General Biotechnology: Lecture Course. Part II - Blinov V.A. 2004

Microbiological production of renewable energy sources

♦ Ethanol production.

♦ Acetone production.

♦ Prospects for hydrogen production.

♦ Biogeotechnology.

Various natural resources are being rapidly depleted. For instance, in the USA, 70% of energy demand is met by three sources: oil (34%), coal (18%), and natural gas (18%). According to estimates, oil and gas reserves will last only until the mid-first century of the third millennium. Therefore, obtaining energy from renewable raw material sources is a highly pressing issue. Renewable raw Materials refer to the plant kingdom, where organic matter is formed from Water and carbon dioxide through Photosynthesis. The Earth's vegetation cover contains 1,800 billion tons of dry matter, which stores 3.0×10²² J of energy. At the same time, the reserves of oil, gas, coal, and uranium are equivalent to 2.5×10²² J. The annual increase in organic matter on the planet is 2×10¹¹ tons, of which 1.2×10¹¹ tons come from lignocellulosic raw materials.

The simplest way to convert biomass into energy is combustion, which provides heat that can be converted into mechanical or electrical energy. If the material is wet, the most efficient method is to produce biogas from it. Energy can be obtained from agricultural crops specially cultivated for this purpose (fast-growing tree plantations, starch- and sucrose-rich plants). Reserve CARBOHYDRATES are extracted from such plant biomass and subjected to Hydrolysis, after which they are converted into alcohol through microbial Fermentation. Depending on the final product, alcoholic, lactic acid, butyric acid, and other Types of fermentation are distinguished. In this process, glucose is transformed into a key product—pyruvic acid—from which acetic acid, propionic acid, butyric acid, lactic acid, acetone, ethanol, butanol, etc., are subsequently formed.

Ethanol production. Alcoholic Fermentation is carried out by saccharomycete Yeasts, certain mycelial Fungi (Aspergillus oryzae), and Bacteria (Erwinia amylovora, Sarcina ventriculi, Zymomonas mobilis, Z. anaerobia). Yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and S. octosporus) are of primary importance in obtaining ethanol.

Yeast strains are subdivided into top-fermenting and bottom-fermenting races, and according to their flocculation ability, into flocculent and powdery types. Top-fermenting races include alcohol, baker's, and certain brewing yeasts; bottom-fermenting races include most wine and brewing yeasts. Cells of both races can be subjected to flocculation.

Powdery yeasts are in a dispersed state during fermentation; they are less resistant to autolysis and ferment the wort more completely. Flocculent yeasts can settle to the bottom or rise to the surface, and they are more pronounced flavor producers. For example, S. cerevisiae is cultivated at pH 3–4 and a Temperature of 30 °C, with the ethanol yield reaching 100% of the maximum. The maximum accumulated alcohol concentration can reach 130 g/L.

Yeasts do not utilize pentoses. Z. mobilis is aerotolerant, while Z. anaerobia is thermotolerant, etc. S. rosei is of great interest. For ethanol production, they utilize Jerusalem artichoke, which contains a high amount of inulin that is hydrolyzed and subsequently fermented into ethanol.

Raw materials used for ethyl alcohol production include grain, potatoes, beet and cane molasses, rice, etc., as well as softwood Cellulose, straw, peat, and sulfite liquors (wastes from the pulp and paper industry). From 1 ton of old cardboard, 150 liters of ethanol can be obtained.

Technological flowchart for ethanol production. First, starch must be transformed into glucose. To achieve this, the raw material is treated with amylolytic Enzymes. Fungal amylase (A. niger, A. oryzae, etc.) or sprouted grain (malt) is typically used.

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Potato, corn, wheat, rice, and other starches are used to produce ethanol. The starch is crushed using roller, hammer, or other crushers. Next, the starch is gelatinized during cooking. For example, wheat and wheat flour completely gelatinize at 68 °C within 30 minutes. Then, the starch is hydrolyzed into lower sugars (monoses, bioses) using enzyme preparations derived from filamentous fungi or malt.

