Enhanced Biogas Production from Glucose and Glycerol by Artificial Consortia of Anaerobic Sludge with Immobilized Yeast

Abstract

Today, there is considerable interest in creating artificial microbial consortia to solve various biotechnological problems. The use of such consortia allows for the improvement of process indicators, namely, increasing the rate of accumulation of target products and enhancing the conversion efficiency of the original substrates. In this work, the prospects for creating artificial consortia based on anaerobic sludge (AS) with cells of different yeasts were confirmed to increase the efficiency of methanogenesis in glucose- and glycerol-containing media and obtain biogas with an increased methane content. Yeasts of the genera Saccharomyces, Candida, Kluyveromyces, and Pachysolen were used to create the artificial consortia. Their concentration in the biomass of consortium cells was 1.5%. Yeast cells were used in an immobilized form, which was obtained by incorporating cells into a cryogel of polyvinyl alcohol. The possibility of increasing the efficiency of methanogenesis by 1.5 times in relation to the control (AS without the addition of yeast cells) was demonstrated. Using a consortium composed of methanogenic sludge and yeast cells of the genus Pachysolen, known for their ability to convert glycerol into ethanol under aerobic conditions, the possibility of highly efficient anaerobic conversion of glycerol into biogas was shown for the first time. Analysis of the metabolic activity of the consortia not only for the main components of the gas phase (CH₄, CO₂, and H₂) and metabolites in the cell culture medium, but also for the concentration of intracellular adenosine triphosphate (ATP), controlled by the method of bioluminescent ATP-metry, showed a high level of functionality and thus, prospects for using such consortia in methanogenesis processes. The advantages and the prospect of using the developed consortia instead of individual AS for the treatment of methanogenic wastewater were confirmed during static tests conducted with several samples of real and model waste.

1. Introduction

Recently, there has been growing interest in the creation of artificial microbial consortia with a heterogeneous composition, especially for bioremediation processes, wastewater treatment, biofuel production, and other processes, analogs of which can be found in natural conditions [1,2,3]. Microbial consortia demonstrate significant advantages in such processes over individual cell strains. This is a result of the emerging possibility of converting a wider range of substrates due to the combination of different microorganisms, catalyzed by their enzymes’ biochemical reactions, which leads to the appearance of intermediate metabolites, the transformation of which ends with an increased accumulation of target products [4,5].

Artificial microbial consortia are, in most cases, created on the basis of isolated, well-characterized cells of microorganisms capable of effectively performing the major stages of biotechnological processes; they are then supplemented with strains possessing catalytic characteristics, thereby improving the process parameters [6,7]. In some cases, artificial consortia are composed of immobilized cells of microorganisms [8], since immobilization of cells allows for their use in the form of intensively functioning and highly concentrated populations in the quorum sensing state, i.e., actively communicating cells [9]. The immobilization of microbial populations by their inclusion in gel matrices imitates the formation of a nature-like stabilized state of cells, similar to what occurs when they are part of heterogeneous biofilms or self-immobilized biosystems.

It is known that consortia involving yeast cells are used to obtain biofuels, various organic compounds, and in winemaking [10,11]. To increase the efficiency of biotechnological processes in terms of the level of accumulation of target products, the possibility of combining cells of different yeast strains with each other and with bacterial cells or mycelial fungi, which often allows for an increase in the level of conversion of the original substrates into the required product, has been shown [12].

Today, among the urgent tasks of ecology and biotechnology are those related to the study of problems of the accumulation of large volumes of organic waste, in particular, in the food, agriculture, and fuel industries, which require rational and safe disposal [13,14]. The anaerobic conversion of such waste enables the production of biogas with a high methane content [13,14,15,16]. Biogas usually forms as a result of the metabolic activity of anaerobic microbial consortia within four main stages of the methanogenic conversion of complex substrates: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. During the first two stages, substrates (carbohydrates, proteins, and lipids) are converted into small organic compounds, including volatile short-chain acids (propionic, lactic, and butyric), which are further converted into acetic acid, CO₂, and H₂ (stage three). Then, methanogenic archaea cells transform H₂, CO₂, and acetate into CH₄ (Figure 1).

Based on generally known catalytic dependencies, a higher concentration of substrate is then available for conversion to the product without inhibiting any effect on the biocatalyst, after which a higher velocity of the process and product accumulation are observed.

Despite the prospects of such a process, there are a number of problems that reduce its efficiency, primarily due to the decrease in the pH of the environment resulting from the accumulation of short-chain fatty acids (SCFA) [16]. The addition of yeast to the AS in the process of methanogenesis allows for an increase in the efficiency of biogas production (

Table 1. The effect of the addition of yeast cells to the AS on the results of methanogenesis.

