IntroductionAt the present stage of its economic development, Russia is going through a crisis of traditional technologies, especially in the fields of ecology and energy. The modern agenda involves new technological solutions for food processing, organic waste recycling, pollution reduction, and alternative energy.Chinese miscanthus (Miscanthus sinensis) (Fig. 1) is a perennial herb in the grass family Poaceae, which can serve as a renewable raw material for biodiesel. One hectare can yield up to 30 tons of crops during 30 years. Plant biomass cultivation improves the environmental situation and prevents soil erosion, not to mention that biodiesel is carbon neutral [1, 2].However, miscanthus is difficult to process because its acid hydrolysates are chemically unstable and tend to accumulate harmful substances, e.g. volatile acids, lignohumic substances, formaldehyde, furfural, oxymethylfurfural, etc. These compounds appear as a result of the combined action of chemical agents and such physical factors as temperature and pressure [3–5].Enzymatic hydrolysis is known to occur in milder conditions than acid hydrolysis. As a rule, enzymatic hydrolysis requires 4.0–6.5 units of active acidity, while the temperature range usually depends on the type of microorganisms and varies from 30 to 60°C. This technological mode converts sugars into the target product and forms no harmful by-products [6–9].Bioethanol production from lignocelluloses requires simultaneous fermentation and saccharification – for the following reasons:cellulose hydrolysis to simple sugars is a lengthy process that can take from 48 to 144 h. Combining the two steps into can significantly reduce time costs;the one-step method possesses a higher microbiological stability and efficiency [14, 15].However, the one-step method is not without flaws. For instance, enzyme catalysis systems have different optimal parameters. Those that catalyze saccharification operate at 40–60°C, while those that catalyze glucose-to-bioethanol reaction operate at 26–28°C [16, 17].Кригер О. В. [и др.] Техника и технология пищевых производств. 2021. Т. 51. № 2 С. 387–394   Figure 1. Chinese miscanthus (Miscanthus sinensis) Nevertheless, biotechnological methods make it possible to obtain strains of microorganisms with an optimal temperature mode. They are applicable both for enzyme preparations that produce simple sugars and for enzyme complexes that catalyze the glucose-to-bioethanol reaction. However, genetically engineered microorganisms have several disadvantages, such as:higher mutation rates;sensitive cultivation conditions, i.e. nutrient media, aseptic conditions, etc.;eventual degradation of ethanol biosynthesis.As a result, biotechnological industries still rely on traditional strains of yeasts and bacteria that are capable of producing simple sugars and target products.Saccharomyces cerevisiae are one of the main producers of bioethanol. However, saccharomycetes do not ferment pentoses that take up 40% of total sugars in the biomass of herbaceous plants, which limits their industrial applicability for lignocellulosic biomass conversion.Thermotolerant yeasts are an alternative to S. cerevisiae in ethanol production. Thermotolerant yeasts consume glucose, xylose, cellobiose, and arabinose. Glucose, fructose, and mannose substrates have better indicators of biomass production. Clostridium, Geobacillus, and Thermoanaerobacter can process a wide range of compounds, including D-glucose, D-xylose, and L-arabinose, at 55–70°C, which makes them especially advantageous for bioethanol production.The present study confirmed the available foreign and domestic research results in that saccharification followed by fermentation of miscanthus cellulose can increase bioethanol processing