Mash fermentation is carried out using pure cultures in batch or continuous mode. Importantly, unfavorable conditions for contaminant microbes are created in the mash. Initially, this is high osmotic pressure caused by sugars and dextrins, followed by ethanol concentration and medium acidity (pH = 3.8–4.0). Acidity can be natural, due to metabolic features, or artificial, following The addition of sulfuric acid. During fermentation, the temperature is maintained between 30 and 38 °C. Mash fermentation is affected by: the type and race of yeast, temperature, pH, Design Features of the fermenters (cooling or heating system), and the method and intensity of agitation. Fermentation duration averages 1.5 to 3 days.

The fermented wash (beer) accumulates from 1–1.5% to 6.5–8.5% ethanol; it is distilled and rectified up to 96%. Fusel oils (a high-boiling fraction at 90–150 °C) and 5–10% aldehydes with esters also accumulate in the wash. Fusel oils are a mixture of isopropyl, n-propyl, isobutyl, n-butyl, and isoamyl alcohols. The share of the last two usually accounts for 50%. Fusel oils also contain β-phenyl- and p-hydroxyphenylethyl alcohols.

Ethyl alcohol production is a large-scale tonnage industry. Ethanol is widely used in various economic sectors as a solvent, a feedstock for chemical synthesis, in medicine, etc. Ethanol can be used as an extractant and antifreeze. It serves as a substrate for many Dyes, Pharmaceuticals, lubricants, adhesives, detergents, plasticizers, explosives, and resins used to manufacture synthetic fibers. Attempts have been made to use ethanol as automotive fuel. In 1980, a mixture of 6–9 parts gasoline and 1 part ethanol ("gasohol") went on sale in the USA; this is one of the promising directions for ethanol utilization.

The wastes of ethanol production are stillage (vinasse) and carbon dioxide. Stillage is used for feeding livestock and poultry, while Carbon dioxide is utilized in the food industry.

It should be emphasized that obtaining ethanol via the fermentation of wood and herbaceous plant hydrolyzates is gaining increasing economic and practical significance; these usually contain 2–3% reducing sugars. Thus, in the combined production of alcohol and fodder yeast from 1 ton of absolutely dry wood, one can obtain: absolute ethanol 175–182 L, methanol 2 kg, fusel oils 0.3 kg, furfural (94%) 3.6 kg, liquid carbon dioxide 70 kg, yeast with 10% residual moisture 32 kg, absolutely dry Lignin 380 kg, and gypsum 225 kg.

Using Introduction/32.html">Genetic Engineering Methods, the Gene encoding xylose isomerase Biosynthesis was introduced into Schizosaccharomyces pombe yeast. This enzyme catalyzes The conversion of D-xylose into D-xylulose. An E. coli "xylose isomerase" plasmid was used as a vector. As a result, direct conversion of xylose to ethanol was achieved, and the yeast cells became capable of simultaneously fermenting glucose and xylose in hydrolyzates derived from various wood species. However, it should be kept in mind that the economic viability of bacterial anaerobic fermentations of plant waste hydrolyzates will only be realistic if the ethanol concentration in the wash is at least 4.5–5%. Furthermore, "hydrolysis" alcohol after rectification contains up to 0.05–0.1% methanol, along with elevated amounts of aldehydes, organic acids, and esters compared to rectified ethanol derived from potatoes and grain.

The specific technological features of obtaining ethanol from wood hydrolyzates, sulfite liquors, molasses, gas, and other sources may be studied independently.

Ethanol production can be increased through the following approaches:

♦ employing continuous fermentation instead of batch fermentation. This increases system productivity by more than 2-fold. Additionally, ethanol yield is enhanced by using flocculent yeast races, biomass recirculation, and Cell immobilization;

♦ conducting vacuum fermentation at a reduced pressure of 32–35 mm Hg to strip away ethanol;

♦ performing flash fermentation, in which a portion of the culture liquid is periodically introduced into a vacuum chamber to remove ethanol;

♦ breeding of ethanol-tolerant microorganism strains capable of producing high alcohol concentrations.