Yeast/Reference	* Conditions	Effects
Saccharomyces cerevisiae (Angel Yeast Co., Ltd., Hubei, China) [17]	Substrate—Food waste Biocatalysts—Sludge from a rural biogas station (80% by VS) and Yeast (2% VS); 37 °C, 60 rpm, 50 days	33.2% increase in biogas production
Brewing yeast [18]	Substrate—Mixture of 50% brewery wastewater and 50% municipal wastewater (400–700 mg/L COD) Biocatalysts—Granular sludge (20% VS) and yeast (0.7%); 37 °C, pH 6.5, 120 rpm, 228 days	Increased biogas production
Brewing yeast [19]	Substrate—Brewery wastewater Biocatalysts—Granular sludge from brewery and yeast (2–22% v/v); 35 °C, pH 6.5, 7 days	50% increase in biogas production
S. cerevisiae NKL [20]	Substrate—Cassava wastes 1% (w/v) with 0.4% urea (w/v) Biocatalysts—Cow rumen (8–20% w/v) and yeast (0.08–0.15% w/v); 26–33 °C, pH 7.0, 16 days	Increased biogas production
S. cerevisiae [21]	Substrate—Anaerobically pre-fermented sorghum biomass (500 g/L) Biocatalysts—Sludge from biogas plant and yeast (105 CFU/g wet substrate weight); 39 °C, 7 days	Enhanced production of total biogas (236–251 mL/g VS) and methane (96–105 mL/g VS)
S. cerevisiae Ragi [22]	Substrate—Tofu wastewater (COD—576 mg/L, total nitrogen—13.5 mg/L, pH 4.2) Biocatalysts—Rumen anaerobic consortium (10% v/v) and Yeast (1 g); pH 8.0, 8 days	150% increase in biogas production
S. cerevisiae [23]	Substrate—Mixture of kitchen wastes (vegetables and fruits)—20 g/L Biocatalysts—Microbial consortia from fresh mixed mammal feces (20–500 g/L) and yeast with cellulolytic activity—400 mg/L (4 × 108 cells); 25, 39 or 55 °C, 96 h	S. cerevisiae improved cellulolytic activity in kitchen waste digestion and provided an increased biogas yield
S. cerevisiae [24]	Substrate—Acid-pretreated free-floating aquatic plant Salvinia molesta—10 g Biocatalysts—Cow rumen anaerobic consortium (25 mL) and yeast (1 g) in 130 mL H2O; pH 7.0, 30 °C, 30 days	Increase in biogas production (up to 113.7 mg/g VS) and CH4 content (up to 84%)
S. cerevisiae [25]	Substrate—Chicken manure—1.2 g Biocatalysts—Sludge from UASB (upflow AS blanket) reactor (5 g) and yeast (0.48–1.07 g/g VS); 35 °C, 120 rpm, 500 h	The highest biogas yield (470.95 mL/g VS) with 77.5% CH4 was observed at a yeast concentration of 0.69 g/g VS (4.3 times higher than the control without yeast cells)
Yarrowia lipolytica H181 from citric acid production [26]	Substrate—Waste frying fat Biocatalysts—AS from biogas plant treated maize silage (0.21–0.45 (VS/VS substrate) and yeast (5% w/w); pH 7.7–8.0, 70 days	Increase in biogas yield (1.45 m3/kg VS) with CH4 content—67 ± 4%

* VS—volatile solids; COD—chemical oxygen demand.

[17,18,19,20,21,22,23,24,25,26]).

The authors of such studies associate this effect with a decrease in the concentration of SCFAs accumulating under the influence of AS, due to the conversion by yeast of part of the substrate mainly into ethanol [17], which is then transformed into acetic acid, which is necessary for archaea at the methanogenic stage and the accumulation of biogas (Figure 1).

Preferably, Saccharomyces cerevisiae yeast cells in the form of a suspension, widely available for practical application and most often used in the food industry, are introduced into the anaerobic reactor [27,28]. Immobilized yeast cells have not been studied in similar studies before.

Among the media suitable for anaerobic methanogenic fermentation, the majority are those containing glucose in the composition of various food industry and agriculture waste. So, the existing interest in the cells of S. cerevisiae is understandable. However, there are many other yeasts cells that are also used in biotechnological processes, and in this regard, it was interesting from a scientific and practical point of view to study the possibility of their use for purposes similar to those indicated for S. cerevisiae, and under the same conditions, in order to compare their effect on the methanogenic conversion of glucose.