time, thus increasing the accumulation of by-products and microbial contamination. In this regard, simultaneous saccharification and fermentation of the initial substrate by an effective microbial consortium can improve the bioethanol production from lignocellulosic raw materials.The research objective was to create such a microbial consortium, as well as to optimize bioethanol production by developing a one-step fermentolysis of technical cellulose obtained by delignification of miscanthus. Study objects and methodsThe research featured Chinese miscanthus (Miscanthus sinensis) of the grass family Poaceae harvested in the Northwestern Federal District of the Russian Federation in 2019. It is a perennial herb that grows as tall as 0.8–2 m and yields 10.1–15.4 t/ha. Its leaf blades are linear, hard, and rough, with a prominent mid-rib, and can reach up to 1.5 cm in width, while its ne-flowered spikelets are arranged into loose panicles with a short axis and can be up to 0.7 cm long.Below are the characteristics of technical cellulose obtained from Miscanthus sinensis using hydrotropic delignification with pertrifluoroacetic acid:mass fraction of α-cellulose = 81.56–82.68%;dynamic viscosity = 22.78–24.33 mPa·s;average polymerization degree = 756.2–977.5;– brightness = 72.6–82.7%;lignin content ≤ 0.2%.The preparation of the plant mass of miscanthus before the delignification process was carried out according to the standard procedure described in related scientific and technical sources.Table 1 introduces the types and strains of microorganisms that were used to create the microbial consortium for the subsequent cellulose bioconversion: The ethanol content in the culture liquid after cultivation was determined using an Agilent 7890B gas chromatograph with a flame ionization detector. The final chromatography time was 10 min; split flow – 100:1; injected sample volume – 1 μl; helium served as the carrier gas; 2 mL of the resulting alcohol sample was extracted with diethyl ether. The sample was then poured into a 15 mL falcon together with 2 mL of diethyl ether and stirred in an orbital shaker for 30 min. After that, the samples were centrifuged at 3900 rpm for 10 min. The upper organic layer was removed into a flask to evaporate diethyl ether. Then, 1 mL of ethyl acetate was added to the concentrated sample, and the resulting solution was placed into a vial and injected into the gaschromatographer.The biocompatibility of the strains was tested by direct co-cultivation in a solid nutrient medium as described by N.A. Glushanova. The culture was grown in a liquid nutrient medium until its optical density reached 0.10–0.11. After that, it was applied onto a solid nutrient medium with a 3 mm inoculating loop. After the drop had been absorbed, a drop of another test culture was applied to the surface of the same medium. When spreading, it covered a half of the first drop. The Petri dishes were incubated for 24 h at 27°C. In the overlapping area, theKriger O.V. et al. Food Processing: Techniques and Technology, 2021, vol. 51, no. 2, pp. 387–394 Table 1. Microorganisms used for one-step bioethanol production from technical cellulose obtained from Chinese miscanthus MicroorganismMediumCultural and morpholo- gical characteristicsPhysiological and biochemical propertiesPichia stipites Y7124Intestinal tractascomycete yeastfacultative anaerobic, capable of fermenting xylose, t = 28–32°С, pH = 4.0–4.5Pachysolen tannophilusY-3269decaying woodyeastfacultative anaerobic, t = 28–32°С,pH = 2.0–2.5; ferments glucose and xyloseCandida shehataeNCL3501decaying woodyeastferments both hexose and pentose sugars; t =28–30°С, pH = 3.5–4.0Kluyveromyces marxianus Y-4290Cultured yoghurtround and oval cells of 4-8 µmferments D-glucose, D-galactose, sucrose, raffinose; t = 25–50°C; pH = 3.0–7.0Clostridium thermocellum B-10909plants, cow and horse manuregram-positive spore- forming bacillusanaerobic, thermophilic, t = 55–60°С,pH = 6.0–8.0; ferments cellulose, glucose, and xyloseClostridium thermohydrosuluricumplants, cow and horse manuregram-positive spore- forming bacillusanaerobic, thermophilic, t = 55–60°C, pH = 4.7–8.0; ferments glucose, xylose, and arabinoseThermoanaerobacter ethanolicus JW 200Yellowstone hot springsnon-spore-forming bacteriathermophilic, anaerobic; ferments sugars, including xylose and arabinose; t = 37–77°C, pH = 4.4–9.9Geobacillus stearothermophilus В-1169bottom sediment of thermal springs,Northern Baikal regiongram-positive bacillusaerobic, thermophilic; t = 60–65°C, pH = 6.5–7.5; converts lignocellulosic biomass into bioethanolBacillus stratosphericus В-11678bottom sediments of Lake Solenoe, Novosibirsk regiongram-positive bacillusaerobic, mesophilic; t = 30–37°C, pH = 6.5–7.5; converts lignocellulosic biomass into bioethanolZymomonas mobilis 113palm winegram-negative, non- spore-forming, polar flagellate bacteriumfacultative anaerobic, ferments glucose, sucrose, xylose; t = 30–37°C, pH = 4.0–6.5 cultures competed with each other. Two drops of one and the same culture were used as control, according to the technique described above. Inhibition of one of the strains was interpreted as antagonism; if one of the cultures surfaced, regardless of the sequence of application, it was interpreted as weak antagonism; fusion of the drops or increased growth of both strains indicated biocompatible cultures.The results were processed using regression analysis. The dependence diagrams were compiled after the obtained data had been processed using the least squares methodand described in Microsoft Excel and MatLAB 6.5. The studies were carried out in 5-fold repetition. The confidence level was 0.95; 0.99 (P ≤ 0.05; P ≤ 0.01). Results and discussionThe research objective was to develop an effective microbial consortium and select the optimal producers for one-step bioethanol production from cellulose of Chinese miscanthus. The technical cellulose obtained by hydrotropic delignification was fermented with all the microbial strains. Table 2. Mass fraction of ethanol in the test samples (P ≤ 0.05) MicroorganismBioethanol yield, %Fermentation time, hFermentation temperature, °СPichia stipites Y71241.20 ± 0.0112.0 ± 0.530 ± 1Pachysolen tannophilus Y-32690.07 ± 0.0112.0 ± 0.530 ± 1Candida shehatae NCL35010.45 ± 0.0112.0 ± 0.530 ± 1Kluyveromyces marxianus Y-42901.80 ± 0.0112.0 ± 0.535 ± 1Clostridium thermocellum B-109090.51 ± 0.0112.0 ± 0.560 ± 1Clostridium thermohydrosuluricum0.46 ± 0.0112.0 ± 0.560 ± 1Thermoanaerobacter ethanolicus JW 2002.03 ± 0.0512.0 ± 0.560 ± 1Geobacillus stearothermophilus В-11692.00 ± 0.0512.0 ± 0.560 ± 1Bacillus stratosphericus В-116780.56 ± 0.0512.0 ± 0.537 ± 1Zymomonas mobilis 1130.31 ± 0.0112.0 ± 0.530 ± 1Кригер О. В. [и др.] Техника и технология пищевых производств. 2021. Т. 51. № 2 С. 387–394 Table 4. Composition of nutrient media ComponentCulture medium number depending on the content of the components, g/l123456Yeast extract0.30.40.5–––Glucose1.05.010.020.025.030.0Sodium chloride–––0.51.01.5Peptone1.05.010.010.05.05.0К2HPO41.01.52.01.51.01.5(NH4)2HPO41.01.51.51.01.51.5KH2P04–––0.51.01.5MgSO40.50.51.00.50.51.0CaC03–––10.05.010.0рН of the medium4.55.26.07.06.87.0 During the enzymatic hydrolysis, the substrate sample in an acetate buffer solution (0.1 g/mL, pH 5.0) was inoculated with a suspension of microorganisms with optical density = 0.60–0.61 in the amount of 5% of the total sample volume. The enzymatic hydrolysis was carried out in an incubator shaker at 100 rpm for 12 h at the optimum temperature for each of the producers. Table 