MASTER OF SCIENCE THESIS WITHIN BIOTECHNOLOGY
Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production Amanda Steen
Performed at SP Processum AB Örnsköldsvik, Sweden Spring 2014 Supervisor: Björn Alriksson, SP Processum AB Examiner: Gen Larsson, KTH School of Biotechnology Division of Bioprocess Technology The Royal Institute of Technology, KTH Stockholm, Sweden, 2014
Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
Abstract The demand of food, and especially protein, is increasing along with the increase of the global human population. Fish is an important source of protein for the global population. The demand for aquatic food is projected to increase with about 20% between the year 2010 and 2020. This increase has to be met by increased aquaculture production. Consequently, there is an increasing demand for fish feed. The favoured protein source in fish feed is fishmeal. The availability of fishmeal is however predicted to decrease. Therefore, there is an increasing demand for alternative high-‐quality protein sources for fish feed. Soybean meal is the most frequently used alternative source for fish feed today. However, vegetable protein sources are associated with challenges, such as antinutritional substances and unfavourable amino acid composition. Single cell protein (SCP) is another alternative protein source that has many benefits, such as a fast production and a favourable amino acid profile. In addition, SCP can be produced from different residual streams derived from industry. This provides the possibility to have a cheap production from renewable and sustainable feedstocks. In this master thesis project the potential to produce SCP from residual streams from the 2nd generation bioethanol production has been investigated. Three different residual streams based on lignocellulosic material (prehydrolysate and stillage of wheat straw, and prehydrolysate of spruce) were utilised and four different microorganisms were evaluated (Paecilomyces variotii, Cunninghamella echinulata, Mortierella isabellina, and Yarrowia lipolytica). Pilot-‐scale cultivation of P. variotii on prehydrolysate of wheat straw and on detoxified prehydrolysate of spruce showed promising results with biomass concentrations of 8-‐10 g/L and with a protein content of around 50%. In addition, the biomass consisted of high levels of β-‐glucans, about 20%. β-‐glucans are an interesting molecule that increasingly is being supplied to fish feed due to their immunostimulatory effect. The high β-‐glucan content could potentially increase the value of the SCP as an ingredient in fish feed. Y. lipolytica grew well on stillage of wheat straw and reached a biomass concentration of 15 g/L with a protein content of over 50% in a pilot-‐scale experiment. An interesting finding was the utilisation of uncharacterised carbon sources within the prehydrolysate and stillage of wheat straw. This indicates that the microorganisms, and especially Y. lipolytica, were able to utilise a broad range of the carbon sources available within the residual streams. This study shows that the utilisation of residual streams from the 2nd generation bioethanol production is an interesting and potential substrate for large-‐scale production of SCP, which warrants further studies.
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
Sammanfattning Världens befolkning ökar och med den ökar också behovet av mat. Fisk är en viktig proteinkälla för en stor del av jordens befolkning. Efterfrågan på fisk förväntas öka med nästan 20% mellan år 2010 och 2020. Denna ökning måste komma från produktion i fiskodlingar, vilket i sin tur innebär en ökad efterfrågan på fiskfoder. Fiskmjöl är generellt den mest använda proteinkällan för fiskfoder, men tillgången av fiskmjöl förväntas minska kommande år. Följaktligen finns det ett ökande behov av alternativa högkvalitativa proteinkällor som ersättningsprodukt för fiskmjöl. Idag är sojamjöl den vanligaste alternativa proteinkällan i fiskfoder. Proteinkällor i form av grödor och andra växter är dock förknippat med vissa nackdelar, såsom antinutrionella ämnen och en ofördelaktig aminosyraprofil. Single cell protein (SCP) är en annan intressant alternativ proteinkälla. SCP har många fördelar såsom en snabb proteinproduktion och en fördelaktig aminosyraprofil. SCP kan dessutom produceras från restströmmar från olika industrier, vilket ger möjligheten till en billig produktion från förnyelsebara och hållbara råmaterial. I detta examensarbete har potentialen att producera SCP från restströmmar från 2:a generationens bioetanolproduktion undersökts. Tre olika restströmmar från bioetanolproduktion från lignocellulosa (förhydrolysat och drank från vetehalm, samt förhydrolysat från gran) och fyra olika mikroorganismer (Paecilomyces variotii, Cunninghamella echinulata, Mortierella isabellina och Yarrowia lipolytica) utvärderades. Odling av P. variotii i pilotskala på förhydrolysat från vetehalm och detoxifierat förhydrolysat från gran gav lovande resultat och resulterade i biomassakoncentrationer på 8-‐10 g/L med ett proteininnehåll på cirka 50%. Dessutom bestod biomassan av cirka 20% β-‐glukan. β-‐glukan är en grupp intressanta molekyler med immunostimulerande egenskaper vilka används i allt större utsträckning inom fiskfoderindustrin. Ett högt innehåll av β-‐glukan kan potentiellt öka värdet på SCP som en ingrediens i fiskfoder. Även Y. lipolytica växte bra på drank från vetehalm och gav biomassakoncentrationer på 15 g/L med ett proteininnehåll på över 50% i odling i pilotskala. Ett intressant resultat från studien är att mikroorganismerna använde andra kolkällor, än de som analyserades och identifierades i restströmmarna från vetehalm. Detta visar att mikroorganismerna, och särskilt Y. lipolytica, har möjligheten att i stor utsträckning använda de källor av kol som finns närvarande i de undersökta restströmmarna. Detta arbete visar att restströmmar från produktion av 2:a generationens bioetanol är intressanta och lovande som substrat för storskalig produktion av SCP, och att vidare studier inom detta område bör genomföras.
