single cell protein 4.1 Introduction 4.2 Conventional protein sources 4.4 Substrates for SCP production 4.5 Micro-organisms for SCP production 4.6 sCP from carbon dioxide 4.7 SCP from carbohydrates 4.8 SCP from hydrocarbons and derivatives 4.9 The Pruteen process-a case study Summary and objectives Resource material
58 Single cell protein 4.1 Introduction 60 4.2 Conventional protein sources 60 4.3 Single cell protein 62 66 67 69 4.4 Substrates for SCP production 4.5 Micro-organisms for SCP production 4.6 SCP from carbon dioxide 4.7 SCP from carbohydrates 74 4.8 SCP from hydrocarbons and derivatives 85 4.9 The Pruteen process - a case study 88 Summary and objectives 106 Resource Material 107
ter 4 Single cell protein 4. 1 Introduction In this chapter we examine the processes that have been developed to produce micro-organisms as a source of food protein. We will examine the reasons why micro-organisms have been considered as alternative protein sources, the substrates on which they have been grown, the various process technologies developed and the comparative economics of these processes. One process will be mined in depth, to illustrate how a team composed of such diverse people as microbiologists, process engineers, patent lawyers and cost analysts work together to develop a marketable product The driving forces behind the development of many single cell protein projects emerged from global e conditions and social concerns of the 1960s. In the 1970s and early 1980s, there were considerable technological advancements associated with single cell rotein process developments and many types of prc ocesses were operated commercially. In this chapter we present technological and economic data derived from and animal feed source. You will see that many important principles underpinning modern process technology are based on the experiences gained in the development of single cell protein processes 4.2 Conventional protein sources essential Animals, including humans, cannot synthesise all the different amino acids they need amino aads and thus require them in their diet. These amino acids are called the essential amino acids. Proteins in food are hydrolysed in the digestive tract and the resulting amino acids are reassembled into proteins within the animals cells. All animals are ultimately dependent on plants for protein, as it is plants that create protein by combining inorganic nitrogen from the soil(as nitrate)with organic molecules derived from carbon from the atmosphere(as CO2) organoleptic For us to remain perfectly healthy the protein in our diet must supply suffidient ropertes quantities of amino acids. We prefer to eat our protein in particular forms, that is in foods having particular textures, tastes and smells(these are called organoleptic properties). Conventional sources of protein are plants, mainly as cereals and pulses, and animals, mainly as meat, eggs and milk. The proportions of such proteins eaten in various parts of the world differ widely(Figure 4.1)
60 Chapter 4 Single cell protein 4.1 Introduction In this chapter we examine the processes that have been developed to produce micro-organisms as a source of food protein. We will examine the reasons why micmrganisms have been considered as alternative protein sources, the substrates on which they have been grown, the various process technologies developed and the comparative economics of these processes. One process will be examined in depth, to illustrate how a team composed of such diverse people as microbiologists, process engineers, patent lawyers and cost analysts work together to develop a marketable product. The driving forces behind the development of many single cell protein projects emerged from global economic conditions and social concerns of the 1960s. In the 1970s and early 198Os, there were considerable technological advancements associated with single cell protein process developments and many types of processes were operated commercially. In this chapter we present technological and economic data derived from these early developments to provide a historical context for single cell protein as a food and animal feed source. You will see that many important principles underpinning modem process technology are based on the experiences gained in the development of single cell protein processes. 4.2 Conventional protein sources essential aminoacids Animals, including humans, cannot synthesise all the different amino acids they need and thus require them in their diet. These amino acids are called the essential amino acids. Proteins in food are hydrolysed in the digestive tract and the resulting amino acids are reassembled into proteins within the animal's cells. All animals are ultimately dependent on plants for protein, as it is plants that create protein by combining inorganic nitmen from the soil (as nitrate) with organic molecules derived from carbon from the atmosphere (as COJ. For us to remain perfectly healthy, the protein in our diet must supply suffiaent quantities of amino acids. We prefer to eat our protein in particular forms, that is in foods having particular textures, tastes and smells (these are called organoleptic properties). Conventional sources of protein are plants, mainly as cereals and pulses, and animals, mainly as meat, eggs and milk. The proportions of such proteins eaten in various parts of the world differ widely (Figure 4.1). organoleqtic PmeS
