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Article

Impact of Granular Activated Carbon on Anaerobic Process and Microbial Community Structure during Mesophilic and Thermophilic Anaerobic Digestion of Chicken Manure

by
Elvira E. Ziganshina
,
Svetlana S. Bulynina
and
Ayrat M. Ziganshin
*
Department of Microbiology, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, Russia
*
Author to whom correspondence should be addressed.
Sustainability 2022, 14(1), 447; https://doi.org/10.3390/su14010447
Submission received: 17 November 2021 / Revised: 15 December 2021 / Accepted: 20 December 2021 / Published: 1 January 2022
(This article belongs to the Special Issue Sustainability and Anaerobic Digestion Technologies)
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Abstract

:
In this work, the impact of granular activated carbon (GAC) on the mesophilic and thermophilic anaerobic digestion of chicken manure and the structure of microbial communities was investigated. These results demonstrated that GAC supplementation effectively enhanced the consumption of produced organic acids in the mesophilic and thermophilic batch tests, accompanied by faster biomethane production in the presence of GAC than from reactors without GAC. However, since the free ammonia level was 3–6 times higher in the thermophilic reactors, this led to the instability of the anaerobic digestion process of the nitrogen-rich substrate at thermophilic temperatures. Bacteroidia and Clostridia were the two main bacterial classes in the mesophilic reactors, whereas the class Clostridia had a competitive advantage over other groups in the thermophilic systems. The archaeal communities in the mesophilic reactors were mainly represented by representatives of the genera Methanosarcina, Methanobacterium, and Methanotrix, whereas the archaeal communities in the thermophilic reactors were mainly represented by members of the genera Methanosarcina, Methanoculleus, and Methanothermobacter. New data obtained in this research will help control and manage biogas reactors in the presence of GAC at different temperatures.

1. Introduction

Anaerobic digestion is an environmentally friendly biotechnological process widely used to treat various organic waste materials while reducing waste generation and producing biomethane as a renewable energy source [1,2]. Other benefits associated with the anaerobic digestion include reducing greenhouse gas emissions, recycling nutrients, generating additional income for farmers, and reducing soil and water pollution [3,4].
However, despite the validity of the anaerobic digestion process, it is difficult to maintain the anaerobic reactors’ stability because of the instability of pH, the accumulation of toxic compounds, which inhibit the growth of microorganisms, and other important factors [3,5]. To better exploit the benefits of anaerobic digestion technology, active efforts have been made to prevent disturbances and improve energy efficiency using different methods [6,7,8,9]. As more and more animal products are required to meet the needs of a growing world population, animal waste’s environmental impact and the need to remove manure become more and more important [10]. Anaerobic digestion is a beneficial and sustainable method for converting these wastes into biomethane and digestate. The digestate can be further used as a bio-fertilizer [1].
Chicken manure, as one of the primary organic waste materials, needs to be effectively utilized. The anaerobic digestion of chicken manure is considered a good choice for minimizing this waste and recovering bioenergy. However, due to the high content of organic nitrogen and the low C/N ratio in chicken manure, ammonia inhibition is the main problem encountered in practical applications. Much research has been done to find out the cause and find a solution to reduce the ammonia inhibition [3,6,9,11,12,13], but more work needs to be done to restore the ammonia-inhibited process.
Process temperature is another important parameter that affects anaerobic digestion. The thermophilic (50–60 °C) anaerobic process has advantages over the mesophilic (35–40 °C) process due to such benefits as the improved breakdown of volatile solids and therefore the improved biomethane production, as well as the removal of pathogens [14]. On the contrary, mesophilic anaerobic digestion provides a stable biomethane yield with lower heat input compared to thermophilic anaerobic digestion. With the advances in technology, thermophilic anaerobic digestion has attracted attention due to its faster reaction rates compared to process performed at mesophilic temperatures, which improves the breakdown of solids and the production of biogas [15,16]. However, thermophilic microorganisms are more sensitive to environmental conditions and high ammonia levels in the nutrient medium than microorganisms growing at mesophilic temperatures [17] and, therefore, require further research.
During the anaerobic digestion process, various organic compounds are transformed in several stages by the activity of anaerobic microorganisms with different metabolic abilities. In the last step of the anaerobic process, methanogenesis, methane is formed through three major pathways: acetoclastic, hydrogenotrophic, and methylotrophic pathways [18]. Besides, direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea has recently been demonstrated in anaerobic reactors to enhance the syntrophic transformation of various reduced organic compounds to methane in the presence of conductive carbon materials (granular activated carbon, biochar, and carbon cloth) [19,20,21,22,23] and iron-bearing minerals [24,25]. However, studies aimed at studying the effect of different concentrations of carbon-based conductive materials on the anaerobic conversion of nitrogen-rich poultry manure, both in mesophilic and thermophilic conditions, are scarce.
The balanced interaction between various anaerobic microorganisms during the substrate conversion process is essential for the continuous transformation of the intermediate products formed and, therefore, for efficient methane production. New knowledge about the bacteria and methanogenic archaea of anaerobic reactors is of practical interest for the subsequent monitoring of the anaerobic digestion process, reducing disturbances in the technological process, and increasing the efficiency of biogas reactors. It is essential to study the microbial community structure, interactions, and relationships with operational conditions to enhance the methane production [23,24,25,26,27].
In this study, the influence of granular activated carbon on the mesophilic and thermophilic anaerobic digestion of chicken wastes was investigated. The reactors’ performance was assessed in terms of the production of organic acids with their further conversion into methane at various temperatures. Bacterial and archaeal 16S rRNA genes were further characterized to clarify the structure of microbial communities in these anaerobic digesters. The experimental results have laid a solid foundation for improving the anaerobic digestion of chicken manure in terms of engineering applications.

