PRODUCTION OF SINGLE CELL PROTEIN

To succeed in producing single cell protein (SCP) in large scale from wood with economic viability, there are many parts that must be perfected, for example: 1 Cheap and available raw materials 2 Efficient and cheap pretreatment processes

3 Efficient and fast growth of the SCP organism 4 Energy-efficient harvesting and drying procedures >>

WRITTEN BY ANDREAS HÖRNBERG

Within the scope of this project, hthe project team has addressed many of these parameters, and calculated the production price for 1 ton of product from forest raw material. In work package 1 (WP1), the raw material (point 1 above) and the pretreatment method (point 2) was evaluated and in WP2 we focused on optimizing the SCP organism (point 3) and investigated the harvesting and drying procedures (point 4).

The available equipment, including the instruments that were purchased within the project, has mostly worked well for their purpose. The experiments were performed in scales from small shake flasks to bioreactors of 3, 50, and 600 liters. Upscaling worked well, but not perfect since the bioreactors in pilot scale was limited in stirring and aeration capacities. The dissolved oxygen in the cultivation media is a very important parameter when growing a filamentous fungus. When the SCP organism reaches high biomass levels, the broth becomes viscous, which results in difficulties in stirring and aeration. This type of SCP organism is easily harvested by filtration and for the larger bioreactor cultivations in this project (50-600 liters) a filter press was used. There are different types of equipment for drying of the harvested material in pilot and demo scale, for example a belt dryer, spray dryer, and a tornado dryer. In the scale used in the present project and with the funding available, manual drying in drying cabinets were used.

AP 2.1 OPTIMIZATION OF SCP-PRODUCTIVITY

To investigate and optimize the productivity of a microorganism, a bioreactor system must be run in continuous mode, where new cultivation media is fed to the reactor at the same speed as cultivated media is harvested. After a few hours or days of continuous operation an equilibrium is reached, and thereafter representative samples of the cultivated media can be harvested until all fresh media is consumed. The equipment (4 bioreactors at 3 liter each, Fig. 1) that was invested in during the project did not perform as planned. The harvesting pumps were unable to pump the highly viscous broth containing the SCP cells out of the reactors, instead removing only the liquid media; this resulted in accumulation of SCP within the reactors. Continuous fermentations (2) were instead run in a 50-liter bioreactor (Fig. 2) with 40 and 30 liter of cultivation media and a dilution rate of 0.1 (i.e. 4- and 3-liter feed/hour respectively). The larger reaction volume meant that larger tubing and pumps could be used. The cultivations were allowed to proceed for 6 and 4 days respectively, and the harvested material was analyzed for biomass, protein content, sugar- and acid concentrations. Practically, the experiments went well overall, but the results did not meet the expectations. Productivity was 1,5 g/L/h at most, and the protein content was only about 43% at the best harvest (the goal of the project was a productivity of 2 g/L/h and protein content at above 60%). The low productivity and protein content is probably the result of low oxygen transfer to the cells due to insufficient stirring and aeration of the cultivation media. Sugar analysis of the media confirmed the decreased growth of the cells, since there was significant amounts residual sugar in the harvested media.

Spent sulphite liquor (SSL) from the Domsjö Fabriker was used in all the productivity optimization experiments since a large volume was needed. The SSL is a well-known model substrate and is used in many applications by RISE Processum.

AP 2.2 INCREASED CELL CONCENTRATION OF THE SCP FUNGUS BY MODIFICATION OF THE CELL MORPHOLOGY

To address the optimization of fast and efficient cell growth of the SCP organism, we focused on increasing the cell biomass (g SCP/ L of cultivation media) through strain development. The fungus was modified either by long-term adaptation or by random mutagenesis with UV-light during the whole project period. Small and compact colonies on agar plates is the preferred morphology of the microorganism and these colonies were cultivated with liquid media in shake flasks. The theory for this selection is that the long filaments (hyphae) of the fungus will likely result in higher viscosity in liquid media,

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FIGURE 1. A bioreactor system in stainless steel containing four reactors of 3 liter each.

FIGURE 2. A 50-liter bioreactor that is working very well for cultivation of the SCP organism.

FIGURE 3. A 3-liter bioreactor system in glass used for the adaption experiments.

and thereby decrease stirring, aeration and oxygen transfer into the media. Since good oxygenation is crucial to the growth of the fungus, higher viscosity leads to decreased overall biomass is the result. A shorter filament should thereby lead to higher possible biomass content.

In the adaptation experiments the filamentous fungus was cultivated in 3-liter bioreactors (Fig. 3) for an extended time (3 weeks), and portions of the cultivation media were replaced with fresh media every 3 days. After the cultivation period, cells were cultivated on agar plates and the size of the resulting colonies were evaluated. The best (small and compact) colonies were used for a second round of adaptation experiments. This procedure was repeated a third time before the evolved strain was analyzed for biomass production. With the final adapted strain, a biomass of 25 g/L of dry cell weight was reached, compared to about 7-8 g/L for the wildtype strain. Mutation experiments with UV-light were also performed during the project and these also resulted in elevated biomass concentrations, but none of the produced strains in these mutation experiments exceeded 20 g/L of SCP. Within this part of the project, a number of other parameters were also investigated to increase the efficiency and decrease the cost of production of SCP in large scale. The explored parameters were cultivation of the microorganism at lower pH, increased concentration of the substrate (spent sulphite liquor), and optimization of the nutrient solution composition.

