Industrial fermentation

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Industrial fermentation is the intentional use of fermentation in manufacturing processes. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical industry. Commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. [1] Moreover, nearly all commercially produced industrial enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as is the case for single-cell proteins, baker's yeast, and starter cultures for lactic acid bacteria used in cheesemaking.

Contents

In general, fermentations can be divided into four types: [2]

These types are not necessarily disjoined from each other, but provide a framework for understanding the differences in approach. The organisms used are typically microorganisms, particularly bacteria, algae, and fungi, such as yeasts and molds, but industrial fermentation may also involve cell cultures from plants and animals, such as CHO cells and insect cells. Special considerations are required for the specific organisms used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature. The rate of fermentation depends on the concentration of microorganisms, cells, cellular components, and enzymes as well as temperature, pH [3] and level of oxygen for aerobic fermentation. [4] Product recovery frequently involves the concentration of the dilute solution.

General process overview

In most industrial fermentations, the organisms or eukaryotic cells are submerged in a liquid medium; in others, such as the fermentation of cocoa beans, coffee cherries, and miso, fermentation takes place on the moist surface of the medium. [5] [6]

There are also industrial considerations related to the fermentation process. For instance, to avoid biological process contamination, the fermentation medium, air, and equipment are sterilized. Foam control can be achieved by either mechanical foam destruction or chemical anti-foaming agents. Several other factors must be measured and controlled such as pressure, temperature, agitator shaft power, and viscosity. An important element for industrial fermentations is scale up. This is the conversion of a laboratory procedure to an industrial process. It is well established in the field of industrial microbiology that what works well at the laboratory scale may work poorly or not at all when first attempted at large scale. It is generally not possible to take fermentation conditions that have worked in the laboratory and blindly apply them to industrial scale equipment. Although many parameters have been tested for use as scale up criteria, there is no general formula because of the variation in fermentation processes. The most important methods are the maintenance of constant power consumption per unit of broth and the maintenance of constant volumetric transfer rate. [3]

Phases of growth

Bacterial growth curve Bacterial growth en.svg
Bacterial growth curve

Fermentation begins once the growth medium is inoculated with the organism of interest. Growth of the inoculum does not occur immediately. This is the period of adaptation, called the lag phase. [7] Following the lag phase, the rate of growth of the organism steadily increases, for a certain period—this period is the log or exponential phase. [7]

After a phase of exponential growth, the rate of growth slows down, due to the continuously falling concentrations of nutrients and/or a continuously increasing (accumulating) concentrations of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration phase. After the deceleration phase, growth ceases and the culture enters a stationary phase or a steady state. The biomass remains constant, except when certain accumulated chemicals in the culture chemically break down the cells in a process called chemolysis. Unless other microorganisms contaminate the culture, the chemical constitution remains unchanged. If all of the nutrients in the medium are consumed, or if the concentration of toxins is too great, the cells may become senescent and begin to die off. The total amount of biomass may not decrease, but the number of viable organisms will decrease.[ citation needed ]

Fermentation medium

The microbes or eukaryotic cells used for fermentation grow in (or on) specially designed growth medium which supplies the nutrients required by the organisms or cells. A variety of media exist, but invariably contain a carbon source, a nitrogen source, water, salts, and micronutrients. In the production of wine, the medium is grape must. In the production of bio-ethanol, the medium may consist mostly of whatever inexpensive carbon source is available.[ citation needed ]

Carbon sources are typically sugars or other carbohydrates, although in the case of substrate transformations (such as the production of vinegar) the carbon source may be an alcohol or something else altogether. For large scale fermentations, such as those used for the production of ethanol, inexpensive sources of carbohydrates, such as molasses, corn steep liquor, [8] sugar cane juice, or sugar beet juice are used to minimize costs. More sensitive fermentations may instead use purified glucose, sucrose, glycerol or other sugars, which reduces variation and helps ensure the purity of the final product. Organisms meant to produce enzymes such as beta galactosidase, invertase or other amylases may be fed starch to select for organisms that express the enzymes in large quantity.[ citation needed ]

