Natural Materials as Carriers of Microbial Consortium for Bioaugmentation of Anaerobic Digesters

Featured Application

Improvement of biogas production reactors by inserting bacterial species conditioned for the decomposition of specific biomass.

Abstract

The production of biogas is achieved during anaerobic digestion (AD) using organic matter as a substrate. In Mediterranean countries, a promising substrate is lignocellulose biomass of perennial grass Miscanthus x giganteus, due to its potentially high biogas yields, which could be comparable to maize silage. During AD, bacteria convert biomass into more minor compounds, which are further converted to methane by methanogenic archaea. The selection of appropriate microbes for the degradation of the substrate is crucial, and the enhancement of this step lies in the immobilization of microbes on biocarriers. Described here, a microbial consortium, de novo isolated and conditioned to degrade the Mischantus biomass, was immobilized onto several natural biocarriers: natural zeolitized tuff, ZeoSand^® (Velebit Agro, Zagreb, Croatia), perlite, and corncob. There was no statistically significant difference in the number of immobilized bacteria across the different materials. Therefore, all proved to be suitable for the immobilization of the consortium. In the consortium, five bacterial species with different shares in the consortium were identified: Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter asburiae, Leclercia adecarboxylata, and Exiguobacterium indicum. After immobilization on each carrier, the share of each species changed when compared to starting conditions, and the most dominant species was E. cloacae (71–90%), while the share for other species ranged from 2 to 23%. The share of E. indicum was 14% at the start. However, it diminished to less than 1% because it was overgrown during the competition with other bacterial species, not due to an inability to immobilize.

1. Introduction

According to the latest statistical report by the European Biogas Association (EBA), gas import dependency in Europe rose from 83% in 2021 to 97% in 2022. Therefore, it is necessary to enhance the production of alternatives to natural gas, and biogas is one of them [1]. One of the ways to produce biogas is through anaerobic digestion using organic matter as a substrate [2]. During the process, lipids, carbohydrates, and proteins are digested into smaller compounds in the absence of oxygen. AD consists of four biochemical steps, hydrolysis, acidogenesis, acetogenesis, and methanogenesis, which are conducted by bacteria and archaea [2]. The most commonly used organic matter in AD is maize silage, an edible component also used as livestock feed [3]. Another option is lignocellulose biomass such as dedicated energy crops with biogas yields comparable to maize silage [4]. In Mediterranean countries, an example of a promising substrate for energy production is Miscanthus x giganteus (MxG) or Arundo donax [5]. MxG was investigated as a potential biomass due to its abundant carbohydrate content, non-edible nature, rapid growth, and ability to be grown on marginal land. Moreover, cultivation is simple due to low nutrient and water requirements [4]. In order to convert MxG crops into biogas, the crucial step is the presence of physiological groups of bacteria that will degrade the biomass to substrates (acetate, H₂, and CO₂) for further methanogenesis.

Immobilization of the selected bacterial species or microbial consortium onto solid materials as carriers has frequently been employed in biotechnological processes, especially in the bioremediation of soil or wastewater and the biodegradation of food waste and different types of silage [6]. Lignocellulose biomass is to a large scale quite resistant to microbial enzymatic biodegradation. Therefore, the immobilization of desired microbes accompanied by bioaugmentation is promising for the enhancement of lignocellulose degradation [3]. The usage of immobilized microbes provides many advantages, such as higher cell density and consequent metabolic activity, great stability, and protection from extreme environmental conditions, subsequently resulting in higher efficiency in bioremediation and biodegradation [7]. However, not all materials are appropriate for the immobilization of microbes and selecting a suitable carrier is crucial for successful immobilization. The properties of an ideal carrier of microbes should be a porous structure with a rough and irregular surface for bacterial colonization, inexpensiveness, and non-toxicity [7]. Numerous types of microbial carriers can be divided into organic and inorganic materials and synthetic and natural materials [8]. The usage of natural materials has many ecological advantages against man-made plastic carriers. Natural organic materials such as corncob, straw, plant fibers, and rice are promising, but their utilization in AD is limited due to low resistance to biodegradation and stability in different pHs [8]. Natural inorganic materials such as zeolite, perlite, and porous glass have high chemical and physical resistance [8].