Another large-tonnage waste product of various processes, including the production of biodiesel fuel, is glycerol [29]. The processing of glycerol into biogas is limited due to the formation of intermediate products, such as long-chain fatty acids and propionic acid [30,31]. In this regard, to increase the efficiency of methanogenesis, glycerol is more often used not as the main substrate, but as a co-substrate in combination with organic sources (waste) rich in nitrogen, while maintaining the optimal C:N ratio [32,33]. Under anaerobic conditions, the conversion of glycerol occurs through the formation of dihydroxyacetone and pyruvate, which are converted into ethanol and acetic acid (Figure 1). Thus, yeasts of the genus Saccharomyces do not directly convert glycerol into ethanol; additional biochemical reactions are required. In this regard, the use of the yeast Pachysolen tannophilus, which has not been previously utilized for these purposes, appears promising in methanogenesis. At the same time, for the P. tannophilus Y-475 yeast cells immobilized in polyvinyl alcohol (PVA) cryogel, it was established that under aerobic conditions, they are capable of providing for the conversion of 25 g/L of glycerol with the formation of 8.3 g/L of ethanol. In addition, the possibility of using these cells in at least 16 working cycles of such a process without reducing the efficiency of operation and the yield of ethanol was established [34].

Since Pachysolen tannophilus cells have not been previously used under anaerobic conditions in immobilized form, such a study seemed very promising as a potential approach to intensifying methanogenesis using glycerol as a substrate. Thus, the aim of this study was to investigate the effect of combining the cultivation of yeasts of different genera (Saccharomyces, Candida, Kluyveromyces, and Pachysolen) in an immobilized form with suspended AS for the conversion of substrates such as glucose and glycerol under methanogenesis conditions.

2. Materials and Methods

2.1. Materials

The components of nutrient media for the accumulation of yeast biomass and their subsequent immobilization (K₂HPO₄, KH₂PO₄ × 2H₂O, MgSO₄ × 7H₂O), citric acid, ascorbic acid, glucose, glycerol, peptone, yeast extract, poly(vinyl alcohol) (PVA) (CAS 9002-89-5), ethanol (99.5% CAS 1516-08-1), and acetic acid (CAS 64-19-7), were purchased from Chimmed (Moscow, Russia). For bioluminescent analysis of intracellular adenosine triphosphate (ATP) concentration, the luciferin–luciferase reagent [35] was used. Dimethyl sulfoxide (DMSO) was purchased from Chimmed (Moscow, Russia) and used for the extraction of ATP from the cells.

The yeast strains Saccharomyces cerevisiae Y-185, Candida maltosa Y-2256, Kluyveromyces marxianus Y-5273, and Pachysolen tannophilus Y-475 were purchased from the All-Russian Collection of Industrial Microorganisms, Moscow, Russia (http://www.genetika.ru/vkpm/ has been accessed on 30 April 2025).

Suspended AS was obtained from a food wastewater treatment plant (Kashira, Moscow region, Russia). Sludge was stored at 35 °C with the addition of 1 g COD/L (1 g/L glucose) every month before its use. The characteristics of this consortium were previously determined by the authors of this study [36].

2.2. Yeast Cells Immobilization

To obtain 4 immobilized biocatalysts based on PVA cryogel and yeast cells, a method successfully tested on the example of Pachysolen tannophilus yeast cells was used [34]. Yeast biomass was accumulated as a result of culturing cells on a medium of the following composition (g/L): glucose—20; yeast extract and peptone—5; K₂HPO₄—3; KH₂PO₄—2; citric acid—1; ascorbic acid—1; MgSO₄—0.5; pH—5.6; at 26 ± 2 °C, 150 ± 10 rpm for 20 ± 1 h. Next, the yeast biomass was separated from the culture broth by centrifugation (Avanti J25, Beckman, Germany) for 15 min at 10,000 rpm and mixed with a 12 wt% PVA solution so that the cell concentration in the suspension was 10% w/w. The resulting suspension was then distributed into the wells of 96-well sterile plates (Paneco Company, Moscow, Russia) and frozen at −20 ± 1 °C to form a polymer cryogel. Before use, the immobilized cells were defrosted at +6 ± 1 °C for 6 ± 1 h.

2.3. Biogas Production by Artificial Consortia

For biogas production, the anaerobic bottles (100 mL) (Mimimed, Bryansk, Russia) with 50 mL of total liquid medium were used. The dose of AS was 10% (v/v) with 2.5 ± 0.1 g of immobilized yeast granules with 10% yeast biomass (Figure 2).

