutrient solubility resulting from HPTH and lipid extraction there are substantial losses. Further investigation will be required to determine the suitability of the solubilised nitrogen and phosphorous as an algal growth supplement. One should also note that there may also be potential to recover the insoluble nutrients, specifically phosphorous which may have precipitated following the digestion. 4. Conclusions HPTH has a significant effect on overall anaerobic degradability and subsequent methane yield of the algae. A cumulative effect is observed when combining HPTH and lipid extraction. The rate of hydrolysis in the anaerobic digestion is not affected by HPTH however lipid extraction does increase the rate. Digestion of algae offers potential for the recovery of nutrients from cultivated algae and may be used to recycle nutrients for further algal growth, but phosphorous solubilisation is apparently limited possibly due to its subsequent precipitation. HPTH and lipid extraction further enhance this nutrient recovery as they assist in biomass breakdown during digestion. Acknowledgements The authors wish to thank Prof. Peer Schenk and the staff of the Algae Biotechnology Group (The University of Queensland, Australia), for providing the algal samples. References American Public Health Association, Eaton, A.D., American Water Works Association, Water Environment Federation, 2005. Standard methods for the examination of water and wastewater. APHA-AWWA-WEF, Washington, DC. Appels, L., Lauwers, J., Degrève, J., Helsen, L., Lievens, B., Willems, K., Van Impe, J., Dewil, R., 2011. Anaerobic digestion in global bio-energy production: potential and research challenges. Renew. Sust. Energ. Rev. 15, 4295–4301. Batstone, D.J., Pind, P.F., Angelidaki, I., 2003. Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate. Biotechnol. Bioeng. 84, 195–204. Batstone, D.J., Tait, S., Starrenburg, D., 2009. Estimation of hydrolysis parameters in full-scale anerobic digesters. Biotechnol. Bioeng. 102, 1513–1520. Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, G.A., 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151, 315–331.
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