In this research, four different natural materials (natural zeolitized tuff, ZeoSand^®, perlite, and crushed corncob) were tested as carriers of a microbial consortium which is intended for bioaugmentation of anaerobic digesters using MxG as a substrate.

2. Materials and Methods

2.1. Isolation of Microbial Consortium

The microbial consortium was previously isolated and conditioned for the bioaugmentation of biogas production reactors that use MxG biomass as a substrate. An amount of 1 g of dried and ground biomass of MxG was weighed using a technical digital balance (Kern & Sohn GmbH Ziegelei, school balance EMB 200-3, Balingen, Germany). The sample was then incubated, with the addition of 1 mL of activated sludge from a wastewater treatment plant, at 37 °C for one week (Memmert GmbH, IN110m, Schwabach, Germany). The incubation was carried out anaerobically by tightly sealing Winkler bottles filled to the top and containing approximately 100 mL of minimal salt medium (MSM) (composition in g L⁻¹ of demineralized water; NaCl 2.5, K₂HPO₄ 0.47, KH₂PO₄ 0.56, MgSO₄ × 7H₂O 0.5, CaCl₂ × 2H₂O 0.1, NH₄NO₃ 2.5, pH 7.0 ± 0.2). In such a system, the MxG biomass served as the sole carbon source. After one week, an aliquot of 0.1 mL was inoculated from the Winkler bottles to MSM agar plates (15 g L⁻¹ of agar) and incubated for one week at 37 °C (Memmert GmbH, IN110m, Schwabach, Germany). After such conditioning, the colonies grown on the agar plates were presumably able to use the MxG biomass as a sole carbon source, and thus could be classified as MxG degraders. Alternating anaerobic/aerobic incubation was deliberately chosen to enable the growth of facultative anaerobic bacteria, which would be much simpler to cultivate in the subsequent steps, important from a biotechnological point of view. From the surface of the plates, the grown biomass was scraped and a bacterial solution was prepared and transferred to a Microbank^TM cryo-storage system (PRO-Lab Diagnostics, Richmond Hill, ON, Canada) for future use. This suspension was labeled as a microbial consortium.

2.2. Immobilization of Microbial Consortium on Carriers

Four different natural materials were tested as carriers. The natural zeolitized tuff (NZ) from a quarry in Donje Jesenje, Croatia, consisted of 50–55% clinoptilolite with lower amounts of celadonite, plagioclase feldspars, opal-CT (10–15% each), and traces of quartz and analcime [9]. The commercial mineral composition of the ZeoSand^® (Velebit Agro, Zagreb, Croatia) was a minimum of 80% clinoptilolite, and other mineral components were not listed in the safety data sheet. The expanded perlite was a commercial product intended for gardening (Special Mix B.V. manufactured by Gold Label, Aalsmeer, The Netherlands). The crushed corncob was from organic farming and not treated with pesticides and herbicides. A size fraction from 0.5 to 1 mm of NZ and ZeoSand^® was used. The zeolites were ground by orbital milling and sieved, to obtain the desired particle size fraction. The particle sizes of the perlite were 1–4 mm, as declared by the manufacturer, and the crushed corncob had irregular particles between 2 and 6 mm in size, as measured by a microruler. All materials were weighed to 0.5 g on a technical digital balance (Kern & Sohn GmbH Ziegelei, school balance EMB 200-3, Balingen, Germany), added to a glass Petri dish, and sterilized in a dry oven (Instrumentaria, Zagreb, Croatia) at 105 °C for 1 h prior to testing.

The consortium was revitalized from the Microbank^TM on Luria–Bertani (LB) agar plates incubated at 37 °C (Memmert GmbH, IN110m, Schwabach, Germany) for 24 h. The grown biomass was scraped from the plate and suspended in 10 mL of sterile saline solution to obtain the consortium suspension. To immobilize the consortium on each individual carrier, 0.5 g of sterilized carrier, 0.5 mL of consortium suspension, and 50 mL of sterile LB medium were added to sterile plastic vials. The control vial lacked the carrier. The vials were tightly sealed and maintained for 24 h in an incubator at room temperature (23 ± 1 °C) on a rotatory shaker (set to 5 rpm). Furthermore, the pure culture of E. indicum was immobilized on each carrier following the same procedure.