Fixed nitrogen sources are required for most organisms to synthesize proteins, nucleic acids and other cellular components. Depending on the enzyme capabilities of the organism, nitrogen may be provided as bulk protein, such as soy meal; as pre-digested polypeptides, such as peptone or tryptone; or as ammonia or nitrate salts. Cost is also an important factor in the choice of a nitrogen source. Phosphorus is needed for production of phospholipids in cellular membranes and for the production of nucleic acids. The amount of phosphate which must be added depends upon the composition of the broth and the needs of the organism, as well as the objective of the fermentation. For instance, some cultures will not produce secondary metabolites in the presence of phosphate. [9]

Growth factors and trace nutrients are included in the fermentation broth for organisms incapable of producing all of the vitamins they require. Yeast extract is a common source of micronutrients and vitamins for fermentation media. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum, and cobalt are typically present in unrefined carbon and nitrogen sources, but may have to be added when purified carbon and nitrogen sources are used. Fermentations which produce large amounts of gas (or which require the addition of gas) will tend to form a layer of foam, since fermentation broth typically contains a variety of foam-reinforcing proteins, peptides or starches. To prevent this foam from occurring or accumulating, antifoaming agents may be added. Mineral buffering salts, such as carbonates and phosphates, may be used to stabilize pH near optimum. When metal ions are present in high concentrations, use of a chelating agent may be necessary.[ citation needed ]

Developing an optimal medium for fermentation is a key concept to efficient optimization. One-factor-at-a-time (OFAT) is the preferential choice that researchers use for designing a medium composition. This method involves changing only one factor at a time while keeping the other concentrations constant. This method can be separated into some sub groups. One is Removal Experiments. In this experiment all the components of the medium are removed one at a time and their effects on the medium are observed. Supplementation experiments involve evaluating the effects of nitrogen and carbon supplements on production. The final experiment is a replacement experiment. This involves replacing the nitrogen and carbon sources that show an enhancement effect on the intended production. Overall OFAT is a major advantage over other optimization methods because of its simplicity. [10]

Production of biomass

Microbial cells or biomass is sometimes the intended product of fermentation. Examples include single cell protein, bakers yeast, lactobacillus, E. coli, and others. In the case of single-cell protein, algae is grown in large open ponds which allow photosynthesis to occur. [11] If the biomass is to be used for inoculation of other fermentations, care must be taken to prevent mutations from occurring.

Production of extracellular metabolites

Metabolites can be divided into two groups: those produced during the growth phase of the organism, called primary metabolites and those produced during the stationary phase, called secondary metabolites. Some examples of primary metabolites are ethanol, citric acid, glutamic acid, lysine, vitamins and polysaccharides. Some examples of secondary metabolites are penicillin, cyclosporin A, gibberellin, and lovastatin. [9]

Primary metabolites

Primary metabolites are compounds made during the ordinary metabolism of the organism during the growth phase. A common example is ethanol or lactic acid, produced during glycolysis. Citric acid is produced by some strains of Aspergillus niger as part of the citric acid cycle to acidify their environment and prevent competitors from taking over. Glutamate is produced by some Micrococcus species, [12] and some Corynebacterium species produce lysine, threonine, tryptophan and other amino acids. All of these compounds are produced during the normal "business" of the cell and released into the environment. There is therefore no need to rupture the cells for product recovery.

Secondary metabolites

Secondary metabolites are compounds made in the stationary phase; penicillin, for instance, prevents the growth of bacteria which could compete with Penicillium molds for resources. Some bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the growth of bacterial competitors as well. These compounds are of obvious value to humans wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics (such as gramicidin S). Fungicides, such as griseofulvin are also produced as secondary metabolites. [9] Typically secondary metabolites are not produced in the presence of glucose or other carbon sources which would encourage growth, [9] and like primary metabolites are released into the surrounding medium without rupture of the cell membrane.