Single cell protein Eggs& Milk Meat VEGETABLE PROTEIN Figure 4. 1 World protein consumption ∏ List three factors you think account for such variations in the sources of proteins between various parts of the world? essentially the answer is history, climate, culture and money! Historically, people to eat the food available locally, and this would be controlled by the local nat
Single cell protein 61 Figure 4.1 World protein consumption List three factors you think account for such variations in the sources of proteins n between various parts of the world? Essentially the answer is history, climate, culture and money! Historically, people had to eat the food available locally, and this would be mntrolled by the local natural
62 Chapter 4 environment. Cultural influences have also led to preferences for certain food types. In more affluent countries foods such as meat, or high-protein feedstuffs on which to rear animals, can be produced or be imported. In less affluent countries such luxuries cannot be afforded, Increasing populations in some countries have overstretched food d so limited the availability of foods. changing There are problems, however, with these conventional sources of protein. Crop demands for oduction is dependent upon a suitable climate and in most countries available arable land is already fully farmed. Fish stocks in the oceans are in danger of becoming depleted. In countries where animal meat forms a high proportion of dietary protein, there are controversies such as whether or not the fats eaten with the protein are healthy,whether or not we are justified in keeping killing animals for food at all.Such animals in the unnatural conditions controversies are leading an increasin g eople to become vege likely that the worlds population will double in the next few decades, yet the United Nations estimate that about one thousand million people are already suffering protein deficiency. It is estimated that between 1980 and 2000 the annual demand for protein as food for humans will increase from 50x 10 tonnes to 79 x 10 tonnes, and the demand for protein as feed for animals will increase from 44 x 10 tonnes to 108 x 10 tonnes Biotechnology is being applied to the rapid improvement of conventional food sources, both plant and animal, in an effort to meet the increased demand in food. Interest has also been shown in growing micro-organisms source of protein and it developments in this area that we are going to examine here in detail 4. 3 Single cell protein Single cell protein, normally called simply sCP, is the term used to describe microbial cells, or proteins from them, which are used as food ( food for humans)or feed (food for farm animals or fish). Although the term micro-organisms covers viruses, bacteria fungi, algae and protozoa, viruses and protozoa are not considered suitable for sCP production. ∏ Why do you think viruses and protozoa are not suitable for sCP production? Both viruses and protozoa are difficult to grow in culture. Viruses need living cells to grow in and their small size makes them difficult to deal with. Protozoa need complex diets of organic materials. Bacteria, fungi and algae are relatively easy to grow in The term SCP is not exactly appropriate, as some filamentous organisms are used as SCP and these organisms are multicellular not unicellular You may be wondering why anyone should ever have considered using micro-organisms as a protein source. Let us consider why this should have been 4.3.1 The advantage of micro-organisms as a protein source Eating micro-organisms is nothing new. You might not have been aware that some foods traditionally eaten by man are in fact micro-organisms. Filamentous blue-green bacteria(often called blue-green algae, or cyanobacteria) were collected from lakes and rivers and eaten by the Aztecs in Mexico, and people inhabiting the shores of lake Chad in Africa still do so. Edible fungi have been collected from the wild for centuries and yeasts farmed throughout the last two. During the two World Wars this century, yeasts (unicellular fungi)were grown on a large scale in Germany and used as food and feed
62 Chapter 4 changing demands for dietary protein single cell and fwd proteinlfood filamentous blue-green bederia environment. Cultural influences have also led to preferences for certain food types. In more affluent countries foods such as meat, or high-protein feedstuffs on which to rear animals, can be produced or be imported. In less affluent countries such luxuries cannot be afforded, Increasing populations in some countries have overstretched food supplies, and so limited the availability of foods. There are problems, however, with these conventional sources of protein. Crop production is dependent upon a suitable climate and in most countries available arable land is already fully farmed. Fish stocks in the oceans are in danger of becoming depleted. In countries where animal meat forms a high proportion of dietary protein, there are controversies such as whether or not the fats eaten with the protein are healthy, whether or not we are justified in keeping animals in the unnatural conditions of some farms, or whether or not we are justified in killing animals for food at all. Such controversies are leading an increasing number of people to become vegetarian. It is likely that the world’s population will double in the next few decades, yet the United Nations estimate that about one thousand million people are already suffering protein deficiency. It is estimated that between 1980 and 2000 the annual demand for protein as food for humans will increase from 50 x lo6 tonnes to 79 x lo6 tonnes, and the demand for protein as feed for animals will increase from 44 x lo6 tonnes to 108 x lo6 tonnes. Biotechnology is being applied to the rapid improvement of conventional food sources, both plant and animal, in an effort to meet the increased demand in food. Interest has also been shown in growing micro-organisms as a source of protein and it is developments in this area that we are going to examine here in detail. 