The technology of hydraulic fracturing, also known as fracking, is currently undergoing intensive development, allowing for a significant increase in oil production volumes. The fluid employed in this process comprises a plethora of organic components, including polysaccharide gel-forming compounds such as xanthan gum, guar gum, cellulose, starch, and their chemical modifications and derivatives. The waste fluids generated during this process pose a threat to the environment, necessitating the exploration of effective disposal methods. One promising approach is the utilization of dark fermentation, which enables the biodegradation of polysaccharides present in waste fluids into a valuable product-biohydrogen. This review delves into the fundamental principles of dark fermentation and elucidates the properties of commonly used gel-forming polysaccharides critical for the hydraulic fracturing process. Additionally, it explores the microorganisms capable of decomposing these polysaccharides, resulting in the production of biohydrogen. Particular attention is paid to exploring potential methods for improving the process of exploiting recalcitrant polysaccharides present in waste fluid, particularly through pre-treatment techniques.

Acidogenesis and acetogenesis are the key steps in the formation of H2 during the anaerobic decomposition of organic matter. Various groups of H2-producing microorganisms, as well as aspects of their practical use for obtaining biohydrogen as an environmentally friendly fuel, have been studied. In terms of H2 production, the most efficient acidogenic bacteria belong to the genera Clostridium, Enterobacter and Escherichia. They use different fermentation pathways, resulting in different H2 yields and soluble metabolite products. Within the framework of the chapter, we summarized our current knowledge of the diversity of H2-producing thermophilic, hyperthermophilic, mesophilic, and psychrophilic microorganisms and compared their metabolic pathways and cultivation conditions. Mono-, co-, and mixed cultures have different productivity depending on the environmental factors. In the case of co-cultivation, it is important to pay attention to symbiotic relationships of microorganisms, changes in microbial communities, and the development of predominant groups. Ways to enhance H2 production by inhibiting hydrogen consumers and the use of bioaugmentation are discussed. Application of different methods of cultivation and their modifications in order to increase the yield of biohydrogen and the degree of decomposition of organic waste are included.

A moderately thermophilic Gram-positive chemo-organotrophic bacterium, strain SP2, was isolated by serial dilutions with crotonate and yeast extract as substrates from a butyrate-degrading methanogenic enrichment obtained from thermophilically digested sludge of the Kuryanoskaya wastewater treatment plant (Moscow, Russia). Cells of strain SP2 are spore-forming rods, sometimes occurring in short chains. The bacterium is an obligate anaerobe that grows at temperatures from 20 to 70 °C (55–60 °C optimum) within a pH range of 3.5–8 (7.5 optimum) and with NaCl concentrations of up to 2.5%. The strain utilized yeast extract and simple sugars as carbon and energy sources. Thiosulfate was used as an electron acceptor when grown on sucrose, resulting in the formation of hydrogen sulfide and the accumulation of elemental sulfur globules inside the cells. Strain SP2 is phylogenetically related to Biomaibacter acetigenes strain SK-G1T as revealed by comparison with the 16S rRNA gene (99.9% identity) and genome (ANI 99%, dDDH 90%) of both strains. It is interesting that strain SP2 was capable of syntrophic conversion of glycerol and lactate when co-cultivated with hydrogenotrophic methanogen, which was not previously shown for the SK-G1T type of strain. The isolation and in-depth study of new facultatively syntrophic microorganisms is important for wastewater treatment ecotechnologies due to their ability to switch to an alternative source of carbon and energy and therefore greater resistance to changing environmental conditions in bioreactors.

The hydrogen-producing bacteria SP4 and SP6 were isolated from the compost and identified by 16S rRNA gene sequencing as Clostridium butyricum and Clostridium beijerinckii, respectively. A comparative study on the biohydrogen-producing activity of the isolated strains was carried out using mono-, di-and tri-saccharides belonging to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose). To assess the biotechnological significance, real wastewater rich in sugars (cheese whey, confectionery wastewater, sugar beet processing wastewater) was also used as a substrate. C. butyricum SP4 fermented sugars with a yield of 0.93-1.52 mol H2/mol hexose (pentose); the maximum yield was obtained from fructose, the minimum from raffinose and cellobiose. The most preferred substrate for C. beijerinckii SP6 was sucrose with a yield of 1.76 mol H2/mol hexose, while cellobiose yielded only 0.64 mol H2/mol hexose. Overall, the efficiency of converting wastewater to H2 by C. butyricum SP4 was also slightly lower (66-93 ml H2/g chemical oxygen demand (COD)) than that of C. beijerinckii SP6 (76-103 ml H2/g COD). Even though the main soluble metabolite products (SMPs) for both isolates were acetate and butyrate, C. butyricum SP4 also produced a significant amount of ethanol (up to 21.5% of SMPs) and formate (up to 32.5% of SMPs), and C. beijerinckii SP6 lactate (up to 25% of SMPs). A distinctive feature of C. beijerinckii SP6 was a significantly lower (almost 2 times) yield of SMPs, while C. butyricum SP4 had a higher rate of H2 production according to the results obtained from the kinetic study using the modified Gompertz equation and the first order equation. Analysis of Spearman's rank correlation coefficients revealed a statistically significant relationship between the kinetic parameters of H2 production and the concentration of butyrate and the final pH of the medium for C. butyricum SP4, and with the concentration of ethanol for C. beijerinckii SP6. These findings provide valuable information on the metabolic capabilities of the most studied hydrogen producing representatives of the Clostridium genus for their use in optimizing the technology for biohydrogen production by dark fermentation of various organic wastes. & COPY; 2023 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