In the early days of the biotechnology industry, most biopharmaceutical products were made in E. coli ; by 2004 more biopharmaceuticals were manufactured in eukaryotic cells, such as CHO cells, than in microbes, but used similar bioreactor systems. [6] Insect cell culture systems came into use in the 2000s as well. [13]

Production of intracellular components

Of primary interest among the intracellular components are microbial enzymes: catalase, amylase, protease, pectinase, cellulase, hemicellulase, lipase, lactase, streptokinase and many others. [14] Recombinant proteins, such as insulin, hepatitis B vaccine, interferon, granulocyte colony-stimulating factor, streptokinase and others are also made this way. [6] The largest difference between this process and the others is that the cells must be ruptured (lysed) at the end of fermentation, and the environment must be manipulated to maximize the amount of the product. Furthermore, the product (typically a protein) must be separated from all of the other cellular proteins in the lysate to be purified.

Transformation of substrate

Substrate transformation involves the transformation of a specific compound into another, such as in the case of phenylacetylcarbinol, and steroid biotransformation, or the transformation of a raw material into a finished product, in the case of food fermentations and sewage treatment.

Food fermentation

In the history of food, ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa, etc., can be dated to more than seven thousand years ago. [15] They were developed long before humanity had any knowledge of the existence of the microorganisms involved. Some foods such as Marmite are the byproduct of the fermentation process, in this case in the production of beer.

Ethanol fuel

Fermentation is the main source[ citation needed ] of ethanol in the production of ethanol fuel. Common crops such as sugar cane, potato, cassava, and maize are fermented by yeast to produce ethanol which is further processed to become fuel.

Sewage treatment

In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria. Solid organic matters are broken down into harmless, soluble substances and carbon dioxide. Liquids that result are disinfected to remove pathogens before being discharged into rivers or the sea or can be used as liquid fertilizers. Digested solids, known also as sludge, is dried and used as fertilizer. Gaseous byproducts such as methane can be utilized as biogas to fuel electrical generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing space needed for dumping. The main disadvantage of bacterial digestion in sewage disposal is that it is a very slow process.

Agricultural feed

A wide variety of agroindustrial waste products can be fermented to use as food for animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility. [16]

Precision fermentation

Precision fermentation is an approach to manufacturing specific functional products which intends to minimise the production of unwanted by-products through the application of synthetic biology, particularly by generating synthetic "cell factories" with engineered genomes and metabolic pathways optimised to produce the desired compounds as efficiently as possible with the available resources. [17] Precision fermentation of genetically modified microorganisms may be used to manufacture proteins needed for cell culture media, [18] providing for serum-free cell culture media in the manufacturing process of cultured meat. [19] A 2021 publication showed that photovoltaic-driven microbial protein production could use 10 times less land for an equivalent amount of protein compared to soybean cultivation. [20]

See also

Related Research Articles

<span class="mw-page-title-main">Nutrition</span> Provision to cells and organisms to support life

Nutrition is the biochemical and physiological process by which an organism uses food to support its life. It provides organisms with nutrients, which can be metabolized to create energy and chemical structures. Failure to obtain the required amount of nutrients causes malnutrition. Nutritional science is the study of nutrition, though it typically emphasizes human nutrition.

<i>Aspergillus niger</i> Species of fungus

Aspergillus niger is a mold classified within the Nigri section of the Aspergillus genus. The Aspergillus genus consists of common molds found throughout the environment within soil and water, on vegetation, in fecal matter, on decomposing matter, and suspended in the air. Species within this genus often grow quickly and can sporulate within a few days of germination. A combination of characteristics unique to A. niger makes the microbe invaluable to the production of many acids, proteins and bioactive compounds. Characteristics including extensive metabolic diversity, high production yield, secretion capability, and the ability to conduct post-translational modifications are responsible for A. niger's robust production of secondary metabolites. A. niger's capability to withstand extremely acidic conditions makes it especially important to the industrial production of citric acid.

<span class="mw-page-title-main">Bioreactor</span> System that supports a biologically active environment

A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres, and are often made of stainless steel. It may also refer to a device or system designed to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical/bioprocess engineering.