The economic advantages of ethanol production depend on A number of conditions: The Structure of agricultural crops and their surpluses; the cost of raw materials extracted from biomass and fuel sources; capital investments in plant construction; and price distribution. For example, landlocked countries are particularly interested in substituting petroleum with alternative fuel materials, etc.

Acetone production. Acetone-butanol fermentation is anaerobic and driven by spore-forming bacteria *Clostridium acetobutylicum* or closely related species of butyric acid bacteria. Acetone and butyl alcohol are obtained by fermenting grain, molasses-grain mashes, molasses, starch, syrups, cellulose hydrolysates of various origins, and sulfite liquors. For instance, when preparing the medium from corn, coarse-ground flour is mixed with water (6–8 kg of flour per 100 L of water). The mash is then cooked for 2 hours under a pressure of 200 kPa and sterilized. Cooled to 37–42 °C, the mass is fermented for 2 days at a medium pH of 5–7. During fermentation, glucose yields a mixture containing 6 parts of butanol, 1 part of ethanol, and 3 parts of acetone. Calculations show that 3 kg of starch yields 1 kg of organic Solvents.

In the initial stage of acetone-butanol fermentation, acetic and butyric acids are formed, accompanied by the release of hydrogen and carbon dioxide. Subsequently, butyric acid is reduced to butyl alcohol. Acetone is formed from acetoacetic acid via its decarboxylation. The inoculum is prepared from fresh spores, with a cultivation temperature of 37 °C. Main fermentations are carried out in batch, semi-continuous, and continuous modes. After 12 hours, the pH drops from approximately 6.0 to 4.5–4.2 and remains at this level until the end of fermentation. It has been established that low pH values activate enzymes that catalyze the conversion of acetoacetyl-CoA into acetone. Simultaneously, this increases the consumption of NADH₂ for the reduction of butyryl-CoA to butanol, the yield of which increases.

Upon completion of the process, the acetone-butanol whole stillage (vinasse) is separated, and the distillate is evaporated by approximately half. Afterward, acetone is separated from ethanol and butanol through fractional distillation at various temperatures. Acetone boils at 56.2 °C, ethanol at 78.4 °C, the butanol-water azeotrope at 93.4 °C, and pure butanol at 117.7 °C.

Acetone and butanol are widely used in the chemical industry and other fields of human economic activity. Production waste products include gaseous hydrogen and carbon dioxide—approximately 30 m³ per 100 kg of sucrose, of which about 70% is CO₂—as well as dense acetone-butanol stillage. These gases are captured and utilized for the synthesis of ammonia and methanol. Stillage is a valuable product containing significant amounts of riboflavin; to obtain feed-grade vitamin B₂, the stillage is concentrated tenfold in multi-stage vacuum evaporators and dried in spray dryers. This yields a dry concentrate containing 60–100 µg/g of riboflavin. The content of dry matter, predominantly nitrogenous compounds, in the stillage ranges from 3–5%. Previously, dried stillage was used directly for livestock feeding. Nowadays, it is employed for cultivating fodder yeast.

Processes for obtaining organic solvents are constantly being intensified and improved. For instance, fermentation of the producer Organism in a fluidized-bed bioreactor with continuous extraction of butanol from a multi-phase medium has been proposed. The process is further intensified by immobilizing producer cells on the Glass surface of the bioreactor, and methods for synthesizing organic solvents based on synthesis gas are currently under development, among others.

Prospects for hydrogen production. The production of hydrogen as a fuel largely remains at the exploratory research stage. Nevertheless, The problem of harnessing this form of energy is highly significant and relies on several prerequisites:

♦ the photolysis substrate (water) is virtually inexhaustible;

♦ the product (hydrogen) can be stored, is non-polluting to the atmosphere, and possesses a high calorific value (29 kcal/g compared to 3.5 kcal/g for Hydrocarbons);

♦ the process is renewable, since the substrate (water) is regenerated after hydrogen extraction;

♦ hydrogen production occurs at normal temperatures without The formation of toxic intermediates.