The ratio of AS to yeast cells was 64:1 (w/w). The ratio of AS to yeast cells was calculated as a ratio of the dry weight of AS to the dry yeast cell biomass in PVA cryogel granules (biomass in granules was 10 ± 0.02% w/w).

The glucose (5 g/L) and glycerol (5 g/L) were used as carbon sources in a 0.2 M phosphate buffer (pH 7.3 ± 0.1) medium. The bottles with the medium and consortia were filled with helium for 20 min to remove oxygen from the gaseous phase.

Methanogenesis was conducted at 36 ± 1 °C for 28 days. The sample with AS cells without yeasts was used as a control.

2.4. Static Test of Artificial Consortia in Methanogenic Treatment of Wastewater

Several artificial consortia were applied for the static methanogenic treatment of the following samples of wastewaters (

Table 2. Characteristics of wastewaters used for the static test of the artificial consortia.

Wastewater	COD, g/L	Content of Main Components, %			pH
		Proteins	Lipids	Carbohydrates
Milk plant	1.4 ± 0.1	27.0 ± 2.5	41.6 ± 2.7	31.4 ± 3.5	6.8
Bagasse hydrolysate	42 ± 2.5	14.1 ± 1.2	11.7 ± 0.0	75.2 ± 3.5	5.0
Meat processing	4.0 ± 0.1	10.0 ± 0.3	35.0 ± 2.5	1.0 ± 0.1	6.0

): real wastewater from a milk processing plant (OOO Ostankinsky Molochny Kombinat, Moscow, Russia), real wastewater from a meat processing plant OOO “Adria” (Moscow region, Russia), and model wastewater prepared using bagasse hydrolysate. Bagasse hydrolysate was obtained as a result of the enzymatic hydrolysis of 50 g/L dry bagasse pieces by Spezyme CP (Dupont, New York, NY, USA) and Novozyme-188 (Novozymes Corp., Copenhagen, Denmark). Bagasse particles with a size of 100–300 µm were prepared using a Mikrosilema IM-450 impeller mill (Techpribor, Schekino, Russia) and suspended in a 0.1 M citrate buffer (pH 5.0). The enzymes were introduced into the reaction medium at a mass ratio of 3:1. Hydrolysis was carried out at 50 °C with constant mixing (250 rpm) for 24 h. The total carbohydrate, lipid, and protein contents were determined using known procedures [37].

Additionally, the medium containing crude glycerol was used in the experiments as a wastewater contaminated with the by-product of biodiesel production from the lipids of filamentous fungi biomass. It was prepared as published earlier [34]. Glycerol feedstock contained 83% glycerol, 6.5% ash, 0.5% methanol, and 10% water. Glycerol concentration was determined using the glycerol assay kit (Megazyme Ltd., Wicklow, Ireland). Ash content was determined using a muffle furnace at 590 ± 10 °C for 24 h. Ash is expressed as a percentage of the difference in weight before and after ashing.

For biogas production from wastewaters, the anaerobic bottles (1 L) (Mimimed, Bryansk, Russia) with 500 mL of total liquid medium were used. The doses of AS and yeast were the same as in the previous experiment (AS 10% (v/v) with 25 ± 1 g of immobilized yeast). The bagasse hydrolysate and glycerol were previously diluted with a 0.2 M phosphate buffer (pH 7.3 ± 0.1). The pH of all samples was adjusted to 7.3 using 10 M KOH (Chimmed, Moscow, Russia). Methanogenesis was conducted at 36 ± 1 °C for 7 days.

2.5. Analytical Methods

The intracellular ATP concentration was measured using the Microluminometer 3560 (New Horizons Diagnostics Co., Baltimore, MD, USA) and a luciferin–luciferase reagent. Samples were mixed with 800 μL of DMSO and kept for 3 h for intracellular ATP extraction. After extraction, the samples were diluted with distilled water (1:10), and 50 µL of the resulting solution was added to the bioluminometer cuvette with 50 µL of the luciferin–luciferase reagent. For analysis of the obtained results, the calibration curve was used for standard ATP solutions (10^–14–10^–8 mole/mL).