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
Table of contents Abstract ............................................................................................................................................... ii Sammanfattning ............................................................................................................................. iii 1. Introduction .................................................................................................................................. 2 2. Background ................................................................................................................................... 5 2.1 Single cell protein and single cell oil ............................................................................................ 5 2.2 Microorganisms for single cell protein and single cell oil production ............................. 9 2.3 Bioethanol production from lignocellulose ............................................................................ 11 3. Materials and methods .......................................................................................................... 15 3.1 Microorganisms and residual streams ..................................................................................... 15 3.2 Preparation of residual streams ................................................................................................. 16 3.3 Initial screening experiments with prehydrolysate and stillage of wheat straw ...... 16 3.4 Detoxification experiments with prehydrolysate and stillage of wheat straw ........... 17 3.5 Initial screening and detoxification experiment with prehydrolysate of spruce ...... 18 3.6 Multifermenter experiment with P. variotii on prehydrolysate and stillage of wheat straw ............................................................................................................................................................ 19 3.7 Pilot scale experiments .................................................................................................................. 22 3.8 Chemical analyses ............................................................................................................................ 30
4. Results ......................................................................................................................................... 32 4.1 Concentrations of monosaccharides, aliphatic acids, and ethanol in the residual streams ....................................................................................................................................................... 32 4.2 Initial screening experiments with prehydrolysate and stillage of wheat straw ...... 33 4.3 Detoxification experiments with prehydrolysate and stillage of wheat straw ........... 37 4.4 Initial screening and detoxification experiments with prehydrolysate of spruce .... 40 4.5 Multifermenter experiment with prehydrolysate and stillage of wheat straw .......... 45 4.6 Pilot scale experiments .................................................................................................................. 46
5. Discussion .................................................................................................................................. 57 6. Future work ............................................................................................................................... 66 Acknowledgement ....................................................................................................................... 67 References ...................................................................................................................................... 68 Appendix I ....................................................................................................................................... 73 Appendix II ..................................................................................................................................... 75 Appendix III .................................................................................................................................... 76 Appendix IV .................................................................................................................................... 77 Appendix V ...................................................................................................................................... 79 Appendix VI .................................................................................................................................... 81
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