4.3 Single cell protein Single cell protein, normally called simply s8, is the term used to describe microbial cells, or proteins from them, which are used as food (food for humans) or feed (food for farm animals or fish). Although the term micro-organisms covers viruses, bacteria, fungi, algae and protozoa, viruses and protozoa are not considered suitable for SCP production. n Why do you think viruses and protozoa are not suitable for SCP production? Both viruses and protozoa are difficult to grow in culture. Viruses need living cells to grow in and their small size makes them difficult to deal with. Protozoa need complex diets of organic materials. Bacteria, fungi and algae are relatively easy to grow in culture. The term SCP is not exactly appropriate, as some filamentous organisms are used as SCP and these organisms are multicellular not unicellular. You may be wondering why anyone should ever have considered using micmaganisms as a protein source. Let us consider why this should have been. 4.3.1 The advantage of micro-organisms as a protein source Eating micro-organisms is nothing new. You might not have been aware that some foods traditionally eaten by man are in fact micro-organisms. Filamentous blue-p;reen bacteria (often called blue-green algae, or cyanobacteria) were collected from lakes and rivers and eaten by the Aztecs in Mexico, and people inhabiting the shores of Lake Chad in Africa still do so. Edible fungi have been collected from the wild for centuries and farmed throughout the last two. During the two World Wars this century, yeasts (unicellular fungi) were grown on a large scale in Germany and used as food and feed
Single ce‖ protein Micro-organisms are rich in protein. Microbial cells can contain as much protein as conventional foods. Bacteria can contain 60-65%(as a of dry weight) protein whereas fungi and algae contain about 40%. In addition, microbial cells can be a rich source of fibre, unsaturated fats, minerals and vitamins. They are low in saturated fats and sodium protein from Micro-organisms create protein Like plants, many micro-organisms can use inorganic nitrogen and can thus be used as an alternative to plants to create protei processes inorganic nitrogen is usually supplied as ammonia(or as ammonin which is readily available and is renewable, as it can be manufactured from atmospheric d can be recycled through the nitr compete with plant s for co, but there is no0gmp如mn is renewable by recycling through the carbon cycle. Other micro-organisms are heterotrophs(organisms which use organic sources of carbon) and can use a wide range of organic carbon sources. These can be materials unsuitable as food sources for animals (for example methanol). Others are waste products from industries or agriculture and have limited uses and can be a problem to dispose of by other means saves SCP processes are efficient on space. sCP production plants can be built on land bN of protein. Also they are much more efficient in terms of amount of protein produced unsuitable for agriculture and so need not compete for space with conventional sources per unit area(figures are quoted later for some processes) rapid growth Micro-organisms grow rapidly. Micro-organisms grow much more rapidly than plants or animals. Bacteria can grow with mean generation times(doubling times )as short as 20-30 minutes. The mean generation times of unicellular algae and fungi are about 1-3 hours, whereas those of multicellular algae and fungi may be longer. This means that micro-organisms have the potential to produce protein far more rapidly than is possible by rearing plants or animals By completing the following calculation you will be able to demonstrate the amazing potential for micro-organism to rapidly produce protein for food In batch culture, when growth is exponential, the number or organisms produced from one organism is given by 2, where n is the number of generations. So after one 2/eration there are 2(ie 2), after two generations 2 (ie 4)and after three generations Starting from a single bacterial cell with a mean generation time(doubling time)of 1 hour, and assuming exponential growth throughout, how many organisms would you have after 48 hours? As the dry weight of a bacterial cell is about 10 g what would the dry weight of these cells be? Assuming these cells to be 50% protein, how much protein would there be? Assuming you are an average person, you require about 70 g of protein in your diet per day. How long would this protein last you? (Do not cheat, try the calculation before reading on). Now repeat the calculation to find out how much protein would have after 72 After 48 hours there would be 248 or 2. x 10 4 cells This represents 2. x 10x10=2 8x10‘ g dry weight cells