The ability of microorganisms to carry out interspecies electron transfer during the degradation of organic substances under anaerobic conditions opens up new possibilities for a controlled increase in the efficiency of the methanogenic decomposition of organic waste. This review presents the main principles of the effects of a direct electric current on the anaerobic degradation of organic substances, process parameters, changes in the composition of the microbial community, and factors affecting the optimization of the hybrid systems comprising microbial electrolysis cell (MEC) and anaerobic digester (AD), i.e., the performance of the MEC-AD system. The research in this field has been analyzed for the subsequent application of electromethanogenesis, which represents a new energy-efficient biotechnology for anaerobic wastewater treatment and organic waste digestion.

The hydrogen-producing bacterium SP-H2 was isolated from a thermophilic acidogenic reactor inoculated with municipal sewage sludge and processing a carbohydrate-rich simulated food waste. Based on the 16S rRNA gene sequence, the bacterium was identified as Thermoanaerobacterium thermosaccharolyticum. The maximum growth rate was observed at 55-60 degrees C and pH 7.5. The H-2-producing activity of the bacterium was studied using mono-, di-and tri-saccharides related to both hexoses (maltose, glucose, mannose, fructose, lactose, galactose, sucrose, raffinose, cellobiose) and pentoses (xylose and arabinose), as well as using real wastewaters (cheese whey, confectionery wastewater, sugar-beet processing wastewater). The highest H-2 yield was observed during dark fermentation (DF) of maltose (1.91 mol H-2/mol hexose or 77.8mmol H-2/L). Themaximum H-2 production rate was observed during DF of xylose (13.3ml H-2/g COD/h) and cellobiose (2.47mmol H-2/L/h). Themain soluble metabolite products were acetate, ethanol and butyrate. The acetate concentration had a statistically significant positive correlation with the H-2 content in biogas and the specificH(2) yield. Based on the results of the correlation analysis, it was tentatively assumed that in the formic acid (mixed-acid) type fermentation, the rate of H-2 production was higher than in the butyric acid type fermentation. With regard to real wastewater, cheese whey and confectionery wastewater were distinguished by a higher H-2 yield (152 ml H-2/g COD) and H-2 production rate (0.57mmol H-2/L/h), respectively. The highest concentrations of confectionery wastewater and cheese whey, at which the DF process took place, were 5915 and 7311mg COD/L, respectively. At the same time, SP-H-2 dominated in the microbial community, despite the presence of indigenous microorganisms in wastewater. Thus, T. thermosaccharolyticum SP-H2 is a promising strain for DF of carbohydrate-rich unsterile wastewater under thermophilic conditions. (C) 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

The composting of a mixture of excess sludge from biological treatment facilities for wastewater from dairy production and vegetable and wood waste in a ratio of 3 : 3 : 4 (by volume) with a high amount of dry substance (27.5%) at extremely high temperatures (75–82°C) made it possible to isolate active mesophilic and thermophilic methanogenic consortia. The dynamics of methane formation upon the liquid-phase fermentation of the studied mixture was studied. Accelerated methanogenesis and the highest methane yield (31.7 ± 2.9 mM L –1 ) were observed with culturing at 55°C. Analysis of the volatile fatty-acid production with liquid-phase fermentation showed that their content was low (<10 mM L –1 ). This amount could not inhibit methanogenesis, regardless of the applied substrates and temperature conditions. The 16S rRNA sequencing of the obtained consortia showed the presence of methanogenic archaea Methanosarcina thermophila , Methanothermobacter thermoautotrophicus , Candidatus ‘Methanogranum caenicola’, and Methanofollis ethanolicus. The consortia also included archaea that represent putative new taxa that are phylogenetically close to Candidatus ‘Methanoplasma termitum’ and Methanomassiliicoccus luminyensis . Of the mentioned methanogenic archaea, the latter four were found in composting organic waste for the first time. The obtained consortia can conduct active methanogenesis and can be applied as an inoculum in the anaerobic treatment of organic wastes, including composting wastes, in order to intensify and increase the biogas yield.