2 describes the content of ethanol in the samples. Table 2 shows that all the microorganisms proved quite active in the cellulose fermentation. The best bioethanol yield belonged to the thermophilic bacteria Geobacillus stearothermophilus B-1169 (2.0%) and Thermoanaerobacter ethanolicus JW (2.03%) and ethermotolerant yeast Kluyveromyces marxianus Y-4290(1.80%) at the optimal culture temperature.Table 3 demonstrates the biocompatibility of the strains.The strains were subjected to cultural, morphological, hysiological, and biochemical analyses. They also underwent an enzymatic hydrolysis of technical cellulose obtained by hydrotropic delignification and a biocompatibility assessment. The experiments resulted in the following bioethanol-producing consortia:Consortium I: facultative anaerobic mesophilic yeasts Pichia stipites Y7124, Candida shehatae NCL3501, and Kluyveromyces marxianus Y-4290 and bacteria Zymomonas mobilis 113 in a ratio of 1:1:1:1;Consortium II: thermophilic anaerobic bacteriaClostridium thermocellum B-10909, Clostridium thermo- Table 3. Biocompatibility of bioethanol producers (P ≤ 0.05)  MicroorganismMicroorganismPichia stipites Y7124Pachysolen tannophilus Y-3269Candida shehataeNCL3501Kluyveromyces Marxianus Y-4290Clostridium thermocellum B-10909Clostridium thermo- hydrosuluricumThermoanaerobacter ethanolicus JW 200Geobacillus stearothermophilus В-1169Bacillus stratosphericus В-11678Zymomonas mobilis 113Pichia stipites Y7124̶BCBCBCBICBICBICBICBICBCPachysolen tannophilus Y-3269BIC̶BICBICBICBICBICBICBICBICCandida shehatae NCL3501BCBIC̶BCBICBICBICBICBICBCKluyveromyces marxianus Y-4290BCBICBC̶BICBICBICBICBICBCClostridium thermocellum B-10909BICBICBICBC̶BCBCBICBCBICClostridium thermohydrosuluricumBICBICBICBICBC̶BCBICBCBICThermoanaerobacter ethanolicus JW 200BICBICBICBICBCBC–BCBCBICGeobacillus stearothermophilus В-1169BICBICBICBICBICBICBIC–BICBICBacillus stratosphericus В-11678BICBICBICBICBICBICBICBIC–BICZymomonas mobilis 113BCBCBCBCBICBICBICBICBC–BC – biocomatibility;BIC – bioincomatibility.Kriger O.V. et al. Food Processing: Techniques and Technology, 2021, vol. 51, no. 2, pp. 387–394 Bioethanol yield, % 10 28 136 4 20 5 10 15 20 25Cultivation time, h5 1Bioethanol yield, % 342 3 20 5 10 15 20 25Cultivation time, h Figure 2. Effect of cultivation time for Consortium I in nutrient media 1 (curve 1), 2 (curve 2),and 3 (curve 3) on bioethanol yield, P < 0.05Figure 3. Effect of cultivation time for Consortium II in nutrient media 4 (curve 1), 5 (curve 2), and 6 (curve 3)on bioethanol yield, P < 0.05  hydrosuluricum, and Thermoanaerobacter ethanolicusJW 200 in a ratio of 1:1:1.The selection criteria were based on:specific properties of the microorganisms;specific properties of the metabolism of microorganisms isolated from various natural sources;biocompatibility of these microorganisms;cultivation conditions, i.e. time, temperature, pH, nutrient medium, etc.The next step was to define the co-cultivation conditions in order to increase the yield of ethanol. The compositions of nutrient media (g/l) were taken from related scientific publications:for yeasts: yeast extract – 0.3; peptone – 1.0; glucose – 1.0; agar – 2;for Zymomonas bacteria: К2HPO4 – 1.0; (NH4)2 HPO4– 1.0; MgSO4 – 0.5; peptone – 10.0; yeast extract – 0.5;glucose – 10.0; for Clostridium and Thermoanaerobacter bacteria:KH2PO4 – 0.5; K2NRO4 – 0.5; MgSO4 0.5; NaCl – 0.5;glucose – 20.0; peptone – 5.0; CaCO3 – 10.0.The optimal compositions of nutrient media were selected by varying the ratio of the components and measuring the yield of ethanol in the resulting culture liquid. Table 4 illustrates the compositions of the culture media.Consortium I, based on the mesophilic facultative anaerobic microorganisms, was cultivated in nutrient media 1–3. These media included different concentrations of yeast extract and glucose in a weak acidic medium, the cultivation temperature being 30 ± 1°C. Consortium II consisted of thermophilic anaerobic bacteria grown in nutrient media 4–6, which contained glucose, at 60 ± 1°C and neutral pH. Figure 2–3 show the effect of the composition of the nutrient medium and cultivation time on the bioethanol yield. 