Single cell protein 63 Micrmrganisms are rich in protein. Microbial cells can contain as much protein as conventional foods. Bacteria can contain 60-696 (as a 96 of dry weight) protein whereas fungi and algae contain about 40%. In addition, microbial cells can be a rich source of fibre, unsaturated fats, minerals and vitamins. They are low in saturated fats and sodium. protein from inorganic nitrogen autotrophs/ heterotrophs 8aV88 agricultural sparapid gmwth Micrmrganisms create protein. Like plants, many micmrganisms can use inorganic nitrogen and can thus be used as an alternative to plants to create protein. In SB processes inorganic nitrogen is usually supplied as ammonia (or as ammonium salts), which is readily available and is renewable, as it can be manufactured from atmospheric nitrogen and can be recycled through the nitrogen cycle. Microorganisms can use alternative carbon sources. Algae are autotrophs using atmospheric COZ (think of them as plants growing in water instead of soil). They compete with plank for COZ but there is not a shortage of COz in the atmosphere and it is renewable by recycling through the carbon cycle. Other micm-organisms are heterotrophs (organisms which use organic sources of carbon) and can use a wide range of organic carbon sources. These can be materials unsuitable as food sources for animals (for example methanol). Others are waste products from industries or agriculture and have limited uses and can be a problem to dispose of by other means. SCP processes are efficient on space. SCP production plants can be built on land unsuitable for agriculture and so need not compete for space with conventional sources of protein. Also they are much more efficient in terms of amount of protein produced per unit area (figures are quoted later for some processes). Micro-organisms grow rapidly. Micrmrganisms grow much more rapidly than plants or animals. Bacteria can grow with mean generation times (doubling times) as short as 20-30 minutes. The mean generation times of unicellular algae and fungi are about 1-3 hours, whereas those of multicellular algae and fungi may be longer. This means that micmrganisms have the potential to produce protein far more rapidly than is possible by rearing plants or animals. By completing the following calculation you will be able to demonstrate the n amazing potential for micmrganism to rapidly produce protein for food. In batch culture, when growth is exponential, the number or organisms produced from one organism is given by 2”, where n is the number of generations. So after one qneration there are 2’ (ie 21, after two generations 2* (ie 4) and after three generations 2 (ie 8) and so on. Starting from a single bacterial cell with a mean generation time (doubling time) of 1 hour, and assuming exponential growth throughout, how many %anisms would you have after 48 hours? As the dry weight of a bacterial cell is about 10- g, what would the dry weight of these cells be? Assuming these cells to be 50% protein, how much protein would there be? Assuming you are an average person, you require about 70 g of protein in your diet per day. How long would this protein last you? (Do not cheat, try the calculation before reading on). Now repeat the calculation to find out how much protein you would have after 721. After 48 hours there would be 2& or 2.8 x 1014 cells. This represents 2.8 x 1014 x lo-’’ = 2.8 x 104g dry weight cells
This represents28x10°x50%=14x10° g protein This represents 1. 4 x 10"/70=200 days worth of protein for one person. After 72 hours there would be 2" or 4.7 1021 cells This represents 4.7x10 0-4.7x 10 g dry weight cells This represents 4.7x 10x50%=2.35x 10 gprotein. This represents 2.35x 10/70=ca 3 x 10 meals, or enough protein to feed the entire population of China for 3 days! You have now demonstrated the capability of exponentially increasing microbial populations to rapidly produce protein. However, although such outputs of protein are possible in theory, they cannot be achieved in practice, since exponential growth cannot be maintained for such periods because bioreactors are limited in size 4.3.2 The disadvantages of micro-organisms as a protein source Let us consider some of the disadvantages of micro-organisms compared to of pr amiprofigd Are hey n tritis] guidelines on th n utritional qualit of sap have been given by profiles and feeding trials in animals. While most microbial cells are rich in protein, many do not contain sufficient quantities of essential amino adids. For instance, algal and fungal cells tend to lack methionine Microbial cells may not be as easy to digest as conventional protein sources, for instance algae have cellulose cell walls which must be digestibility broken up if the proteins within the cell are to be easily digested by humans. The requirements for food are more strict than those for feed like to eat? Humans are their food. Microbial cells have little taste or smell, or even smell or taste unpleasantly to some people. The texture may not be the same as in conventional foods, particularly with unicellular organisms. These draw-backs can be overcome by adding a proportion of SCP to manufactured foods. However, even when SCP is incorporated into manufactured foods it may not have suitable characteristics such as stability, ability to bind water or fats, or ability to form gels, emulsions or foams. SCP for feed does not have to meet such strict requirements adverse What happens after you have eaten them? Even if a micro-organism is palatable it may effects not necessarily be acceptable to the human digestive system, and if eaten in quantity can produce indigestion, flatulence, nausea, vomiting or diarrhoea. As little as 15 g yeast cells per day can produce such effects in humans cost How much do they cost? sCP must compete in price with conventional protein foods and feeds. In itries where protein foods are readily available they can be relatively cheap. It has not always been possible to produce SCP at competitive prices Are they safe to eat? Micro-organisms which are pathogenic or toxic obviously can not be used as sCP sources. In addition most microbial cells have a higher content of nucleic acid, particularly RNA, than conventional foods. When such cells are digested by animals these nucleic acids are metabolised to uric acid Unlike most other mammals humans do not possess uricase, which oxidises uric acid to soluble allantoid for