1. Introduction In a time when the global human population is increasing, the demand on food, and especially protein is a hot topic given high focus. In the year 2050 the global human population is expected to have reached 9 billion (The World Bank, 2013). Fish is an important source of nutritious food and protein for many of the world’s humans. In the year of 2010 the level of aquatic food consumption was 128 million tonnes. An increase of 23 million tonnes is estimated to be required until 2020, if the current level of per-‐capita consumption of aquatic foods is to be maintained. Today, the majority of the marine fish stocks are exploited, overexploited, or depleted and there is no room for further expansion. The decreasing fish stocks of the oceans imply that the predicted increased demand of aquatic food will have to be met by production through aquacultures and not from captured fish (FAO Fisheries and Aquaculture Department, 2012). Today, almost 50% of the global food fish supply comes from aquaculture production (FAO Fisheries and Aquaculture Department, 2012). During the last three decades the aquaculture production has increased from 5 million to 63 million tonnes. It is expected that the aquaculture production further will expand substantially, to almost 94 million tonnes by the year 2030 (The World Bank, 2013). Currently the aquaculture sector grows with an average rate of 8 to 10 percent per year. Almost 50% of the global aquaculture productions are reliant of addition of fish feed. In order to keep up with the increasing aquaculture production, the supply of feed sources (e.g. protein) will have to grow with a similar rate (Tacon et al., 2011). Fishmeal is the favoured protein source for many aquaculture species. However, it is predicted that the use of fishmeal in fish feed will decrease with about 7% until 2015 (compared to 2012), partly due to reduction in the supplies of caught fish due to tighter quotas, stricter control of unregulated fishing, and enlarged utilisation of more low-‐cost dietary fishmeal substitutes (FAO Fisheries and Aquaculture Department, 2012). As a consequence, there is an increasing demand for alternative high-‐quality protein sources for fish feed. Vegetable protein sources are today commonly used in fish feed. The most frequent alternative protein source used in aquaculture feed is soybean meal (FAO Fisheries and Aquaculture Department, 2012). Feed of plant origin can however only be used to a limited extent due to their amino acid composition being different compared to that of fishmeal protein. In addition, plant-‐derived materials can contain various antinutritional substances. These substances can have a negative effect on the health and productivity of animals (Francis et al., 2001). Another possible alternative protein source is single cell protein (SCP). SCP consists of microorganisms such as filamentous fungi, yeast, algae, and bacteria that are rich in protein. SCP has many benefits. It is a very fast way of producing protein compared to the production of protein through cultivation of agricultural crops or animal farming. The amino acid profile of many SCP is favourable and very similar to that of fishmeal (Nitayavardhana et al., 2013)(Alriksson et al., 2014)(UniBio A/S, 2014). SCP can be produced from residual streams from different industries giving the possibility of a cheap production (Almeida e Silva et al., 1995)(Alriksson et al., 2014). In addition, SCP production can be performed in bioreactors and does not hold up agricultural land. Production of SCP may very well fit into the request of a sustainable high-‐quality alternative to fishmeal since the production can be performed using
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production renewable and sustainable feedstocks such as residual streams from 2nd generation bioethanol production. The 2nd generation bioethanol production (i.e. production from lignocellulosic materials) is predicted to increase in the future, resulting in large volumes of residual and waste streams (Limayem and Ricke, 2012)(Nitayavardhana and Khanal, 2012). These residual streams are commonly considered to be used as substrates for biogas production (Ekman et al., 2013) (SEKAB, 2014). SCP production is an interesting alternative to biogas production, possibly with a higher economic value. However, the usage of residual streams from the 2nd generation bioethanol is associated with several challenges. The complexity of the residual streams is usually quite high with different types of sugars and degradation products of lignocellulose. Some of the degradation products can inhibit the growth of microorganism used for SCP production. It is essential to find microorganisms suitable for the specific residual stream to be used, microorganisms that are able to utilise as much as possible of the different carbon sources present. In addition, counteractions regarding the inhibitors present in the residual streams have to be considered. Except protein, some microorganisms can also produce microbial oils and lipids, referred to as single cell oil (SCO). As well as there is a need of alternative protein sources in fish feed, is there also a need for alternative lipid sources that can substitute fish oils (FAO Fisheries and Aquaculture Department, 2012). Analogously to SCP and fishmeal, SCO production offers an interesting alternative to fish oils. The aim with this master thesis project has been to investigate the potential of producing SCP and SCO from different residual streams, coming from the lignocellulosic ethanol industry. Three different residual streams from bioethanol production from agriculture-‐ and forestry materials have been investigated as carbon source for production of SCP or SCO: i) prehydrolysate from a bioethanol process based on wheat straw, ii) stillage from a bioethanol process based on wheat straw, iii) prehydrolysate from a bioethanol process based on spruce wood chips. The different microorganisms investigated were three filamentous fungi, Paecilomyces variotii, Cunninghamella echinulata, Mortierella isabellina, and the yeast Yarrowia lipolytica. Figure 1 displays a typical process scheme for the production of ethanol from lignocellulosic materials. The residual streams used as substrates in this study are shown as the red arrows in the figure.