1 Bioethanol yield, % 10 39 2876540 5 10 15 20 25Cultivation time, h8Bioethanol yield, % 7 365 24 1320 5 10 15 20 25Cultivation time, h Figure 4. Effect of cultivation time on bioethanol yield for Consortium I: 1 – cultivation temperature = 25°C,2 – cultivation temperature = 32°C,3 – cultivation temperature = 35°С, P < 0.05Figure 5. Effect of cultivation time on bioethanol yield for Consortium II: 1 – cultivation temperature = 50°C,2 – cultivation temperature = 60°C,3 – cultivation temperature = 65°C, P < 0.05Кригер О. В. [и др.] Техника и технология пищевых производств. 2021. Т. 51. № 2 С. 387–394 Figure 2–3 made it possible to select the optimal compositions of nutrient media for the cultivation of the two microbial consortia with the maximal bioethanol yield. For Consortium I, it was nutrient medium 2 (g/l): yeast extract – 0.4; glucose – 5.0; peptone – 5.0; К2HPO4 – 1.5; (NH4)2 HPO4 – 1.5; MgSO4 – 0.5; pH – 5.2. ForConsortium II, it was nutrient medium 4 (g/l): glucose –20.0; peptone – 10.0; К2HPO4 – 1.5; (NH4)2 HPO4 – 1.0;KH2PO4 – 0.5; MgSO4 – 0.5; CaCO3 – 10.0; pH – 7.0.In all the samples of nutrient media, the maximalaccumulation of bioethanol occurred after 16 h of cultivation. It was 9.4% for Consortium I and 5.1% for Consortium II, which significantly exceeded the bioethanol yield obtained by using individual strains.The next stage involved selecting the optimal temperature values for the cultivation process of microbial consortia that provide the maximal bioethanol yield. The consortia were cultivated in the selected nutrient media at various temperature conditions. Consortium I, which united mesophilic microorganisms, was cultivated at 28–35°C, while Consortium 2, which consisted of thermophilic microorganisms, was cultivated at 50–70°C (Fig. 4–5).Figure 4–5 show the optimal temperature cultivation modes: for Consortium I, it was 35°C (maximal bioethanol yield – 9.8%); for Consortium II, it was 65°C (maximal bioethanol yield – 7.4%).Therefore, microbial consortium with enzyme complexes for cellulose bioconversion and subsequent fermentation of sugars increased the bioethanol yield bymore than 50%, in comparison with individual strains. The consortium that united thermotolerant yeasts of the genera Pichia, Candida, and Kluyveromyces with Zymomonas bacteria proved to be the most effective one. ConclusionThe research produced a highly effective consortium of microorganisms for a one-step fermentation of technical cellulose obtained from Miscanthus sinensis by hydrotropic delignification. It also improved the conditions for the cultivation of microorganisms that provide the maximal bioethanol yield. The new consortium included mesophilic facultative anaerobic yeasts Pichia stipites Y7124, Candida shehatae NCL3501, and Kluyveromyces marxianus Y-4290 and bacteria Zymomonas mobilis 113 in a ratio of 1:1:1:1. The optimal cultivation modes were the following: temperature = 35 ± 1°C, pH = 5.2, cultivation time = 16 ± 1 h. The optimal nutrient medium had the following composition (g/l): glucose – 5.0; peptone – 5.0; yeast extract – 0.4; К2HPO4 – 1.5; (NH)2 HPO4 – 1.5; MgSO4 – 0.5. ContributionAll the authors equally contributed to the research development, processing, data analysis, and manuscript design. Conflict of interestThe authors declare that there is no conflict of interests regarding the publication of this article.