The number of bacteria immobilized onto each carrier was determined as follows: A supernatant was decanted from vials, and the carriers were washed twice with sterile saline solution (0.3%) to eliminate bacteria not attached to the carriers. Then, 20 mL of sterile saline was added, and the vials were vigorously shaken on a vortex (45 Hz/3 min). This way, the immobilized bacteria detach from the carrier and remain as planktonic cells in the supernatant [10]. The supernatant was serially diluted, inoculated on LB agar, and kept in an incubator (Memmert GmbH, IN110m, Schwabach, Germany) at 37 °C for 24 h. The grown colonies were counted, and the number of immobilized bacteria was expressed as log CFU g⁻¹ of the dry carrier.

2.3. Scanning Electron Microscopy Analysis (SEM)

To perform the SEM analysis, after the incubation and rinsing with sterile saline solution, several particles of each carrier with immobilized bacteria were transferred to sterile vial containing 2% paraformaldehyde solution and kept in a refrigerator at 4 °C/24 h for cell fixation. Next, the samples were washed with sterile saline and then dehydrated using ethanol in a stepwise manner: 2 min in 30% ethanol, 2 min in 50% ethanol, 5 min in 70% ethanol, 5 min in 96% ethanol, and twice in 100% ethanol for 5 min. After dehydration, the samples were dried for 30 min at 50 °C in dry oven (Instrumentaria, Zagreb, Croatia). For imaging, a single bioparticle was coated with a plasma of gold and palladium for 180 s and imaged by a TESCAN Vega3 EasyProbe SEM (TESCAN Group, Brno, Czech Republic) at an electron beam energy of 7 keV.

2.4. Identification, Antibiotic Susceptibility Testing, and Biochemical Characterization of Bacterial Species in the Microbial Consortium

The consortium suspension was diluted and inoculated onto LB agar plates. On certain plates, single colonies were visible, and several types of morphologically different colonies were distinguishable (Figure 1). By using this method, it is not possible to claim that these were the only bacterial species in the consortium, but it is safe to presume that these bacterial species were dominant in the consortium, since they remained in notable numbers after serial dilution. All the colonies that were clearly morphologically different were isolated in pure culture and subsequently identified with the MALDI-TOF mass spectrometry method (software version 3.0, Microflex LT, Bruker Daltonics, Billerica, MA, USA).

By counting the colonies on comparable plates, the share of each identified species in the consortium was determined and compared between the consortium suspension, the control group and the bacteria immobilized on the carriers (Figure 1).

All the experiments were done in triplicate and statistically compared by Student’s t-test with the significance margin set at p = 0.05.

Biochemical determination, including catalase and oxidase tests, was conducted for each bacterial species. For the catalase test, biomass was suspended in 3% H₂O₂ on a microscope slide, and the appearance of oxygen bubbles was considered a positive result. The oxidase test was conducted by spreading biomass on strips (Microbiologie Bactident^® Oxydase, Sigma-Aldrich Canada Co., Oakville, ON, Canada) and the appearance of a purple color was considered a positive result.

Susceptibility testing of E. cloacae, E. asburiae, K. pneumoniae, and L. adecarboxylata to imipenem was determined by a EUCAST standard disc diffusion assay [11]. The bacterial suspensions (0.5 McFarland units/10⁸ CFU mL⁻¹) were spread evenly over Mueller–Hinton Agar plates (MH) using a sterile swab. A disc of 10 µg imipenem (Liofilchem^®, Roseto degli Abruzzi, Italy) was placed on the surface of the agar and plates were incubated for 24 h at 37 °C. After the incubation, antibiotic cut-off points were determined following the EUCAST protocol.

3. Results

3.1. Immobilization of Microbial Consortium on Carriers

The bacterial consortium showed the highest affinity for the immobilization onto perlite, resulting in mean value of 1.44 × 10¹⁰ CFU g⁻¹ (Figure 2). The immobilization was in high numbers also on the crushed corncob with a value of 2.7 × 10⁹ CFU g⁻¹, followed by ZeoSand^® and NZ with 1.02 × 10⁹ and 6.12 × 10⁸ CFU g⁻¹, respectively. However, there was no statistically significant difference between carriers (p-value > 0.05) and therefore all materials can be considered as suitable for the immobilization of the microbial consortium.