<span class="mw-page-title-main">Ethanol fermentation</span> Biological process that produces ethanol and carbon dioxide as by-products

Ethanol fermentation, also called alcoholic fermentation, is a biological process which converts sugars such as glucose, fructose, and sucrose into cellular energy, producing ethanol and carbon dioxide as by-products. Because yeasts perform this conversion in the absence of oxygen, alcoholic fermentation is considered an anaerobic process. It also takes place in some species of fish where it provides energy when oxygen is scarce.

Cellulosic ethanol is ethanol produced from cellulose rather than from the plant's seeds or fruit. It can be produced from grasses, wood, algae, or other plants. It is generally discussed for use as a biofuel. The carbon dioxide that plants absorb as they grow offsets some of the carbon dioxide emitted when ethanol made from them is burned, so cellulosic ethanol fuel has the potential to have a lower carbon footprint than fossil fuels.

<span class="mw-page-title-main">Growth medium</span> Solid, liquid or gel used to grow microorganisms or cells

A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.

Acidogenesis is the second stage in the four stages of anaerobic digestion:

Microbial metabolism is the means by which a microbe obtains the energy and nutrients it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe's ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.

<span class="mw-page-title-main">Fermentation</span> Metabolic process producing energy in the absence of oxygen

Fermentation is a metabolic process that produces chemical changes in organic substances through the action of enzymes. In biochemistry, it is broadly defined as the extraction of energy from carbohydrates in the absence of oxygen. In food production, it may more broadly refer to any process in which the activity of microorganisms brings about a desirable change to a foodstuff or beverage. The science of fermentation is known as zymology.

The Pasteur effect describes how available oxygen inhibits ethanol fermentation, driving yeast to switch toward aerobic respiration for increased generation of the energy carrier adenosine triphosphate (ATP). More generally, in the medical literature, the Pasteur effect refers to how the cellular presence of oxygen causes in cells a decrease in the rate of glycolysis and also a suppression of lactate accumulation. The effect occurs in animal tissues, as well as in microorganisms belonging to the fungal kingdom.

Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion". It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels.

Single-cell proteins (SCP) or microbial proteins refer to edible unicellular microorganisms. The biomass or protein extract from pure or mixed cultures of algae, yeasts, fungi or bacteria may be used as an ingredient or a substitute for protein-rich foods, and is suitable for human consumption or as animal feeds. Industrial agriculture is marked by a high water footprint, high land use, biodiversity destruction, general environmental degradation and contributes to climate change by emission of a third of all greenhouse gases; production of SCP does not necessarily exhibit any of these serious drawbacks. As of today, SCP is commonly grown on agricultural waste products, and as such inherits the ecological footprint and water footprint of industrial agriculture. However, SCP may also be produced entirely independent of agricultural waste products through autotrophic growth. Thanks to the high diversity of microbial metabolism, autotrophic SCP provides several different modes of growth, versatile options of nutrients recycling, and a substantially increased efficiency compared to crops. A 2021 publication showed that photovoltaic-driven microbial protein production could use 10 times less land for an equivalent amount of protein compared to soybean cultivation.

Syngas fermentation, also known as synthesis gas fermentation, is a microbial process. In this process, a mixture of hydrogen, carbon monoxide, and carbon dioxide, known as syngas, is used as carbon and energy sources, and then converted into fuel and chemicals by microorganisms.

<span class="mw-page-title-main">Cofactor engineering</span> Modification of use and function of cofactors in an organisms metabolic pathways

Cofactor engineering, a subset of metabolic engineering, is defined as the manipulation of the use of cofactors in an organism’s metabolic pathways. In cofactor engineering, the concentrations of cofactors are changed in order to maximize or minimize metabolic fluxes. This type of engineering can be used to optimize the production of a metabolite product or to increase the efficiency of a metabolic network. The use of engineering single celled organisms to create lucrative chemicals from cheap raw materials is growing, and cofactor engineering can play a crucial role in maximizing production. The field has gained more popularity in the past decade and has several practical applications in chemical manufacturing, bioengineering and pharmaceutical industries.