64 Chapter 4 This represents 2.8 x 1d x 50% = 1.4 x ldg protein. This repments 1.4 x 1$/70 = 200 days worth of protein for one person. After 72 hours there would be 2n or 4.7 x Id' cells. This represents 4.7 x Id' x lo-'' = 4.7 x 1O"g dry weight cells. ms represents 4.7 x IO" x 50% = 2.35 x 1o"g protein. This represents 2.35 x 10"/70 = ca. 3 x lo9 meals, or enough protein to feed the entire population of China for 3 days! You have now demonstrated the capability of exponentially increasing microbial populations to rapidly produce protein. However, although such outputs of protein are possible in theory, they cannot be achieved in practice, since exponential growth cannot be maintained for such periods because bioreactors are limited in size. 4.3.2 The disadvantages of micro-organisms as a protein source Let us consider some of the disadvantages of micro-organisms compared to conventional sources of protein. amino acid profiles digestibility importance of organoleptic properties edverse effects cost =w uric acid Are they nutritious? Guidelines on the nutritional quality of s8 have been given by the Protein Advisory Group (PAG) of the United Nations, and are based on amino acid profiles and feeding trials in animals. While most microbial cells are rich in protein, many do not contain sufficient quantities of essential amino acids. For instance, algal and fungal cells tend to lack methionine. Microbial cells may not be as easy to digest as conventional protein sources, for instance algae have cellulose cell walls which must be broken up if the proteins within the cell are to be easily digested by humans. The requirements for food are more strict than those for feed. What are they like to eat? Humans are particular about the organoleptic properties of their food. Microbial cells may have little taste or smell, or even smell or taste unpleasantly to some people. The texture may not be the same as in conventional foods, particularly with unicellular organisms. These draw-backs can be ovexrome by adding a proportion of SCP to manufactured foods. However, even when SCP is incorporated into manufactured foods it may not have suitable characteristics such as stability, ability to bind water or fats, or ability to form gels, emulsions or foams. SCP for feed does not have to meet such strict requirements. What happens after you have eaten them? Even if a micro-organism is palatable it may not necessarily be acceptable to the human digestive system, and if eaten in quantity can produce indigestion, flatulence, nausea, vomiting or diarrhoea. As little as 15 g yeast cells per day can produce such effects in humans. How much do they cost? SCP must compete in price with conventional protein foods and feeds. In countries where protein foods are readily available they can be relatively cheap. It has not always been possible to produce SCP at competitive prices. Are they safe to eat? Micmrganisms which are pathogenic or toxic obviously can not be used as SCP sources. In addition most microbial cells have a higher content of nucleic acid, particularly RNA, than conventional foods. When such cells are digested by animals these nucleic acids are metabolised to uric acid. Unlike most other mammals, humans do not possess uricase, which oxidises uric acid to soluble allantoid for
Single cell protein excretion, and so uric acid can build up in the blood and may deposit as crystals in the joints, causing gout and arthritis. Thus, SCP used as food is usually processed to reduce he RNA content. Pag guidelines recommend that for humans the daily intake of nucleic acid should not be more than 4g, of which not more that 2 g should be obtained from sCP If SCP has a nucleic acid content of 15%, how much of that sCP could be safely commended human daily requirement (of 70 gprotein) does this represent ingested per day? If the SCP contains 50% protein, what proportion of th Our calculation 2g nucleic acid would be present in +100 581338C At 50% protein this represents 13.3 x 100=6.65 g protein This corresponds too.65 x 100=9.5 of the daily requirement. SAQ 4.1 Which of the following factors supports the use of micro-organisms rather than higher plants for the production of protein food? 1) Plants are more difficult to digest than micro-organisms 2) Micro-organisms can be used to convert organic wastes into proteins 3) Micro-organisms grow more quickly than plants gher plants need CO as a carbon source ) Micro-organisms can use inorganic nitrogen SAQ 4.2 Suggest ways of overcoming or bypassing the following disadvantages of CPAs 1) Unpalatability 2) Indigestibility 3) Poor amino acid profile 4) Toxicity We have seen that only certain micro-organisms that conform to nutritional and safety requirements are suitable for food or feed, and that food has more strict requirements than feed. In addition for use as food, SCP should have a reduced nucleic acid content and should be palatable. Most often this means that its use is limited to processed foods, in which food technologists can produce acceptable tastes, smells and texture