Chemical and electrochemical methods for hydrogen production are uneconomical. Therefore, utilizing microorganisms capable of evolving hydrogen is highly promising. Such an ability is possessed by aerobic and anaerobic chemotrophic bacteria, Purple and green phototrophic bacteria, cyanobacteria, various Algae, and certain Protozoa. The enzymes required for hydrogen production are Hydrogenase and Nitrogenase.

Hydrogenase, an enzyme containing Fe-S centers, catalyzes the following reaction:

However, hydrogenase is unstable, and its activity is rapidly inhibited by hydrogen (the reaction product) and oxygen. To increase hydrogen yield, it is necessary to identify hydrogenases that are less sensitive to oxygen. One such enzyme is the hydrogenase from *Alcaligenes* bacteria.

Three conditions must be met during hydrogen production: 1) there must be a tight coupling between the electron carrier and the hydrogen-producing catalyst; 2) an efficient interaction must be established between the chloroplast membranes and the electron carrier; 3) the electron carrier must not be easily oxidized. The stability of hydrogenase can be enhanced through its immobilization, which also reduces the enzyme's sensitivity to oxygen.

Numerous variants of model systems capable of catalyzing hydrogen generation from water using light energy are known. As a rule, all of them contain METABOLISM/14.html">Chloroplasts or chlorophyll isolated from them, as well as reduced nicotinamide NUCLEOTIDES. When such systems evolve oxygen alongside hydrogen, the process is referred to as water photolysis. An example is the system: chloroplast – ferredoxin – hydrogenase. Here, ferredoxin acts as an intermediate electron carrier from the photosynthetic chain of chloroplasts to hydrogenase.

Hydrogen is also produced using whole microbial cells, whose stability increases upon immobilization. Excellent hydrogen producers are purple phototrophic bacteria, such as *Rhodopseudomonas* sp., which, when immobilized in agarose gel, yield up to 180 µmol of hydrogen per hour per 1 mg of bacteriochlorophyll.

Another enzyme that catalyzes hydrogen evolution is nitrogenase. In all microorganisms, nitrogenase consists of two components: a Mo-Fe protein and an Fe protein. The primary function of nitrogenase is the reduction of molecular nitrogen:

In the absence of the primary substrate (N₂), nitrogenase catalyzes the energy-dependent reduction of H⁺ to form H₂. Switching the enzyme from one operating mode to another presents a technological challenge: it is necessary to obtain microbial strains with a nitrogenase that does not assimilate nitrogen.

Combining the production of hydrogen with other valuable products is also feasible. For instance, Representatives of the genus *Clostridium* yield organic solvents while simultaneously possessing active hydrogenase. If no outlet for the evolved hydrogen is provided in a bioreactor containing *Clostridium saccharoperbutylacetonicum*, H₂ synthesis is inhibited, while the production of butanol, acetone, and ethanol is enhanced.

Alternative energy conversion mechanisms can also be utilized. For example, the halophilic bacterium *Halobacterium halobium* is capable of utilizing light energy captured by Bacteriorhodopsin in its cell membranes. This pigment generates a proton concentration gradient—a proton Electrochemical Potential—which ultimately drives energy production.

Thus, various systems for biological hydrogen production have been proposed. However, the Practical significance of this biotechnological process has yet to be fully evaluated.

Enhancing the efficiency of Photosynthetic Systems. The theoretical efficiency of photosynthesis—namely, the conversion rate of light energy into the chemical energy of Organic compounds—is 15%. However, in practice, even the most productive cultivated crops store no more than 1.5–2% of light energy. To improve photosynthetic efficiency in crops, the following approaches are employed:

♦ the balanced Application of phytohormones and the transplantation of regulatory genes;

♦ increasing plant growth rates through the optimization of water and mineral Nutrition;

♦ increasing the number of chloroplasts per cell unit per leaf area;

♦ raising the solar energy conversion rate to 4–5% by expanding leaf surface area and accelerating early leaf development. This is achieved, in part, through The Use of effective microorganism preparations (EM technology);

♦ establishing an optimal ratio between functioning chlorophyll Reaction Centers and intermediate electron carriers, such as Cytochromes;

♦ increasing The rate of electron transfer between Photosystems 1 and 2, as well as the coupling efficiency between Electron Transport and ATP synthesis.