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
Lignocellulose' Wheat'straw' or'spruce'
Pre8 treatment'
Separa"on' solid/liquid'
Enzyma"c' hydrolysis'
Fermenta"on' SFF'
Dis"lla"on'
S"llage'
Prehydrolysate' liquid'frac"on'
Separa"on'' solid/liquid' SCP' produc"on'
Combined'heat8' and'power'plant'
S"llage'' liquid'frac"on'
SCP' produc"on'
Dissolved' material'
Solid'fuel'
Energy'
Ethanol'
Biogas'
Anaerobic' treatment'
Biofuel'
Figure 1 – Outline for a typical process scheme for the production of ethanol from lignocellulosic materials. The red arrows indicate the residual streams used as substrates in this study (modified from Galbe and Zacchi, 2012).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
2. Background The following sections will give a background regarding the concept of SCP, and also SCO, along with a short description about the history of SCP production, and microorganisms used for SCP and SCO production. Information about the usage of fish feed is also provided, together with knowledge regarding possible toxins and immunostimulants in microorganisms for SCP and SCO production. Furthermore, a general description of the process of bioethanol production, yielding the residual streams like the ones used in this study, is given, including information regarding the biomass used, inhibitors that can be produced during the process, and possible counteractions that can be taken to overcome the problem with inhibitors.
2.1 Single cell protein and single cell oil SCP is the definition for dried cells originated from single-‐celled organisms intended to be used as a protein source in human foods or animal feeds. The type of microorganisms used includes bacteria, algae, yeasts, molds, and other fungi (Litchfield, 1983)(Nasseri et al., 2011). Despite the name, SCP, the microbial cells do not exclusively consist of protein. Microbial cells also consist of lipids, carbohydrates, vitamins, minerals, and nucleic acids (Litchfield, 1983). The interest in SCP started already some time before World War I (Ugalde and Castrillo, 2002). During the World War I Germany tried to supplement their protein supply in animal feed by using Baker’s yeast. They managed to replace as much as half of all the protein sources imported at that time with yeast (Ugalde and Castrillo, 2002). The yeast was cultivated on molasses as carbon source and ammonium salts were used as nitrogen source (Litchfield, 1983). After the end of the World War I the interest in yeast as fodder declined but arose again when World War II started. At this point yeast had been included into the army diets, and after some time also into the diets of civilians. However, the high ambition to produce more than 100 000 tonnes of yeast per year was by far never reached (Ugalde and Castrillo, 2002). The yeast of interest was Candida utilis (Torula yeast) and it was cultivated on sulphite waste liquor from the pulp and paper industries and on wood sugar derived from acid hydrolysis of wood (Litchfield, 1983). The production of Torula yeast continued after the World War II in the United States as part of a larger program for utilisation of natural sources for fodder (Ugalde and Castrillo, 2002). In the early 60’s various companies started to investigate the possibility to produce SCP that could be used as protein source, as a response to the concept of the protein gap, which had been brought forward by The Food and Agriculture Organisation of the United Nations (FAO). Between the mid 60’s and the 80’s the SCP industry looked very promising but due to technical and political developments in the 80’s the expansion levelled off. Instead of an increased SCP production the agricultural production increased as a result of improved production and distribution knowledge. Many of the processes for SCP were ceased as a direct result of being outcompeted by the cheap agricultural crops. Although, a very successful example of a SCP process, that has taken the step into being a commercial product, is the production of Fusarium venenatum, which is sold under the trademark QuornTM. QuornTM constitute a fungal-‐based protein source produced for human consumption (Ugalde and Castrillo, 2002). SCO refers to microbial oils or lipids synthesised by microorganisms (Ratledge, 2004)(Zeng et al., 2013). Some microorganisms, oleaginous microorganisms, are able to accumulate lipids up
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production to levels of 20-‐80% of their cell dry weight (Zeng, et al., 2013). All living organisms synthesise lipids for membranes and other structures but these lipids corresponds to a relatively low share of the cell weight. Some microorganisms can however also produce and accumulate lipids as reserve storage and these microorganisms are typically useful for production of SCO. This type of accumulation is found in some yeasts and fungi, along with a small number of algae (Ratledge, 2004). A requirement for SCO accumulation is a cultivation medium with an excess carbon source and limiting nitrogen source. The accumulation of SCO starts first when the nitrogen is depleted (Ratledge, 2004). The excess carbon source can after depletion of nitrogen continue to be assimilated and is then directed into lipid synthesis, thereby building up triacylglycerols in the form of small oil droplets. The capability to accumulate oil cannot only be explained by the fatty acid biosynthesis. This conclusion is based on that non-‐oleaginous species do not accumulate any oil if placed in a nitrogen-‐limited medium. Oleaginous microorganisms have two features making the lipid accumulation possible. First, they are able to continuously produce acetyl-‐CoA directly in the cytosol (acetyl-‐CoA being an important precursor for fatty acid synthase). Secondly, they are able to produce a sufficient amount of NADPH, which is an essential reductant used in fatty acid biosynthesis (Ratledge 2004). The acetyl-‐CoA is formed from citrate and CoA using ATP by the enzyme ATP:citrate lyase (Reaction [R1]), which does not seem to be present in non-‐oleaginous microorganisms (Ratledge, 2004). 𝐶𝑖𝑡𝑟𝑎𝑡𝑒 + 𝐶𝑜𝐴 + 𝐴𝑇𝑃
!"#:!"#$%!" !"#
%$𝑎𝑐𝑒𝑡𝑦𝑙 − 𝐶𝑜𝐴 + 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝐴𝐷𝑃 + 𝑃!