Single cell protein 6!5 excretion, and so uric acid can build up in the blood and may deposit as crystals in the pints, causing gout and arthritis. Thus, SCP used as food is usually processed to reduce the RNA content. PAG guidelines recommend that for humans the daily intake of nucleic add should not be more than 4g, of which not more that 2 g should be obtained from SCP. If SCP has a nucleic acid content of 1596, how much of that s8 could be safely ingested per day? If the SCP contains 50% protein, what proportion of the mommended human daily requirement (of 70 g protein) does this represent? Try to work these out for yourself before reading our answers. n Our calculation: 29 nucleic acid would be present in 2 x 15 g =13.3 g SCP At 50% protein this represents 13.3 x 100 = 6.65 g protein This corresponds to - x 100 = 9.5 76 of the daily requirement. 100 50 6.65 70 Which of the following factors supports the use of micro-organisms rather than higher plants for the production of protein food? 1) 2) 3) 4) 5) Plants are more difficult to digest than micro-organisms. Micro-organisms can be used to convert organic wastes into proteins. Micmrganisms grow more quickly than plants. Higher plants need CG as a carbon source. Micmrganisms can use inorganic nitrogen. Suggest ways of overcoming or bypassing the following disadvantages of SCP as food. 1) Unpalatability 2) Indigestibility 3) Poor amino acid profile 4) Toxicity We have seen that only certain micro-organisms that conform to nutritional and safety requirements are suitable for food or feed, and that food has more strict requirements than feed. In addition, for use as food, SCP should have a reduced nucleic acid content and should be palatable. Most often this means that its use is limited to processed foods, in which food technologists can produce acceptable tastes, smells and textures
4.4 Substrates for SCP production For a micro-organism to grow it must be supplied with all the nutrients required for cell material and energy pre The physiological types of organism used in sCP production and their corresponding shown in Table 4.1. Photosynthetic bacteria utilise CO from the atmosphere and nitrate in inorganic salts or natural ground water media. algae are similar, growing on nitrate, ammonia or ammonium salt as nitrogen source. Some can also be grown as heterotrophs, in the dark, using sugars as sources of carbon and energy. Heterotrophic bacteria and fungi for SCP are grown on a variety of organic substrates, serving as both carbon and energy sources. Some organisms have additional requirement for growth factors, such as vitamins. For yeast, the substrate is in the form f sugars, as yeast cells cannot break down polysaccharides, whereas filamentous fungi hemiceulb may in addition be able to use starch oy secreting, amylases ), pectin(by secreting pectinases)and cellulosic material (by secreting cellulases and hemicellulases). Waste hemicellulases containing cellulosic material is in solid rather than in liquid form. Processes have also been developed with yeasts growing on n-paraffins or ethanol, and with bacteria growing on methanol Inorganic nitrogen is supplied in such processes as ammonia, or as ammon Physlological Carbon Energy Nir。ga source source Autotroph enc Atmos H4 cO Heterotroph As carbon IH3, NH4 (sugars) Heterotroph Carbohydrate As carbon NH3, NH4 sugars, starch culosis As carbon NH3 NH4* (n-paraffins, Bacteria Heterotroph Hydrocarbon As carbon NH3, NH4 (methylotroph) derivatives (methanol Table 4.1 Organisms and substrates in SCP production
66 Chapter 4 4.4 Substrates for SCP production For a micrmrganism to grow it must be supplied with all the nutrients required for ceII material and energy production. The physiological types of organism used in SCP production and their corresponding substrates are shown in Table 4.1. Photosynthetic bacteria utilise CQ from the atmosphere and nitrate in inorganic salts or natural ground water media. Algae are similar, growing on nitrate, ammonia or ammonium salt as nitrogen source. Some can also be grown as heterotrophs, in the dark, using sugars as sources of carbon and energy. Heterotrophic bacteria and fin@ for SCP are grown on a variety of organic substrates, serving as both carbon and energy sources. Some organisms have additional requirement for growth factors, such as vitamins. For yeast, the substrate is in the form of sugars, as yeast cells cannot break down polysaccharides, whereas filamentous fungi may in addition be able to use starch (by secreting amylases), pectin (by secreting pectinases) and cellulosic material (by secreting cellulases and hemicellulases). Waste containing cellulosic material is in solid rather than in liquid form. Processes have also been developed with yeasts growing on n-paraffins or ethanol, and with bacteria growing on methanol. Inorganic nitrogen is supplied in such processes as ammonia, or as ammonium salt. pectinases cellulases and hfli&lulmes Organisms Physiological Carbon Energy Nltrogen type source source source Blue-green Autotroph Atmospheric bacteria c02 Algae Fungi Autotroph Atmospheric Heterotroph Carbohydrate Heterotroph Carbohydrate con (sugars) (sugars, starch, pectin, cellulosics) Hydrocarbons and derivatives (n-paraff ins, ethanol) Bacteria Heterotroph Hydrocarbon (methylotroph) derivatives (methanol) Sunlight Sunlight As carbon source As carbon source As carbon source As carbon source N03- NH3, NH4+ N03- NH3, NH4+ N03' NH3, NH4+ NH3, NH4+ NH3, NH4+ Table 4.1 Organisms and substrates in SCP production