For potentiometric measurements, the Corning Pinnacle 530 pH meter (Corning Incorporated, Corning, NY, USA) was used. During methanogenesis, the biogas content (H₂, CH₄, and CO₂) was analyzed using a Crystallux-4000M gas chromatograph (RPC ‘Meta-chrom’, Yoshkar-Ola, Russia) equipped with a katharometer. Argon was used as the carrier gas, the flow rate was 20 mL/min, and the column temperature was 51 ± 1 °C. The gas phase sample volume was 0.2 mL. A gas mixture of H₂-CH₄-CO₂ (5%) in argon was used as a standard (PGS-Service, Moscow, Russia). The obtained data was analyzed using NetChrom software (Meta-chrom ver. 2.1, Yoshkar-Ola, Russia).

COD was analyzed using the Merck Cod Test Kit (Merck Life Science LLC (Moscow, Russia).

Methanogenesis efficiency was estimated based on biogas accumulation and the following equations:

% = (Biogas _experimental/Biogas _theoretic) × 100

Biogas _theoretic = (C × V _{liquid phase}) × 0.5 × 1000

where C—concentration of organic matter, g COD/L; V _{liquid phase}—0.05 L; and 0.5—volume of biogas formed from 1 g COD at 273 K.

The accumulation rate of CH₄ (mL/day) was calculated as the ratio of the total volume of methane to the duration of the process.

The concentrations of methanol, ethanol, and acetic acid were determined using a Shimadzu GC-15A (Shimadzy Corporation, Kyoto, Japan) gas chromatograph equipped with an FID. Argon was used as the carrier gas. The temperature of the injector, column thermostat, and evaporator was 200, 220, and 240 °C, respectively. The sample volume was 1 μL. The 0.1% (v/v) solutions of methanol, ethanol, and acetic acid were used as standards.

The standard deviation (±SD) of data obtained from three independent experiments conducted in this work was not more than 5%. Statistical analysis was performed using SigmaPlot (ver. 12.5, Systat Software Inc., San Jose, CA, USA).

3. Results

As the main components of various wastes, including food, agricultural, biodiesel production, and palm oil [38,39], glucose and glycerol, which can be utilized by yeast as the main substrate, were chosen as substrates for methanogenesis. Since various components of complex media containing glucose and glycerol can have an ambiguous effect on methanogenesis, it was decided to use as substrates in this work not just any production wastes, but pure substances (glucose and glycerol) for a more objective interpretation of the results obtained.

According to the previously known publications, the increase in the efficiency of methanogenesis when combining yeast cells with AS occurs due to the conversion of the main substrates by these yeasts primarily into ethanol and acetic acid [17].

With this in mind, their accumulation in the liquid phase of the reaction methanogenic medium was monitored during the initial period of glucose and glycerol conversion by yeast consortia with AS. Analyses were carried out during the first six days, but only on the second and fourth days of the process was it possible to register changes in the accumulation of these metabolites, since their concentrations only decreased after that as they underwent conversion into biogas (Figure 3).

When using glucose as the main substrate, the presence of ethanol (in some variants, about 2 g/L) and acetic acid in samples with cells of Saccharomyces, Candida, and Kluyveromyces was noted in the medium after 96 h.

The conversion of glycerol with the accumulation of ethanol in the reaction medium by these same consortia proceeded less dynamically, and in most cases, the current concentration of ethanol did not exceed 0.25 g/L.

However, a very high concentration of ethanol (1.8 ± 0.1 g/L) in comparison with consortia containing different yeast cells was revealed in the variant that contained P. tannophilus yeast. By the 96th hour, the concentration of ethanol in the medium with this consortium was 7.2 times higher than in the others.

This result was undoubtedly due to the fact that these yeasts are capable of highly efficiently converting glycerol into ethanol [34]; however, it was revealed for the first time in this study that these yeasts can carry out such conversion not only under aerobic but also under anaerobic conditions.

The current concentration of acetic acid was comparable with the analogous parameter for consortia with S. cerevisiae and C. maltosa cells, established on the media with glucose. In the medium with the consortium, which contained P. tannophilus cells, the level of acetic acid in the medium with glycerol was 25% higher than in other variants of the consortia, which clearly indicates that this consortium on the medium with glycerol is a clear favorite, and the yeast takes an active part in the processes that occur within the consortium.

Throughout the entire period of culturing the consortia under anaerobic conditions, the efficiency of methanogenesis was analyzed in each variant, as well as in the control (AS without yeast cells) for biogas yield in relation to the theoretically possible level. An analysis of the current composition of the gas phase in the methanogenic process was also carried out, determining the proportions of gases (CO₂, H₂, CH₄) present in the biogas (Figure 4).