3.2. SEM Analysis

The SEM analysis revealed bacterial cells attached to the surface of the different carriers. In the ZeoSand^® and NZ images, it was difficult to distinguish the cells clearly due to extracellular substances (Figure 3A,B). In the case of perlite, the cells are more clearly distinguishable and visible in microcolonies scattered all over the perlite surface and in the micropores (Figure 3C). The SEM analysis of crushed corncob revealed that the surface of the particles is full of outgrowths (Figure 4A), which are covered with attached cells (Figure 4B), thereby potentially increasing the surface area available for bacterial immobilization.

3.3. Share, Antimicrobial Resistance Profile and Biochemical Characteristics of Bacterial Species in Microbial Consortium

After cultivating the consortium on agar plates that were diluted enough to have single colonies clearly separated from each other, five types of morphologically different bacterial colonies were observed. These were re-inoculated to grow pure cultures, at least three colonies of each type. All of these were identified by the MALDI-TOF MS method, and with unambiguity (MALDI score above 2.0, interpreted as High Confidence Identification), five species were identified and reasonably reliably ascribed to a specific colony type: Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter asburiae, Leclercia adecarboxylata, and Exiguobacterium indicum. Results revealed that in consortium suspension, the dominant species was E. cloacae, followed by E. indicum and L. adecarboxylata, and in smaller share E. asburiae and K. pneumoniae. During immobilization, the shares change in favor of E. cloacae, which becomes an even more dominant species with 72–88% presence, and shares for other species range from 2 to 23% (Figure 5).

Interestingly, the share of E. indicum changed from 14% in consortium suspension to <1% during immobilization, and this was the same for all the tested carriers. The share of E. indicum in the control suspension was less than 1%, indicating that it was overgrown in competition with other bacteria and not because of an inability to immobilize. This was further confirmed in the experiment where a pure culture of E. indicum was immobilized on NZ (2.18 × 10⁷ CFU g⁻¹), ZeoSand^® (2.15 × 10⁸ CFU g⁻¹), perlite (4.48 × 10⁸ CFU g⁻¹), and corncob (2.93 × 10⁸ CFU g⁻¹) (Figure 6). There was no statistically significant difference between carriers (p-value > 0.05).

Based on biochemical tests, these bacterial species are catalase-positive and oxidase-negative, confirming they are facultative anaerobes. Since several of the identified species belonged to the Enterobacterales group, and are potential opportunistic pathogens, the antibiotic susceptibility to carbapenems was determined. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard readings for inhibition zone diameter, K. pneumoniae (24 mm), E. asburiae (22 mm), and L. adecarboxylata (22 mm) were categorized as susceptible to imipenem. The one diameter for E. cloacae was 21 mm and it was described as susceptible with increased exposure.

4. Discussion

Based on the results, all materials proved to be promising biocarriers for the microbial consortium intended to be used in biogas production. Perlite, an aluminosilicate material with high porosity, previously demonstrated great potential as a biocarrier for immobilization of phosphate-accumulating bacteria Acinetobacter junii, resulting in a high number of immobilized bacteria (12.65 × 10⁹ CFU g⁻¹) [12]. Its efficiency in augmenting biogas-producing reactors using olive waste as a substrate was also reported. A high number of immobilized bacteria on perlite suggests its suitability for various applications in biogas production [3]. There was no statistical difference in the number of immobilized bacteria between the NZ and ZeoSand^®. The most obvious difference between the two zeolite samples is in the fraction of clinoptilolite mineral, 50–55% in NZ and >80% in ZeoSand^®, suggesting that clinoptilolite content in the zeolitized tuff does not influence the immobilization of bacteria, already shown for other NZ samples [10]. On NZ, a great rate of immobilization was reported for bacteria such as A. junii (1.27 × 10¹⁰ CFU g⁻¹), Escherichia coli (4.53 × 10⁸ CFU g⁻¹), and Enterococcus faecalis (1.3 × 10⁹ CFU g⁻¹) [9]. Immobilization of microorganisms on NZ has proven to be beneficial for biogas production. In the anaerobic digestion of grass silage, bacteria immobilized onto NZ enhanced the enzymatic activity of xylanase, which is responsible for the degradation of xylan, a cell wall component in grass biomass [13].