Single cell protein In a culture medium for the growth of heterotrophs what do you think the carbon: nitrogen(C: N)ratio should be? 10:1 C: N ratio The C N ratio should be about 10: 1. The organic carbon in the medium provides both a source of a sour。e of carbon Cells contain more carbon that nitrogen. The correct ratio of C: N is about 10: 1, although this differs slightly between organisms. This means that a medium containing 3%w/v sugar should be supplied with ammonia at about 0.3%w/v. At a C: N ratio of 1: 1 most of the ammonia would not be incorporated into cells(it is present in excess)and would be wasted. At C N ratios more than 10: 1 the ammonia would be completely used up before all the sugar, reducing the biomass output and wasting the sugar onid-substrate The cost of substrates used in sCP production may represent 40-75% of the total fermentations production cost. Ammonia contributes 5-15% of the substrate cost but the major portion is the carbon source. Atmospheric Co is free, but costly energy is needed for agitation to dissolve it into dense algal cultures. Wastes from agriculture and industry can be plentiful and relatively cheap, but may still represent 20-30% of the total production costs. Solid agricultural wastes, especially cellulosic ones, may also need expensive pre-treatment before they can be used in solid-substrate fermentations. Industrial wastes in the form of effluents can have high levels of BOd(Biological Oxygen Demand), which means they could cause pollution if disposed of in water without treatment Using themas substrates for sCP production can reduce the BOD by as much as 70-80% and so save on treatment costs. Such agricultural and industrial wastes are derived from biomass(plant material)which is renewable and likely to remain plentiful and relatively cheap Hydrocarbons and their derivatives can represent from 30-70% of total production costs. They are derived from oil or natural gas which are non-renewable, will not remain as plentiful as at present and will become increasing expensive. They also have alternative uses as fuels and petrochemicals and availability is often influenced by political issues 4.5 MIcro-organlsms for SCP production The physiological groups of organisms used in sCP production have been given in Table 4. 1. We have examined the characteristics an organism should and should not have in order to be suitable as food or feed in Section 43. When selecting an organism for a particularproduction process, factors relating to growth of the organism also need to be considered Listed below are characteristics in culture of an organism you are intending to use in an seP process you are developing. Consider whether each characteristic is an advantage or disadvantage to you. Tick the appropriate box, or if you think thecharacteristicis an advantage on the one hand but a disadvantage on the other tick both boxes
Single cell protein 67 CN ratio ~lii-aubstrate fermentations renewable In a culture medium for the growth of heterotrophs what do you think the n carbon : nitrogen (CN) ratio should be? 1:l 1O:l 1oo:l 1Ooo:l The CN ratio should be about 101. The organic carbon in the medium provides both a source of energy and a source of carbon. Cells contain more carbon that nitrogen. The correct ratio of CN is about lO:l, although this differs slightly between organisms. This means that a medium containing 3% w/v sugar should be supplied with ammonia at about 0.3% w/v. At a CN ratio of 1:l most of the ammonia would not be incorporated into cells (it is present in excess) and would be wasted. At CN ratios more than 10:l the ammonia would be completely used up before all the sugar, ducing the biomass output and wasting the sugar. The cost of substrates used in SCF' production may represent 40-7596 of the total production cost. Ammonia contributes 515% of the substrate cost but the major portion is the carbon source. Atmospheric COZ is free, but costly energy is needed for agitation to dissolve it into dense algal cultures. Wastes from agriculture and industry can be plentiful and relatively cheap, but may still re resent 20-3096 of the total roduction pretreatment before they can be used in solid-substrate fermentations. Industrial wastes in the form of effluents can have high levels of BOD (Biological Oxygen Demand), which means they could cause pollution if disposed of in water without treatment Using them as substrates for XP production can reduce the BOD by as much as 70-801 and so save on treatment costs. Such agricultural and industrial wastes are derived from biomass (plant material) which is renewable and likely to remain plentiful and relatively cheap. Hydrocarbons and their derivatives can represent from 3G7096 of total production costs. They are derived from oil or natural gas which are non-renewable, will not remain as plentiful as at present and will become increasingly expensive. They also have alternative uses as fuels and petrochemicals and their availability is often influenced by political issues. costs. Solid agricultural wastes, especially ce 2 ulosic ones, may also n ee8 expensive 4.5 Micro-organisms for SCP production The physiological gruups of organisms used in XP production have been given in Table 4.1. We have exarmned ' the characteristics an organism should and should not have in order to be suitable as food or feed in Section 4.3. When selecting an organism for a particular production process, factors relating to growth of the organism also need to be considered. Listed below are characteristics in culture of an organism you are intending to use in an s8 process you are developing. Consider whether each characbistic is an advantage or disadvantage to you. Tick the appm riate box, or if you think tick both boxes. n the characteristic is an advantage on the one hand but a B 'sadvantage on the other