It was found that the addition of any of the four yeast strains used in the study to AS resulted in an increase in the efficiency of methanogenesis by 110–155% (biogas accumulation) during the conversion of both substrates compared to the AS in the control (without yeast). The efficiency of methanogenesis during the conversion of glucose was higher than that of glycerol. This was probably due to the faster conversion of glucose consortia by cells (Figure 1) and a higher COD level in the medium with glycerol (6.15 g/L) compared to the COD level in the glucose-containing medium (5.35 g/L).

In this work, the efficiency of methanogenesis during glycerol conversion in the consortium of AS with Pachysolen yeast was the highest among other studied samples of consortia and the anaerobic methanogenic sludge itself. It should be noted that in the first 7 days, CO₂ and H₂ predominated in the composition of the resulting biogas, and they composed up to 90% of the total gaseous phase volume. Then, a gradual increase in the proportion of CH₄ in the biogas was observed. At the same time, the methane content in the biogas produced by the consortia of AS with yeast was significantly higher than in the gas phase with sludge without yeast.

During glucose conversion, the highest amount of CH₄ was observed in samples with a consortium of sludge and S. cerevisiae yeast cells, followed by consortia obtained by combining methanogenic sludge and C. maltose or K. marxianus yeasts at almost the same level across all parameters (methane concentration in biogas and its accumulation rate) (

Table 3. Accumulation rate of CH₄ in methanogenesis carried out for 28 h in glucose and glycerol media using different variants of AS-based consortia.

Consortia	CH4, mL/day
	Glucose	Glycerol
AS (control without yeast)	1.2 ± 0.06	1.1 ± 0.06
AS + S. cerevisiae	2.8 ± 0.14	1.9 ± 0.10
AS + C. maltosa	2.7 ± 0.13	2.2 ± 0.11
AS + Kluyveromyces marxianus	2.4 ± 0.12	2.0 ± 0.10
AS + Pachysolen tannophilus	1.6 ± 0.06	2.9 ± 0.15

).

During glycerol methanogenesis, the maximum in terms of methane accumulation rate in biogas and its accumulation level in the composition was observed when studying the functioning of a consortium of sludge with P. tannophilus yeast cells. Moreover, in media with glucose, this consortium with P. tannophilus yeast exceeded the control level (sludge without yeast) but was not as successful as other variants of the consortia.

Thus, it is obvious that the presence of immobilized yeast cells in the consortia with AS was exclusively positive in media containing both substrates (glucose and glycerol), but at the same time, obvious preferences were revealed among the consortia due to the presence of different yeast cells in them in relation to these substrates.

During the study of methanogenesis, the metabolic activity of microorganisms in the consortia was also monitored by assessing the change in the concentration of intracellular ATP in the samples of mixed cells (Figure 5).

When using glucose as a substrate, an increase in the level of intracellular ATP was observed in all consortia during the first seven days of the process, and then it decreased slightly, which was associated with a gradual decrease in the concentration of the main substrate in the reaction medium. Thus, in consortia with K. marxianus and P. tannophilus yeast cells, the ATP concentration in the analyzed samples, cultured in a medium with glucose, decreased below the initial level by an average of 40% during the process itself.

In the medium with glycerol, in samples of three consortia with the yeasts S. cerevisiae, C. maltosa, and K. marxianus, a similar decrease in ATP from the initial level was recorded during the entire process of methanogenesis. At the same time, the consortium with P. tannophilus yeast cells demonstrated a very high level of ATP in the medium with glycerol, which was predetermined by the known efficiency of these cells in the utilization of glycerol as the main substrate.

ATP plays a central role in metabolism and provides energy for many biochemical reactions in all living cells. The synthesis and hydrolysis of ATP are used by microorganisms to generate energy during various biochemical transformations and physiological processes; therefore, changes in the concentration of ATP in cells confirm their viability and the high level of metabolic activity. The concentration of ATP can be quickly and easily determined using the bioluminescent method of ATP-metry [36,40].

In general, all cells of all consortia were characterized by a high intracellular level of ATP after the end of the process, which allows us to expect their at least repeated use in similar processes. According to known data [40], the absence of significant (more than 3–4 times) fluctuations in the controlled level of ATP during the functioning of the consortia indicates a favorable “working” state of the cells in the studied processes.

Various samples of real and model wastewater from industrial and agricultural enterprises were processed under the action of artificial anaerobic consortia formed for this study, based on AS and immobilized yeast cells under conditions of a static methanogenic process (

Table 4. Static methanogenic tests of various wastewaters using developed anaerobic consortia for seven days.