[R1]
2.1.1 Fish feed from single cell protein SCP is a potential protein source for use in fish feed, as well as SCO is a potential lipid source. Today, the search for alternative protein sources for fish feed is a hot topic since the use of fishmeal is predicted to decline while the aquaculture production at the same time increases (FAO Fisheries and Aquaculture Department, 2012). Feed used for aquaculture production is categorised into three main groups: animal nutrient sources, plant nutrient sources, and microbial nutrient sources. Animal nutrient sources include both aquatic and terrestrial animals. Among the microbial nutrient sources are algae, yeasts, fungi, bacteria and/or microbial SCP sources. Today, the only microbial derived feed source available in commercial quantity is yeast-‐derived products, such as brewer’s yeast and extracted fermented yeast products (FAO Fisheries and Aquaculture Department, 2012). The most common alternative protein source used today is soybean meal, 23% (by weight) of the total compounds in fish feeds were made out of soybean meal in 2008 (FAO Fisheries and Aquaculture Department, 2012). Feed with plant origin can however only be used to a limited extent due to their amino acid composition being different compared to fishmeal protein. In addition, plant-‐derived materials contain various antinutritional substances, substances which interfere with the utilization of feed and that affect the health and production of animals (Francis et al., 2001).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production As described in the introduction (Section 1) the benefits of SCP as an alternative protein source in fish feed are many. Microorganisms have a short generation time and grow much faster than many of the agricultural or animal protein sources. Another benefit with using SCP is the possibility to have the production based on industrial residual and waste streams, an aspect which is attractive from both an economical and environmental point of view. Today there is a great need for a sustainable fishery industry (FAO Fisheries and Aquaculture Department, 2012). The choice of ingredients for aquaculture feed should not only be based upon nutrient level, digestibility, and cost, but also upon the sustainability and environmental impact of the production. Such criteria come somewhat hand in hand since using a high-‐quality feed source will contribute to less nutrient loss and feed wastage, which will minimize the negative impacts on the environment and the ecosystem (Tacon et al., 2011). An important aspect to consider when using alternative protein sources for fishmeal is to utilize sources with similar amino acid profile. Alternative protein sources may have a different amino acid composition than the desired one, and thereby causing negative effects on the growth of the fish. It is therefore of importance not only looking at the crude protein level, but also at the protein composition (Tacon, 1987). The amino acids can be divided into essential and non-‐ essential amino acids. The essential amino acids in fish feed are: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Requirements of essential amino acids for fish at varying dietary protein levels are displayed in Table 1 (Tacon, 1987). Alriksson et al., (2014), showed that P. variotii cultivated on spent sulphite liquor permeate consisted of similar amounts of the important amino acids as fishmeal with comparable protein content. Table 1 – Dietary essential amino acid (EAA) requirements (% of dry diet) of fish at varying dietary protein levels (Tacon, 1987).
Amino acid
Dietary protein level (%) 45 50 55 1.94 2.15 2.37 0.31 0.35 0.38 0.82 0.91 1.00 1.26 1.40 1.54 2.30 2.55 2.81 2.66 2.96 3.25 0.87 0.96 1.06 1.31 1.45 1.60 1.04 1.15 1.27 1.45 1.61 1.77 0.27 0.30 0.33 1.50 1.66 1.83
Arginine Cystine* Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine* Threonine Tryptophan Valine *Non-‐essential amino acid.
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production 2.1.1.1 Toxins from microorganisms One important aspect of using SCP in animal feed is safety. Some microorganisms can produce mycotoxins. Mycotoxins are a toxic secondary metabolite expressed by the kingdom of fungi. Mycotoxins are associated with diseases and they may also be carcinogenic. The major mycotoxins include aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-‐2 toxin, ochratoxin, and certain ergot alkanoids (Richard, 2007). The European Food and Safety Authority have set up guidance values for the maximum content of mycotoxins in μg/kg allowed in animal feed (Table 2) (The Commission of the European Communities, 2006)(The Commission of the European Communities, 2003). In order to be able to utilise SCP as a protein source in fish feed the content of mycotoxins have to be analysed. In the study performed by Alriksson et al., (2014), the concentrations of mycotoxin in P. variotii cultivated on spent sulphite liquor permeate were well below the recommended maximum values given by the European Food and Safety Authority. Table 2 – Directive and recommendation from the European Union Commission regarding maximum values of some mycotoxins in products animal feed.