It is potentially beneficial to cultivate the cells at lower pH, since many pretreatment reactions (in this case others) are performed under acidic conditions; therefore the amount of base needed to neutralize the media before cultivation is decreased. Cultivation at low pH would then improve the economics by decreasing the cost of chemicals, while also decreasing the amount of chemicals used which is beneficial for the environment. Before the project was initiated, the optimal pH for this fungus was around 6, but after optimization and adaptation during this project, the microorganism could be cultivated at pH 4.5 with no loss in productivity. Cultivation at even lower pH was also investigated, down to pH 3.0, but at these conditions the microorganism grew slower and produced less biomass. Experiments to cultivate at a higher substrate concentration were also performed. The initial concentration of the spent sulphite liquor was 50% (diluted with water, nutrient solution and inoculation culture) and experiments using 75-94% of substrate were performed. Many experiments with increased substrate concentrations were successful, but not all, which indicates that more research must be done to get consistently better performance. It is important to be able to cultivate the microorganism at as high concentration as possible to reduce the water consumption and to get increased economic viability.

Another interesting parameter to optimize is the nutrient solution composition (mainly nitrogen and phosphorus content). The goal with the optimization is to determine the lowest amount of added chemicals that will maintain good cell growth and high protein content. The first experiments indicated small differences in the composition would result in slightly higher biomass, but more experiments are needed to evaluate and confirm these results. It is important for a future factory to minimize additions of chemicals, both for the cost and for the environment.

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FIGURE 4. A pilot-sized filter press.

FIGURE 5. Shredder used for grinding SCP before drying.

FIGURE 6. A set of drying cabinets to produce the final dry SCP product.

AP 2.3 HARVEST, WASHING AND DRYING

In order to investigate the options for harvesting and drying of the cellular SCP biomass, a filter press (Fig. 4), a shredder (Fig. 5) and five drying cabinets (Fig. 6) in pilot scale, was purchased, installed and used during the project. Many separation and washing experiments were performed in the filter press to optimize the harvesting of SCP. The filter press has a built-in function to squeeze the filter cakes after all the material is filtered. This squeezing is done first with water pressure (the filter cakes are protected by the filter membranes) and then by air to increase the dry content from around 20 up to 35-40%. This function results in much drier and cleaner filter cakes, and a slightly higher protein content was also detected when compared to harvesting in a conventional filter press.

After the harvesting procedure with the filter press, filter cakes with dimensions of 30 x 30 cm and up to 4 cm thick are obtained. These filter cakes are broken into smaller pieces with a shredder, and the shredded material was subsequently distributed onto metal sieve trays and dried in a drying cabinet for around 6 hours. The drying cabinets purchased within the project are equipped with several excellent functions, such as an efficient fan system within the cabinets, removal of the moisture, temperature interval up to 150 ˚C, and temperature control with a timer. The filter press, shredder, and the drying cabinets are all working very well.

Additionally, the digestibility of the SCP product was investigated by an in-vitro method to evaluate the effect of drying conditions. Digestibility is an important parameter in the feed industry since it is an indication of how well the animals can absorb the feed nutrients in their gut. Drying at three different temperatures was studied (60 ˚C, 75 ˚C, and 90 ˚C) and out of these the lowest temperature gave the best result, i.e. the protein in the material dried at the lower temperature was the most digestible.

Within the scope of the project, different techniques for large scale harvesting and drying has been performed in cooperation with equipment producers. One of the techniques, a belt drier, was found to work very well together with the SCP organism used in the project. In the pilot/ demo facility that is under construction in Örnsköldsvik, the acquisition of a demo-scale belt dryer is planned as a result of the FISK-project. .

AP 2.4 DEMONSTRATE PRODUCTION OF SCP IN PILOT SCALE USING PRETREATED TOPS AND BRANCHES AS RAW MATERIAL

Within the project, spent sulphite liquor was used in optimization studies for cellular biomass and productivity, but pretreated tops and branches from birch and pine were also evaluated and used as a substrate for SCP production. In WP 1, PFI sorted and selected representative samples of birch and spruce, which were used in pretreatment experiments to produce a fermentable substrate with high sugar content and with low concentrations of inhibitors. These samples were sent to Processum to evaluate the fermentability of the media using the SCP microorganism. The first samples contained too low amount of sugar for the organism to grow at high biomass content, but the inhibitor content was also negligible. PFI continued with optimizing the pretreatment experiments and produced a slurry with high sugar content and low amounts of inhibitors after a concentration step. The microorganism grew well on the concentrated substrate from tops and branches (birch 14.9 g/L and spruce 18.0 g/L SCP). An upscaled experiment was also performed using this material with 2-liter

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cultivation media in 3-liter bioreactors using tops and branches from both birch and spruce, and with a substrate composed of theoretical amounts of monomeric sugars present in the trees as a control substrate. The microorganism grew as well or even better in the pretreated material of tops and branches than in the control media (Fig. 7).

The questions that have been addressed within WP 2 of the project are growth and productivity of the SP-organism, and also optimization of the harvesting and drying procedures. During the project, the biomass production of the cultivated filamentous fungus has increased three times and the productivity has also been increased, but to a lesser extent. Future plans might be to investigate how well the fungus would grow at optimal conditions with perfect aeration and stirring and an optimized nutrient solution. To reach economically viable production of SCP, the microorganism must be able to grow on substrates containing fermentable carbon sources of at least 50 g/L to get a biomass content of 25 g/L. The protein content also needs to be increased in order to have an economic production process. Harvesting and drying have worked very well in the scale examined in this project, but for a large-scale facility another type of drying equipment is more suitable, i.e. a belt dryer with 2-4 segments with different drying temperatures.

Biomass (g/l) 20

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0 GROT Spruce GROT Birch Modeling sugar Spruce Modeling sugar Birch

FIGURE 7. An experiment with the comparison between tops and branches from birch and spruce and control samples.