Chapter ad vant disadvantage ii) High biomass yield coefficient. iii) Filamentous growth iv) Tolerance to broad range of temperatures v) Tolerance to broad range of pH vi) High spontaneous mutation rate vii) Low aeration requirement. put i Disadvantage. High growth rate is needed for high output(weight of biomass roduced per unit of time). The only advantage could be that as the RNa content of cells is generally proportional to the growth rate, growth at low growth rate could result in a product with lower nucleic acid content. 1) Advantage. The biomass yield coefficient (weight of cells produced per unit of ubstrate consumed)should be high in order to give a high output. It also ensures efficient utilisation of the(expensive )substrate. Advantage/Disadvantage Compared to unicellularorganisms, filamentous ones are easier (and cheaper) to recover from fermentation media( by sieving or rotar vacuum filtration)and have a more fibrous texture. However dense broths filamentous organisms can be difficult to aerate and wall growth can cause problems such as clogging of pipes and valves o Advantage. Temperature increases can occur during fermentations, as growth processes are exothermic. The ability of an organism to tolerate raised temperature would reduce the need for cooling. The ability of an organism to grow at ambient temperatures also overcomes the need for heating and cooling The broader the temperature range tolerated, the less the need for temperature v) Advantage. The pHof a medium tends to change during fermentation. Most often media are buffered, and the fermentor is fitted with pH control. However, the bility to tolerate a wide range of ph can overcome the need for pHoontrol Fungi generally grow at lower pH than bacteria Use can sometimes be made of this by operating fungal processes at very low pH, preventing bacterial growth. This means that an aseptic process (using sterilising procedures to maintain a pure culture)will be less prone to contamination if aseptic procedures fail. In some circumstances non-aseptic (non-sterile) processes can be operated, saving Disadvantage Organisms for SCP production require a high degree of genetic stability. We have been considering the characteristics an organismmust have for sCP production. These characteristics are under genetic control and any mutation
68 Chapter 4 advantage disadvantage i) Low growth rate. ii) High biomass yield coefficient. iii) Filamentous growth. iv) Tolerance to broad range of temperatures. v) Tolerance to broad range of pH. vi) High spontaneous mutation rate. vii) Low aeration requirement. Output i) Disadvantage. High growth rate is needed for high output (weight of biomass produced per unit of time). The only advantage could be that as the RNA content of cells is generally proportional to the growth rate, growth at low growth rate could result in a product with lower nucleic acid content. Advantage. The biomass yield coefficient (weight of cells produced per unit of substrate consumed) should be high in order to give a high output. It also ensures efficient utilisation of the (expensive) substrate. wall growth iii) AdvantageDisadvantage. Compared to unicellularorganisms, filamentous ones are easier (and cheaper) to recover from fermentation media (by sieving or rotary vacuum filtration) and have a more fibrous texture. However, dense broths of filamentous organisms can be difficult to aerate and wall growth can cause problems such as clogging of pipes and valves. Advantage. Temperature increases can occur during fermentations, as growth processes are exothermic. The ability of an organism to tolerate raised temperature would reduce the need for Cooling. The ability of an qanism to grow at ambient temperatures also overcomes the need for heating and cooling. The broader the temperature range tolerated, the less the need for temperature control. Advantage. The pH of a medium tends to change during fermentation. Most often media are buffered, and the fermentor is fitted with pH control. However, the ability to tolerate a wide range of pH can overcome the need for pH control. Fungi generally grow at lower pH than bacteria. Use can sometimes be made of this by operating fungal processes at very low pH, preventing bacterial growth. This means that an aseptic process (using sterilising procedures to maintain a pure culture) will be less prone to contamination if aseptic procedures fail. In some circumstances non-aseptic (non-sterile) processes can be operated, saving sterilisation costs. Disadvantage. Organisms for SCP production quire a high degree of genetic stability. We have been considering the characteristics an organism must have for s8 production. These characteristics are under genetic control and any mutation ii) iv) v) non-aseptic processes vi)