Substance Aflatoxin B1 Deoxynivalenol Zearalenone Ochratoxin A Fumonisin B1 + B2
Maximum content in μg/kg 5-‐20 900-‐12 000 100-‐3000 50-‐250 5000-‐60 000
2.1.1.2 β-‐glucan, an immunostimulant present in microorganisms Microbial protein may need additional properties, than just constituting a protein source, in order to compete with vegetable protein as an alternative protein source in fish feed. Microorganisms can, in addition to protein and lipids, also contain other substances that can improve the quality of the fish feed. β-‐glucans are a group of molecules generally called “biological response modifiers” due to their physiological activeness. β-‐glucans constitutes a structural component in the cell walls of fungi, yeast, bacteria, seaweed and some plants (Vetvicka et al., 2013). It is a homopolysaccharide consisting of glucose molecules linked together by glycosidic bonds (Meena et al., 2013). The cell wall further consists of chitin, other hemicelluloses, and mannans (Kyanko et al., 2013). β-‐glucans are interesting molecules due to their bioactive and medicinal properties. Some of them are anti-‐microbial, anti-‐viral, and immune stimulating (Kyanko et al., 2013). Within the aquaculture industry the risks and occurrence of diseases and infections increases along with the increasing production. Due to the concern regarding use of antibiotics alternative strategies are requested. Alternative approaches can include use of vaccine, dietary supplement of probiotics, prebiotics, and immunostimulants. Immunostimulants is an effective tool to enhance the resistance against infectious diseases through improvement of the immune system. Immunostimulants can enhance the innate humoral and cellular defence mechanism. β-‐glucans
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production have shown to be one of the most promising immunostimulants within aquaculture (Meena et al., 2013). Studies with β-‐glucan have shown to affect the growth, survival, resistance and protection against pathogen, antibody production, and immune-‐related gene expression in many fish species. It has been shown that the dosages, quality, time of administration and duration of treatments of β-‐glucan is of great importance for the enhancement of various parameters related to growth, survival, and immunity (Meena et al., 2013). β-‐glucans is today routinely used in commercial aquaculture production (Vetvicka et al., 2013). The potential of using β-‐glucan as prebiotics (a non-‐digestible feed ingredient which stimulate growth and activity of beneficial bacteria in the gastro intestinal tract) is an interesting aspect for future investigation (Meena et al., 2013). Kyanko et al., (2013) studied the content of total dietary fibre, and especially the amount of β-‐ glucan in thirty-‐seven different filamentous fungi, including P. variotii. P. variotii obtained the highest amount of total dietary fibre (51.7%) and the highest content of β-‐glucan (23.8%), values being far higher than earlier reported values in Basidiomycetes and yeast. Today, the company Biofeed Technology Inc. produces β-‐glucan by production and derivation from specific Paecilomyces ssp., Saccharomyces cerevisiae and Ganoderma lucidum (Biofeed Technology Inc., 2014).
2.2 Microorganisms for single cell protein and single cell oil production Various bacteria, yeasts, fungi and algae have been tested and investigated for production of SCP and SCO throughout the years. The four microorganisms used in this study are described below.