Diauxic growth, diauxie or diphasic growth is any cell growth characterized by cellular growth in two phases. Diauxic growth, meaning double growth, is caused by the presence of two sugars on a culture growth media, one of which is easier for the target bacterium to metabolize. The preferred sugar is consumed first, which leads to rapid growth, followed by a lag phase. During the lag phase the cellular machinery used to metabolize the second sugar is activated and subsequently the second sugar is metabolized.

Single cell oil, also known as Microbial oil consists of the intracellular storage lipids, triacyglycerols. It is similar to vegetable oil, another biologically produced oil. They are produced by oleaginous microorganisms, which is the term for those bacteria, molds, algae and yeast, which can accumulate 20% to 80% lipids of their biomass. The accumulation of lipids take place by the end of logarithmic phase and continues during station phase until carbon source begins to reduce with nutrition limitation.

Solid state fermentation (SSF) is a biomolecule manufacturing process used in the food, pharmaceutical, cosmetic, fuel and textile industries. These biomolecules are mostly metabolites generated by microorganisms grown on a solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes.

<span class="mw-page-title-main">Yeast assimilable nitrogen</span> Form of nitrogen available to wine yeast to use during fermentation

Yeast assimilable nitrogen or YAN is the combination of free amino nitrogen (FAN), ammonia (NH3) and ammonium (NH4+) that is available for a yeast, e.g. the wine yeast Saccharomyces cerevisiae, to use during fermentation. Outside of the fermentable sugars glucose and fructose, nitrogen is the most important nutrient needed to carry out a successful fermentation that doesn't end prior to the intended point of dryness or sees the development of off-odors and related wine faults. To this extent winemakers will often supplement the available YAN resources with nitrogen additives such as diammonium phosphate (DAP).

<i>Rhizopus oryzae</i> Species of fungus

Rhizopus oryzae is a filamentous heterothallic microfungus that occurs as a saprotroph in soil, dung, and rotting vegetation. This species is very similar to Rhizopus stolonifer, but it can be distinguished by its smaller sporangia and air-dispersed sporangiospores. It differs from R. oligosporus and R. microsporus by its larger columellae and sporangiospores. The many strains of R. oryzae produce a wide range of enzymes such as carbohydrate digesting enzymes and polymers along with a number of organic acids, ethanol and esters giving it useful properties within the food industries, bio-diesel production, and pharmaceutical industries. It is also an opportunistic pathogen of humans causing mucormycosis.

<span class="mw-page-title-main">Industrial microbiology</span>

Industrial microbiology is a branch of biotechnology that applies microbial sciences to create industrial products in mass quantities, often using microbial cell factories. There are multiple ways to manipulate a microorganism in order to increase maximum product yields. Introduction of mutations into an organism may be accomplished by introducing them to mutagens. Another way to increase production is by gene amplification, this is done by the use of plasmids, and vectors. The plasmids and/ or vectors are used to incorporate multiple copies of a specific gene that would allow more enzymes to be produced that eventually cause more product yield. The manipulation of organisms in order to yield a specific product has many applications to the real world like the production of some antibiotics, vitamins, enzymes, amino acids, solvents, alcohol and daily products. Microorganisms play a big role in the industry, with multiple ways to be used. Medicinally, microbes can be used for creating antibiotics in order to treat infection. Microbes can also be used for the food industry as well. Microbes are very useful in creating some of the mass produced products that are consumed by people. The chemical industry also uses microorganisms in order to synthesize amino acids and organic solvents. Microbes can also be used in an agricultural application for use as a biopesticide instead of using dangerous chemicals and or inoculants to help plant proliferation.