Corncob is a material mostly made of lignin, cellulose, and hemicellulose. It has a high bulk density and provides a large amount of biodegradable carbon to immobilized bacteria [14]. Moreover, SEM visualization revealed a perforated structure with outgrowths that increased the surface area available for the attachment of bacteria. Corncob was shown to be suitable carrier for ureolytic bacteria due to its wrinkled and porous structure that allows bacteria to immobilize and survive [14]. However, it was confirmed that corncob is not an appropriate carrier for plant growth-promoting rhizobacteria due to high carbon content and poor adherence [15]. Furthermore, corncob was not ideal for enriching anammox bacteria, but it could be used in research on denitrifying bacteria [16].

In general, the success of bacterial immobilization onto carriers is affected by several physical and chemical properties of the materials, mostly the particle size, as it has been observed that an increase in particle size leads to a decrease in the number of immobilized bacteria [7]. In further research with materials used here, the particle size of NZ and ZeoSand^® could be reduced to a size of <0.125 mm, which has proven effective in achieving a higher number of immobilized bacteria [10]. In SEM images of perlite, aside from the outer surface, bacteria are visible in the micropores, which is consistent with the fact that the porous structure of perlite allows greater efficiency in the immobilization of bacteria [3]. In this research, expanded perlite was used, which is confirmed to influence the extent of bacterial immobilization compared to semi-expanded and non-expanded perlite [12]. To achieve the higher rate of immobilization, it is important for the carrier to have an irregular surface for bacterial colonization, as evidenced by many specific outgrowths on the SEM images of corncob. Further, previous research has examined the effect of the charge, expressed as zeta potential, of zeolite and perlite on the rate of immobilization. Bacterial cells are negatively charged, and the tested materials are natural and therefore also negatively charged, but not enough to be repulsive to the bacterial cells [10,11,12]. Therefore, surface charge could show a particular, but not crucial, effect on the rate of immobilization. The hydrophobicity presumably also did not affect the immobilization, since it was previously shown that bacteria, which are generally naturally hydrophobic, interact with both hydrophilic and hydrophobic surfaces and successfully immobilize onto NZ [7].

In view of this research, the isolated microbial consortium should not be considered a biogas producer but a biomass degrader. In the complex anaerobic digestion system, methane production is performed by methanogenic archaea, which were not a part of the described consortium. This consortium’s role was to help biodegrade complex organic biomolecules contained in MxG biomass, such as lignocellulose and cellulose, and to provide end products that methanogens could metabolize. In this manner, it is very well known that microbial consortia, compared to single species, are more advantageous for the degradation of complex compounds [17]. Microbial consortia can be either isolated or constructed. For construction, a bottom-up approach is to determine the genomes of individual species of a potential consortium and reconstruct metabolic networks and microbial interactions. This should allow the design of microbiomes with specific properties, such as distributed pathways, modular species interactions, and resilience, that would be optimized for specific ecosystem function and stability [17]. For actual bioaugmentation on an industrial scale, the approach must be as simple as possible and ready to be performed in a basic microbiological laboratory. This was the focus of the approach described here—to isolate the targeted consortia that can be adjusted to biodegrade various types of biomass, but in a simple and reproducible way, by focusing only on the bacteria that can be cultivated with ease, repeatedly for each new experiment. Using this parameter, we ended up with a consortium of five bacterial species that gave reproducible results and were identified with high certainty.

Detected species in the microbial consortium were E. cloacae, K. pneumoniae, E. asburiae, L. adecarboxylata, and E. indicum. These bacterial species are catalase-positive and oxidase-negative, meaning they are facultative anaerobic microbes [18,19]. Facultative anaerobic bacteria should be completely metabolically functional in the anaerobic digesters, and at the same time large amounts of biomass can be easily grown in aerobic conditions, without the use of specific anaerocultivators. The individual bacterial species from the bacterial suspension did not exhibit the same affinity for a specific carrier, whereas E. cloacae demonstrated an ability to be immobilized on each tested carrier in huge percentages (72–88%). The difference probably arises from either carrier affinity for E. cloacae or the best adaptation of this species to competition with other species in tested conditions. In a study by Dermayati et al. [20], a microbial consortium consisting of Enterobacter cloacae, Bacillus sp., and Bacillus licheniformis was immobilized on perlite, zeolite, silica, and vermiculite. The consortium was successfully immobilized on carriers, including E. cloacae, although with a lower cell loading capacity on zeolite (5.10 × 10⁶ CFU g⁻¹) and perlite (5.37 × 10⁶ CFU g⁻¹). The same microbial consortium immobilized on perlite improved the degradation of crude oil by 25% as compared to free-living cells [21].