2.2.1 Paecilomyces variotii Paecilomyces variotii is a filamentous fungus belonging to the order Eurotiales within the phylum of Ascomycota. The specie is commonly found in soil, wood, and food (Houbraken et al., 2010). P. variotii has a history of being used for SCP production, and it has the ability to grow in various complex residual streams from different industries (Almeida e Silva et al., 1995). In the 70’s, the Pekilo process was started in Finland. In a continuous fermentation process the filamentous fungi was fed with spent sulphite liquor from a pulp mill in order to produce SCP. The protein-‐rich fungus was even approved as animal feed in Finland (Romantschuk, 1976), although the process is currently not running (Ugalde and Castrillo, 2002). The investigation of using P. variotii for SCP production has continued, even though the interest has been quite low the last decades. Bajpai and Bajpai (1987) investigated SCP production form rayon pulp mill waste, Almeida e Silva et al., (1995) investigated using eucalyptus hemicellulose hydrolysate as substrate for production of SCP. In a recent study by Alriksson et al. (2014), spent sulphite liquor permeate was used as substrate for production of SCP. P. variotii has shown to consume both hexoses (C6 sugars) and pentoses (C5 sugars), and it can also metabolise aliphatic acids like acetic acid and formic acid (Alriksson et al., 2014). Cultivation is commonly performed at 30°C and at pH 6.0 (Bajpai and Bajpai, 1987)(Almeida e Silva et al., 1995)(Alriksson et al., 2014).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
2.2.2 Cunninghamella echinulata and Mortierella isabellina Cunninghamella echinulata and Mortierella isabellina are two oleaginous filamentous fungi belonging to the order of Mucorales within the phylum Zygomycetes (Fakas et al., 2009)(Chatzifragkou et al., 2010). Strains from the phylum Zygomycota are considered to be potential producers of SCO that contain γ-‐linolenic acid (GLA) (Chatzifragkou et al., 2010). It has been reported that these two strains show variations within their way to regulate the lipid accumulation process when cultivated in nitrogen-‐limited media. It has also been shown that depending on what type of sugars that are metabolised the fatty acid composition of the lipids can vary (Chatzifragkou et al., 2010). The genera of Cunninghamella may also be considered as a relevant microorganism for production of SCP (Ugalde and Castrillo, 2002). C. echinulata and M. isabellina are normally cultivated at 28°C and at pH 6.0 (Fakas et al., 2009)(Chatzifragkou et al., 2010). Zeng et al. (2013) showed that M. isabellina preferable use C6 sugars compared to C5 sugars. They also found M. isabellina to be more sensitive to degradation products from lignin than to other inhibitory compounds commonly formed during pretreatment of lignocellulose. Aliphatic acids like acetic acid and formic acids even improved the growth and lipid production at low concentration (Zeng et al., 2013). Zeng et al., also showed that up to 12.6 g/L of mycelium containing 34% lipids could be obtained when cultivating M. isabellina on wheat straw hydrolysate (pretreated with dilute sulphuric acid) for 6 days.
2.2.3 Yarrowia lipolytica The oleaginous yeast strain Yarrowia lipolytica is strictly aerobic and it produces many important metabolites and has a high secretory activity. It is considered to be non-‐pathogenic and the Food and Drug Administration (FDA, USA) has classified many processes based on this microorganisms as generally regarded as safe (GRAS) (Coelho et al., 2010). Y. lipolytica can be described as an industrial workhorse, being used for a broad range of applications. Some products that can be produced using Y. lipolytica are for example lipase and different organic acids (e.g. citric acid) (Coelho et al., 2010). Considering SCP and SCO, Y. lipolytica is mainly considered as an SCO producer but can also be used for SCP production (Ugalde and Castrillo, 2002). Cultivation is usually performed at a temperature between 28°C and 30°C and at pH 5.5-‐ 6.0 (Makri et al., 2010)(Katre et al., 2012). Y. lipolytica can utilize several different hexoses as carbon source, for example glucose, fructose, and mannose. The yeast can also degrade acids like acetic, lactic, propionic, malic, succinic, citric, and oleic acid and use those as the sole carbon and energy source. Additionally, ethanol and glycerol can also be used as carbon source (Coelho et al., 2010). Ethanol concentrations up to 3% can be used as carbon source before it becomes too toxic (Barth and Gaillardin, 1997). Other carbon sources that can be utilized are n-‐alkenes and 1-‐alkenes (Barth and Gaillardin, 1997). Tsigie et al., (2011), showed that some strains of Y. lipolytica can utilize pentoses as a carbon source. They also showed that biomass formation could be inhibited by degradation products commonly found in lignocellulose hydrolysates, such as HMF and furfural (Tsigie et al., 2011).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production
2.3 Bioethanol production from lignocellulose Production of ethanol from lignocellulose is usually performed through pretreatment and hydrolysis of the lignocellulosic material, followed by fermentation and distillation. Lignocellulosic biomass is an interesting feedstock due to its abundance and its lower cost compared to other substrates, such as sugar and starch from agricultural crops (Ruan et al., 2012). In addition, lignocellulosic biomass has fewer competing uses, compared to crops or grains, and does not compete with food supply (Huang et al., 2013).