References

  1. ↑ Yusuf C (1999). Robinson RK (ed.). Encyclopedia of Food Microbiology (PDF). London: Academic Press. pp. 663–674. ISBN   978-0-12-227070-3.
  2. ↑ Stanbury PF, Whiitaker A, Hall SJ (1999). Principles of Fermentation Technology (Second ed.). Butterworth-Heinemann. ISBN   978-0750645010.
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  7. 1 2 "Bacterial Growth". Bacanova. Archived from the original on 29 October 2013.
  8. ↑ Liggett RW, Koffler H (December 1948). "Corn Steep Liquor in Microbiology". Bacteriological Reviews. 12 (4): 297–311. doi:10.1128/MMBR.12.4.297-311.1948. PMC   180696 . PMID   16350125.
  9. 1 2 3 4 Stanbury PF (2007). "Chapter 1: Fermentation Technology" (PDF). In Walker JM, Rapley R (eds.). Molecular Biology and Biotechnology. Royal Society of Chemistry. pp. 1–24. ISBN   978-1-84755-149-8. Archived from the original (PDF) on 2012-12-02.
  10. ↑ Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CK (2017-01-06). "Strategies for Fermentation Medium Optimization: An In-Depth Review". Frontiers in Microbiology. 7: 2087. doi: 10.3389/fmicb.2016.02087 . PMC   5216682 . PMID   28111566.
  11. ↑ "Algae harvesting – Industrial fermentation – Separators". Alfalaval.com. Archived from the original on 2015-06-02. Retrieved 2015-06-02.
  12. ↑ Kinoshita S, Udaka S, Shimono M (December 2004). "Studies on the amino acid fermentation. Part 1. Production of L-glutamic acid by various microorganisms". The Journal of General and Applied Microbiology. 50 (6): 331–43. PMID   15965888.
  13. ↑ Drugmand JC, Schneider YJ, Agathos SN (2012). "Insect cells as factories for biomanufacturing". Biotechnology Advances. 30 (5): 1140–57. doi:10.1016/j.biotechadv.2011.09.014. PMID   21983546.
  14. ↑ De Lourdes M, Polizeli TM, Rai M (2013). Fungal Enzymes. CRC Press. ISBN   978-1-466-59454-8.
  15. ↑ Humphrey AE, Lee SE (1992). Industrial Fermentation: Principles, Processes, and Products. pp. 916–986. doi:10.1007/978-94-011-7691-0_24. ISBN   978-94-011-7693-4.{{cite book}}: |journal= ignored (help)
  16. ↑ Albores S, Pianzzola MJ, Soubes M, Cerdeiras MP (2006). "Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed". Electronic Journal of Biotechnology. 9 (3). doi:10.2225/vol9-issue3-fulltext-2. hdl: 1807/48850 . Retrieved 2015-06-02.
  17. ↑ Teng, Ting Shien; Chin, Yi Ling; Chai, Kong Fei; Chen, Wei Ning (2021). "Fermentation for future food systems: Precision fermentation can complement the scope and applications of traditional fermentation". EMBO Reports. 22 (5): e52680. doi:10.15252/embr.202152680. ISSN   1469-221X. PMC   8097352 . PMID   33908143.
  18. ↑ Tubb, Catherine; Seba, Tony (2020). Rethinking food and agriculture, 2020-2030: the second domestication of plants and animals, the disruption of the cow and the collapse of industrial livestock farming (1st ed.). United States: RethinkX. ISBN   978-0-9970471-7-2. OCLC   1257489312.
  19. ↑ Singh, Satnam; Yap, Wee Swan; Ge, Xiao Yu; Min, Veronica Lee Xi; Choudhury, Deepak (2022). "Cultured meat production fuelled by fermentation". Trends in Food Science & Technology. 120: 48–58. doi:10.1016/j.tifs.2021.12.028. S2CID   245491693.
  20. ↑ Leger, Dorian; Matassa, Silvio; Noor, Elad; Shepon, Alon; Milo, Ron; Bar-Even, Arren (2021-06-29). "Photovoltaic-driven microbial protein production can use land and sunlight more efficiently than conventional crops". Proceedings of the National Academy of Sciences of the United States of America. 118 (26): e2015025118. Bibcode:2021PNAS..11815025L. doi: 10.1073/pnas.2015025118 . ISSN   0027-8424. PMC   8255800 . PMID   34155098.

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