The most significant proportion of L. adecarboxylata was on perlite (23%), which is a higher result compared to the proportion in the consortium suspension (13%). This indicates that perlite could be an appropriate carrier for L. adecarboxylata, but information regarding the immobilization on carriers or its potential implication in bioremediation is still lacking.

K. pneumoniae and E. asburiae could be used in the form of immobilized or free-living cells in the process of bioaugmentation. It was reported that E. asburiae is a promising approach for bioremediation of organochlorine pesticides in surface water [22]. K. pneumoniae could be used for the pre-treatment of food waste prior to anaerobic co-digestion with straw to decrease lipid content and enhance methane production [23].

Although the proportion of E. indicum in the bacterial suspension was 14%, it was not detected after the immobilization of the consortium on any carrier. This bacterial species was present in significant amounts in the original consortium suspension but was absent after incubating the consortium in nutrient media without the carrier. However, after incubation of the pure axenic culture, it was successfully multiplied and immobilized. Therefore, the assumption is that E. indicum vanishes in competition with the other four species of the consortium.

One thing to consider in biotechnology is that it is important not to use potentially pathogenic bacteria due to the risk of them leaking into the environment. Although K. pneumoniae was characterized as susceptible to imipenem, it is well known as a member of the ‘ESCAPE’ pathogens, a group of clinically significant and multi-drug-resistant bacteria [24]. Instead of using potentially pathogenic bacteria, the microbial consortium can be adjusted to avoid risking public health. For further research, K. pneumoniae could be removed or replaced with other bacterial species.

To verify that the carriers described here could be successfully used for bioaugmentation of large-scale anaerobic digesters, the capacity for biogas yield will be tested for each carrier in subsequent research. Encouraging results have already been obtained when a bacterial consortium obtained in a similar manner was used to bioaugment reactors with olive residues as a substrate and perlite as a carrier [3]. Next, the long-term stability and performance of the carriers in the reactors need to be tested. Since all the carriers except for the crushed corncob are minerals, no degradation in the anaerobic sludge is expected. In theory, the bioparticles would act as seeds for the added organisms for a longer period of time; however, the duration needs to be determined.

When considering large-scale usage of the described biocarriers, environmental impact is very important. Therefore, the chosen carriers are completely natural and have no negative impact on the environment, making them safely disposable along with the surplus sludge. This was one of the reasons these carriers were chosen. Another reason is that all of the carriers are easily commercially obtainable and feasible, so scaling up the described technology to an industrial scale should not be a challenge. Probably the highest risk is maintaining the bacterial consortium. Therefore, all procedures were designed to be easily replicable; the consortium is kept in a Microbank and revitalized for each replicate experiment, proving its stability. The growth occurred on standard, non-fastidious nutrient media, and in aerobic conditions or tightly sealed containers, without the need for sophisticated anaerobic cultivation equipment. The five species in the consortium are only a small part of the natural diversity of the microbial consortiums in the anaerobic sludge, but according to the shown experiments, these bacterial species were targeted for the degradation of a specific substrate, in this case the Mischantus grass. If added in large enough numbers in the reactors, attached to the biocarriers, they presumably should increase the biogas yield. However, following research must pair cultivation experiments with metagenomics, to obtain a larger and more comprehensive picture on how bioaugmentation affects the composition of naturally found microbiota. Hopefully, all the mentioned factors should make scaling-up possible and feasible.

5. Conclusions

This research unequivocally demonstrated that the microbial consortium, consisting of several species intended for bioaugmentation of anaerobic digestion process, could successfully be immobilized onto natural biocarriers. All the tested materials showed a similar capacity for bacterial immobilization and can be considered promising for the reinforcing of biogas production. The experimental setup was designed to be used in real-life systems for anaerobic digestion, using easily cultivable species.

In this manner, further steps should be to identify the potential of each individual species for biomass degradation and maybe use principles of systemic biology to construct an optimal bacterial consortium immobilized onto various biocarriers.

Presumably, this technology could significantly increase the biogas yield by optimizing the degradation of complex organic compounds within the biomass, thereby enhancing the efficiency and rate of biogas production.