Sample of Wastewater	Initial COD, g/L	Type of Anaerobic Biocatalyst *	COD Removal, %	Efficiency of Biogas Production, %	CH4 Content, %
Wastewater from milk plant	1.4 ± 0.1	AS	72 ± 2	47 ± 2	41 ± 2
		AS + K	84 ± 3	63 ± 3	50 ± 3
Wastewater with bagasse hydrolysate	8.5 ± 0.3	AS	49 ± 2	41 ± 2	32 ± 1
		AS + S	67 ± 3	65 ± 3	52 ± 3
Wastewater with crude glycerol	7.0 ± 0.2	AS	32 ± 1	22 ± 1	20 ± 1
		AS + P	54 ± 2	47 ± 3	45 ± 2
Wastewater from meat processing	4.0 ± 0.1	AS	58 ± 2	42 ± 2	37 ± 1
		AS + C	75 ± 3	64 ± 3	53 ± 2

* AS—anaerobic sludge, K—K. marxianus, S—S. cerevisiae, C—C. maltose, P—P. tannophilus.

).

To compare the results obtained in similar experiments, AS was used without the addition of yeast cells. Since the task of the study was precisely to obtain comparative estimates of the functioning of AS itself and its consortia with yeast in wastewaters, the testing was carried out for only seven days. According to the data obtained from the analysis of intracellular ATP concentration at the previous stage of the study (Figure 5), this time period was favorable for consortium cells, and their metabolic activity during this time period was presentable.

According to the literature data [41], such static methanogenic testing is indicative for evaluating the prospects of the proposed process. It should be noted that in this study, additional nutrients and trace elements were not introduced into the wastewater, but the composition of the media components was used, which was predetermined by the conditions of their production (real samples) or preparation (model samples). It was found that the use of consortia of AS with immobilized yeast cells provided stable increased COD removal (12–22%) and efficiency of biogas production (16–25%). This effect was found in all samples of the wastewaters studied.

4. Discussion

The information presented in Table 1 clearly confirms the relevance of the existing scientific and practical interest in the processes of methanogenic treatment of various wastes from the food industry and agriculture, focusing on the production of biogas by using combined biocatalysts consisting of anaerobic activated sludge and yeast cells.

It should be noted that the authors of the conducted studies (Table 1) clearly note improved indicators for the yield of accumulating biogas with an increased content of methane. This is despite the differences in the compositions of the processed substrates, the variability of the conditions for methanogenesis, and the ratio of components in the composition of the used biocatalyst combining AS and yeast.

At the same time, the analysis of the data in Table 1 also shows that, in fact, only S. cerevisiae yeast cells are used to create artificial consortia for the purpose of converting most protein–carbohydrate substrates into biogas for methanogenic sludge. Only in the case of processing fat-containing waste (waste frying fat), the possibility of using other yeasts, such as Yarrowia lipolytica, capable of converting lipid-containing substrates, was shown [12]. In this regard, the results achieved in this work are new, since they demonstrate the success of using yeast cells of different genera (Candida, Kluyveromyces, and Pachysolen) in artificial consortia with methanogenic sludge to obtain an increased yield of biogas with an improved rate of methane accumulation and enhanced methanogenesis efficiency in comparison with the same parameters typical of AS itself.

Thus, significantly improved indicators for the conversion of both glucose- and glycerol-containing media under the action of different variants of artificial consortia with yeast cells have been demonstrated. It should be noted that among those media that are subject to methanogenic treatment, especially in the food industry and agriculture, there are many such media; therefore, the potential prospects of the results shown in this work for their practical application can be assumed. Of particular importance are those preferences identified for the use of yeast cells in consortia that were identified when carrying out the process in media with glucose and glycerol.

The results shown with the use of Pachysolen cells in a consortium with sludge are outstanding in terms of the parameters achieved (the rates and levels of methane accumulation in biogas and the degrees of glycerol conversion into biogas), and, to the best of our knowledge, have no comparable analogs, since, as noted above, glycerol is a substrate that is difficult to utilize for methanogenic sludge and is therefore introduced into the environment for obtaining biogas not as the main substrate, but as a co-substrate [42].

It should also be noted that this work demonstrates for the first time the possibility of combining Pachysolen yeasts with methanogenic sludge for co-cultivation and conversion of glycerol under anaerobic conditions, whereas it was previously shown that it is under aerobic conditions that these yeasts are effective producers of ethanol from such an unusual substrate as glycerol.

The reason for this is that anaerobic conversion of glycerol into biogas under the action of methanogenic sludge proceeds slowly through the formation of dihydroxyacetone and pyruvate, which are converted into acetic acid and ethanol, as well as other metabolites. It is obvious that the presence of Pachysolen yeasts in artificial consortia significantly accelerated the overall process of glycerol conversion into biogas, although the proportion of yeast cells in the overall consortium was insignificant, since the ratio of AS to yeast cells was 64:1 (w/w).