The aim of this work was a comparative study of biohydrogen production from cheese whey and confectionary wastewater by a newly isolated thermophilic microbial strain Thermoanaerobacterium thermosaccharolyticum SP-H2. Experimental results showed that the fermentative hydrogen was successfully produced with the highest hydrogen yield of 3.9 mL H2/mL cheese whey or 80 mL H2/g chemical oxygen demand. The profile of soluble metabolite products showed that hydrogen generation by a new isolate was mainly acetate-type fermentation in the case of confectionary wastewater and mixed ethanol-acetate-lactate type fermentation in the case of cheese whey. The more optimal metabolic pathway of confectionary wastewater fermentation was confirmed by the better kinetic characteristics according to the Gompertz model.

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 degrees C. Peptone and sunflower oil were characterized by the lowest production of H2, namely 5.0 and 2.3 ml H2/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H2/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H2/g COD or 143.4 ml H2/g carbohydrates; from pig feed - 32.1 ml H2/g COD or 91.6 ml H2/g carbohydrates; and from sewage sludge 9.3 ml H2/g COD or 98.0 ml H2/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H2, its content in biogas, as well as the ratio of H2/ soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate. (c) 2021 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

A methanogenic enrichment growing on a medium with methanol was obtained from a petroleum reservoir (Republic of Azerbaijan) and stored for 33 years without transfers to fresh medium. High-throughput sequencing of the V4 region of the 16S rRNA gene revealed members of the genera Desulfovibrio, Soehngenia, Thermovirga, Petrimonas, Methanosarcina, and Methanomethylovorans. A novel gram-positive, rod-shaped, anaerobic fermentative bacterium, strain 1933PT, was isolated from this enrichment and characterized. The strain grew at 13–55 °C (optimum 35 °C), with 0–3.0% (w/v) NaCl (optimum 0–2.0%) and in the pH range of 6.7–8.0 (optimum pH 7.0). The 16S rRNA gene sequence similarity, the average nucleotide identity (ANI) and in silico DNA–DNA hybridization (dDDH) values between strain 1933PT and the type strain of the most closely related species Soehngenia saccharolytica DSM 12858T were 98.5%, 70.5%, and 22.6%, respectively, and were below the threshold accepted for species demarcation. Genome-based phylogenomic analysis and physiological and biochemical characterization of the strain 1933PT (VKM B-3382T = KCTC 15984T) confirmed its affiliation to a novel species of the genus Soehngenia, for which the name Soehngenia longivitae sp. nov. is proposed. Genome analysis suggests that the new strain has potential in the degradation of proteinaceous components.

— Anaerobic digestion of municipal and other organic waste is a microbial process for conversion of complex organic substances to biogas (a renewable energy source) comprising a mixture of methane and CO 2 , and a stabilized sludge, which may be used as an organic fertilizer. Diverse groups of the methanogenic microbial community degrade complex organic compounds into simple fermentation products such as hydrogen, formate, acetate, short-chained volatile fatty acids, ethanol, etc. These low-molecular mass products act as the substrates and carriers involved in biogas production by syntrophic bacteria and methanogenic archaea at the methanogenesis stage, the last stage of the anaerobic process. The present review discusses syntrophic interactions between the microorganisms involved in anaerobic degradation of organic substances, as well as two types of interspecies electron transfer (IET): indirect IET (IIET, Indirect Interspecies Electron Transfer) and direct IET (DIET, Direct Interspecies Electron Transfer). DIET-based syntrophic interactions between microorganisms may be stimulated by adding conductive materials into anaerobic digesters, which may have the potential for practical applications.