2.3.1 Lignocellulose Lignocellulose consists mainly of cellulose, hemicellulose, lignin, extractives, and ash (Figure 2). Depending on the origin of the lignocellulose both the chemical and the structural composition varies (Table 3). Examples of lignocellulose sources are grasses, hardwood, softwood, and agricultural residues such as wheat straw and sugarcane bagasse (Balat, 2011). Cellulose is a linear homopolysaccharide consisting of β-‐D-‐glucopyranose units linked together by 1,4-‐β-‐glycosidic bonds. Parallel cellulose polymers are held together via hydrogen bonds and form microfibrils, which in turn form fibrils that build up the cellulose fibres (Sjöström, 1993) (Berg et al., 2007). The fibres support the plant cell wall and give it its strength and rigidity. Cellulose is the major constituent in plant biomass and make up around 30-‐45% of the dry weight (Balat, 2011). Hemicellulose, around 20-‐40% of the dry weight of lignocellulose, is a short and highly branched heteropolysaccharide consisting of both pentoses (five-‐carbon sugar) and hexoses (six-‐carbon sugar). The most dominant monosaccharides in hemicellulose are xylose, arabinose, which are pentoses, and galactose, glucose and mannose, which are hexoses (Balat, 2011). Xylose is the most abundant sugar in the hemicellulose of agricultural residues while mannose is the most abundant sugar in the hemicellulose of softwoods (Balat, 2011). Lignocellulose consists of approximately 15-‐30% lignin (Balat, 2011). Lignin is an aromatic polymer consisting of phenylpropane units where the residues are linked together to a very complex structure. Precursors to lignin are p-‐coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Sjöström, 1993). Extractives are non-‐structural components and include e.g. phenols, tannins, fats, and sterols (Martínez et al., 2005). Non-‐extractives present in lignocellulose mainly consist of ash components, such as silica and alkali salts. The amount of ash in straw can be as high as up to 10% while the amounts are commonly very low in wood materials, below 1% (Klinke et al., 2004).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production Table 3 – Composition for different types of lignocellulosic materials (% dry weight) (Balat, 2011).
Material Cellulose Grasses 25-‐40 Hardwoods 45 ± 2 Softwoods 42 ± 2 Wheat straw 37-‐41
Hemicelluloses Lignin 25-‐50 10-‐30 30 ± 5 20 ± 4 27 ± 2 28 ± 3 27-‐32 13-‐15
Extractives -‐ 5 ± 3 3 ± 2 7 ± 2
Ash -‐ 0.6 ± 0.2 0.5 ± 0.1 11-‐14
2.3.2 Hydrolysis and pretreatment Hydrolysis of lignocellulose can be performed chemically or biologically (i.e. enzymatic hydrolysis). In the hydrolysis, hemicellulose and cellulose are degraded into monosaccharides. The two main methods for chemical hydrolysis are concentrated-‐acid hydrolysis and dilute-‐acid hydrolysis (Galbe and Zacchi, 2002). Both the concentrated-‐ and dilute-‐acid hydrolysis methods use acid as catalyst (e.g. H2SO4, SO2) along with high temperatures. In dilute-‐acid hydrolysis acid concentrations below 4% and temperatures ranging from 140-‐200°C are commonly used. The acid works as a catalyst by breaking the up the structure of the cellulose, hemicelluloses, and lignin, thereby making the hydrolysis with water easier. Chemical hydrolysis can also be performed using an alkali catalyst (e.g. NaOH, NH3, Ca(OH)2) (Galbe and Zacchi, 2012). Hydrolysis of the lignocellulose can also be performed enzymatically. In the process of enzymatic hydrolysis a mixture of different enzymes, consisting mainly of cellulases and hemicellulases, is used (Galbe and Zacchi, 2012). To enable efficient enzymatic hydrolysis of the lignocellulose a pretreatment step is typically required. The aim with the pretreatment is to open the structure of the lignocellulose, and make the hemicellulose and cellulose more accessible for degradation into monosaccharides (Almeida et al., 2007). Pretreatment can be divided into four categories: physical, chemical, physio-‐chemical, and biological. The thermochemical method “steam explosion” is a commonly used method. Steam explosion is typically performed at temperatures between 160-‐240°C for a short time, 1-‐20 minutes (Galbe and Zacchi, 2012). The residual streams used in this project were collected from a process based on steam explosion with acid catalyst. Depending on the type of pretreatment and hydrolysis method, different types and amounts of degradation products are generated (Figure 2). Some of these products can act as fermentation inhibitors. The severity of the pretreatment effects the formation of degradation products (Klinke et al., 2004).
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Production of Single Cell Protein from Residual Streams from 2nd Generation Bioethanol Production Macromolecules+
Cellulose'30*40%'
Sugar+ components+
Fermenta2on+ inhibitors+
Glucose' Mannose' Galactose'
Xylose' Arabinose' Hemicellulose' 20*30%' Uronic'acid' Ace:c'acid'
Lignin'20*30%'
Extrac:ves'