In this work, the immobilized form of different yeasts introduced into the consortia together with AS demonstrated clear advantages in comparison with the results obtained in similar studies conducted earlier by us using bacterial cells as components of artificial consortia created on the basis of the same methanogenic sludge.

Such experiments with anaerobic consortia based on sludge and individual bacterial cultures of Clostridium acetobutylicum, Pseudomonas sp., or Enterococcus faecalis were carried out by us earlier, with the introduction of bacterial cells in a ratio of 9:1 (w/w) to methanogenic sludge. Bacterial cells were also used in a form immobilized in PVA cryogel, which led, as in the case of yeast, to an improvement in the characteristics of methanogenesis. The use of the same support for the immobilization of different cells (bacteria and yeast) was dictated by the well-known positive characteristics of this polymer carrier [43]. In the work with bacterial participants in the methanogenic consortium, enzymatic hydrolysates of Jerusalem artichoke stems and sugar beet pulp with a significantly higher level of COD in the medium (10.5 g COD/L) were used instead of pure glucose [44], which ensured intensive methanogenesis, leading to an increase in the level of accumulated methane in the biogas by 1.7 times in relation to the control.

When discussing the potential practical application of the results obtained, based on an easily implemented technical solution consisting of combining methanogenic sludge and yeast cells in an anaerobic process, it is necessary to emphasize that large volumes of wastewater with high concentrations of organic pollutants are regularly neutralized during the process of methanogenesis. In this regard, the construction of artificial consortia based on AS and yeast can be an effective approach to improving the performance of wastewater treatment-oriented processes at various enterprises [18,26,45].

As noted earlier, S. cerevisiae yeast cells were mainly added to the AS in various methanogenic processes [17,18,19,20,21,22,23,24,25], while yeast cells of other genera were practically not considered as participants. Although judging by the results of this study, they may have a clear technological potential. In this regard, the prospects of this work lie in expanding the range of different types of yeast introduced into the composition of AS, which may be a by-product of various industries, for example, K. marxianus [46].

The resistance of immobilized cells to the possible presence of various toxicants (heavy metals, antimicrobial agents, etc.) in wastewater certainly provides advantages in using yeast as part of an artificial consortium in this form. This allows for the methanogenic treatment of toxic effluents, which include, for example, effluents from oil desulfurization that are subject to methanogenic treatment [47]. The use of artificial consortia with yeast cells may be extremely interesting here from the point of view of improving the characteristics of the process. Since there is a practical interest in the use of artificial consortia based on AS for the methanogenic conversion of various lignocellulose wastes containing antibiotics and pesticides [44], in this case, for the treatment of wastewater containing substrates hydrolysable to glucose, the obtained artificial consortia may be of undoubted practical interest.

However, this result was achieved with a completely different ratio of cells participating in the consortium. Thus, yeast produced an increase in methane yield by 1.5 times in this work on a glucose-containing medium, when yeast cells made up only 1.5% of the biomass of all cells in the consortium, while the bacteria, with which the comparison is being conducted, made up 10% of the consortium.

5. Conclusions

The creation of artificial consortia comprising anaerobic methanogenic sludge and various types of yeast in immobilized form is aimed at stimulating biogas production by improving the quality of biocatalysts for this process. Methanogenesis, with the addition of yeast, showed a higher rate of CH₄ accumulation due to the formation of ethanol and acetic acid, which can be easily used by methanogens, thereby increasing biogas production and ensuring stable operation of the entire consortium. The maximum efficiency of methanogenesis was over 90%, while the methane content in biogas reached 60–65% during glucose conversion by the consortium of AS with S. cerevisiae, C. maltose, and K. marxianus yeasts.

The addition of yeast cells of the genus Pachysolen proved to be a very effective approach to solving the problem of converting glycerol-containing substrates. The consistently high level of intracellular ATP in the consortium cells confirmed their high metabolic activity and the potential for reusing artificial consortia as biocatalysts. In general, the approach to creating such artificial methanogenic consortia is relevant and promising for obtaining biogas from a wide variety of substrates. The additional immobilization of cells makes it easy to regulate their composition and metabolic activity, helping to maintain their intensive functioning.

A demonstration of the work of the obtained consortia in different wastewaters confirmed the identified advantages of combining AS with different yeast cells and the prospects for their practical application in the methanogenic treatment of sewage of various origins.