Biofuel cells. These devices are capable of converting the chemical energy of a substrate into electrical energy. For instance, fuel cell models have been developed based on The oxidation of methanol to formic acid using Alcohol dehydrogenase, formic acid to CO₂ via formate dehydrogenase, and glucose to gluconic acid using glucose oxidase. Whole cells—such as E. coli, B. subtilis, Ps. aeruginosa, and others—are also utilized in glucose oxidation reactions. The oxidation of the substrate occurs at the electrode (anode), with a biocatalyst acting as a mediator between the substrate and the anode. There are two pathways for electron transfer to the electrode: mediated electron transfer and direct electron transfer.

Enzyme electrodes are utilized not only in biofuel cells. They serve as a core component of biological sensors (biosensors), which are widely applied in the chemical industry, medicine, biotechnological process control, analytical Applications, and beyond. Typically, these systems employ biocatalysts immobilized On the surface of a membrane electrode. For example, the immobilization of penicillinase on a conventional pH electrode yields a sensitive biosensor capable of detecting penicillin concentration. Biosensors have already been developed for the rapid determination of Blood glucose concentrations in patients, which is crucial for the Diagnosis of Diabetes Mellitus.

Biogeotechnology. Globally, the demand for raw materials and environmental conservation challenges associated with the growth of the metallurgical and mining industries are continuously increasing. However, the cost of conventional metal extraction methods makes it economically unviable to recover metals from low-grade ores or secondary waste.

Applications of biotechnology include ore mining, beneficiation, and Processing; the Separation and concentration of metals from wastewater as secondary raw materials; and the extraction of residual oil from depleting reservoirs—all of which fall within The Scope of biogeotechnology. Certain microorganisms can catalyze specific redox reactions, such as the oxidation of iron and manganese in water, the oxidation of sulfur-containing compounds, and the Oxidation and reduction of nitrogen-containing compounds. Aerobic bacteria can mobilize iron, copper, and sulfates. Bacteria, fungi, and algae are capable of accumulating and thereby concentrating copper, uranium, silver, and other metals. Established methods include the leaching of vanadium, in situ leaching of uranium in mines, and gold leaching, with large-scale biotechnological processes planned for the recovery of nickel, zinc, tin, molybdenum, and other metals from low-grade ores. Biological methods for treating cyanide-containing wastewater are well established, and technologies for recovering heavy metals from industrial effluents using algae have also been developed.

Various bacteria possess The ability to convert metals into soluble compounds (bioleaching of metals from ores). For instance, Thiobacillus ferrooxydans leaches iron, copper, zinc, and uranium by oxidizing them with sulfuric acid, which is produced by the bacterium from sulfides. Chromobacterium violaceum dissolves gold According to the scheme Au → Au(CN)₂

The technology for such processes is quite straightforward: to extract residual metals from mining "waste rock," the material is sprayed with water, and the resulting runoff—containing soluble microbial metabolic products (CuS_4, UO;²⁺₂, etc.)—is collected.

Microbial Surfactants are utilized as emulsifiers and wetting agents to enhance oil recovery and facilitate pipeline transportation. Traditional oil recovery extracts only about 30% of reservoir reserves. The use of surfactant-producing microorganisms makes the operation of depleted wells economically viable. Microbial preparations have proven effective in remediating oil spills on land and at sea, as well as in the degradation and emulsification of heavy crude fractions. For example, xanthan, an extracellular polysaccharide produced by the bacterium Xanthomonas campestris, is used to extract petroleum from depleting reservoirs. Demulsifying bacteria, such as Nocardia sp. and Rhodococcus rhodochrous, separate the aqueous and oil phases, which is applied in oil concentration and wastewater remediation. Furthermore, genetically engineered strains (such as pseudomonads) capable of utilizing crude oil allow, firstly, for biomass production using untreated petroleum and, secondly, for the Prevention of environmental oil pollution by clearing oil slicks from The surface of seas and oceans.

Microorganisms can also be employed to remove methane from coal seams in underground mines.