Syngas is a substrate for the anaerobic bioproduction of fuels and valuable chemicals. In this study, anaerobic sludge was used for microbial enrichments with synthetic syngas and acetate as main substrates. The objectives of this study were to identify microbial networks (in enrichment cultures) for the conversion of syngas to added-value products, and to isolate robust, non-fastidious carboxydotrophs. Enrichment cultures produced methane and propionate, this last one an unusual product from syngas fermentation. A bacterium closely related to Acetobacterium wieringae was identified as most prevalent (87% relative abundance) in the enrichments. Methanospirillum sp. and propionate-producing bacteria clustering within the genera Anaerotignum and Pelobacter were also found. Further on, strain JM, was isolated and was found to be 99% identical (16S rRNA gene) to A. wieringae DSM 1911T. Digital DNA-DNA hybridization (dDDH) value between the genomes of strain JM and A. wieringae was 77.1%, indicating that strain JM is a new strain of A. wieringae. Strain JM can grow on carbon monoxide (100% CO, total pressure 170 kPa) without yeast extract or formate, producing mainly acetate. Remarkably, conversion of CO by strain JM showed shorter lag phase than in cultures of A. wieringae DSM 1911T, and about four times higher amount of CO was consumed in 7 days. Genome analysis suggests that strain JM uses the Wood-Ljungdahl pathway for the conversion of one carbon compounds (CO, formate, CO2/H2). Genes encoding bifurcational enzyme complexes with similarity to the bifurcational formate dehydrogenase (Fdh) of Clostridium autoethanogenum are present, and possibly relate to the higher tolerance to CO of strain JM compared to other Acetobacterium species. A. wieringae DSM 1911T grew on CO in medium containing 1 mM formate.

A new species of the genus Trichococcus, strain Art1T, was isolated from a psychrotolerant syntrophic propionate-oxidizing consortium, obtained before from a low-temperature EGSB reactor fed with a mixture of VFAs (acetate, propionate and butyrate). The 16S rRNA gene sequence of strain Art1T was highly similar to those of other Trichococcus species (99.7-99.9 %) but digital DNA-DNA hybridization values were lower than those recommended for the delineation of a novel species, indicating that strain Art1T is a novel species of the genus Trichococcus. Cells of strain Art1T are non-motile cocci with a diameter of 0.5-2.0 µm and were observed singularly, in pairs, short chains and irregular conglomerates. Cells of Art1T stained Gram-positive and produced extracellular polymeric substances . Growth was optimal at pH 6-7.5 and cells could grow in a temperature range of from -2 to 30 °C (optimum 25-30 °C). Strain Art1T can degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate and ethanol. The genomic DNA G+C content of strain Art1T is 46.7 %. The major components of the cellular fatty acids are C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c. Based on genomic and physiological characteristics of strain Art1T, a new species of the genus Trichococcus, Trichococcusshcherbakoviae, is proposed. The type strain of Trichococcusshcherbakoviae is Art1T (=DSM 107162T = VKM B-3260T).

Soehn.ge'ni.a. N.L. fem. n. Soehngenia named in honor of Nicolas L. Soehngen, founder and first head (1911–1937) of the Laboratory of Microbiology of Wageningen University, The Netherlands, where this strain was isolated and described. Firmicutes / “Clostridia” / Clostridiales / incertae Sedis ‐ Family I / Soehngenia Rod‐shaped cells , 0.5–0.7 × 2–11 µm (Figure 1). Gram‐stain‐positive . In the early exponential phase of growth, cells are slightly motile by means of peritrichous flagella (Figure 2); older cells lose their motility. Colonies are rhizoid, resemble a snowflake, dark, and creamy. Rare terminal and subterminal spore formation. Mesophilic . Anaerobic, but aerotolerant ; it can grow with 50% air in the gas phase. Fixes molecular nitrogen. Chemo‐organotrophic . Saccharolytic and weakly proteolytic. Growth substrates are a wide range of carbohydrates and some other carbon sources, including yeast extract, cysteine, and serine. Isolated from a laboratory anaerobic digester sludge with benzaldehyde as the substrate. DNA G + C content ( mol %): 43.0 ( T m ). Type species : Soehngenia saccharolytica Parshina, Kleerebezem, Sanz, Lettinga, Nozhevnikova, Kostrikina, Lysenko and Stams 2003, 1797 VP .

Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain Groll(T) together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes.

Desulfotomaculum nigrificans and D. carboxydivorans are moderately thermophilic members of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. They are phylogenetically very closely related and belong to ‘subgroup a’ of the Desulfotomaculum cluster 1. D. nigrificans and D. carboxydivorans have a similar growth substrate spectrum; they can grow with glucose and fructose as electron donors in the presence of sulfate. Additionally, both species are able to ferment fructose, although fermentation of glucose is only reported for D. carboxydivorans. D. nigrificans is able to grow with 20% carbon monoxide (CO) coupled to sulfate reduction, while D. carboxydivorans can grow at 100% CO with and without sulfate. Hydrogen is produced during growth with CO by D. carboxydivorans. Here we present a summary of the features of D. nigrificans and D. carboxydivorans together with the description of the complete genome sequencing and annotation of both strains. Moreover, we compared the genomes of both strains to reveal their differences. This comparison led us to propose a reclassification of D. carboxydivorans as a later heterotypic synonym of D. nigrificans.