2. Materials and Methods

2.1. Experimental Setup

Chicken manure with total solids (TS) of 70.0 ± 0.56% and volatile solids (VS) of 60.1 ± 0.68% was collected from a local chicken farm (the Republic of Tatarstan, Russia). The inoculum to start up the anaerobic reactors was the digested cattle manure at mesophilic and thermophilic temperatures. The inoculum for the mesophilic experiments had the TS content of 4.99 ± 0.10% and the VS content of 3.65 ± 0.09%, while the inoculum for the thermophilic experiments had the TS content of 5.22 ± 0.14% and the VS content of 4.02 ± 0.12%. Coconut-based granular activated carbon Silcarbon K835 (0.5–2.38 mm particle size; Germany) was used in this study.
Biochemical methane potentials of chicken manure under both conditions (38 °C and 55–50 °C) were determined with Automatic Methane Potential Test Systems (AMPTS II Light, Bioprocess Control, Sweden) for 30–40 days. The released biogas was initially passed via a 3M NaOH solution to remove carbon dioxide and hydrogen sulfide gases. After that, the methane production was estimated by using a gas flow meter system. The mesophilic experiments were conducted in 2 L flasks with a working volume of 1.6 L, including 71.25 g of chicken manure, inoculum, and distilled water (at a TS concentration of 6.8%). The ratio of inoculum to substrate was 42.82 g/42.82 g (calculated as VS). Thermophilic experiments were performed in 2 L flasks with a working volume of 1.6 L, 70.95 g of chicken manure, inoculum, and distilled water (at a TS concentration of 6.6%). The inoculum to substrate ratio was 42.64 g/42.64 g (calculated as VS).
Granular activated carbon at concentrations of 5.00 g L−1 and 10.00 g L−1 was separately added to mesophilic experimental reactors (labeled as M3_GAC_5, M4_GAC_5, M5_GAC_10, and M6_GAC_10) and thermophilic experimental reactors (labeled as T3_GAC_5, T4_GAC_5, T5_GAC_10, and T6_GAC_10). Control mesophilic and thermophilic reactors were operated in the absence of GAC (marked as M1_C, M2_C, T1_C, and T2_C). Due to the addition of granular activated carbon to the experimental reactors, their working volume and final TS content were slightly higher than those observed in the control reactors. Blank rectors (with inoculum and water) were additionally used to compensate for the level of CH4 produced by the inoculum itself. All anaerobic reactors were stirred at 60 rpm for 2 min with a 2-min rest interval.

2.2. Analytical Methods

Process parameters and analytical techniques were measured as described previously [23,25]. Briefly, the methane values generated from AMPTS II Light instruments were normalized to standard conditions. Biogas was periodically sampled, and its composition was quantified by using a gas chromatograph Clarus 580 (Perkin Elmer, Singapore). TS and VS were analyzed at the beginning and the end of the experiment using a drying oven and a muffle oven, respectively. pH was measured with a Starter ST300 pH meter (OHAUS Corporation, Shanghai, China). The acid capacity and the ratio of volatile organic acids (VOA) to total inorganic carbon (TIC) were investigated by titration using a bottle-top burette Titrette (Brand, Wertheim, Germany). Total ammonia nitrogen (TAN) was measured by Nessler’s reagent photometry (Sigma-Aldrich, St. Louis, MO, USA) using a Lambda 35 Spectrophotometer (Perkin Elmer, Singapore). Free ammonia nitrogen (FAN) was calculated from TAN values [28]. All these analyses were measured in triplicate, and the average values are presented together with the standard deviations. The ANOVA or T-test were used to compare differences, and p values < 0.05 were considered to indicate statistical significance.

2.3. Microbial Community Analysis

The composition of bacterial and archaeal communities in chicken wastes, inocula, and mesophilic and thermophilic anaerobic batch reactors was investigated by molecular methods targeting 16S rRNA genes, as explained previously [23,29]. Briefly, the chicken manure and inocula samples were collected before the experiments’ start, while the effluent sample of each digester was collected on day 7 of the experimental period (when differences in gas level were observed). Each sample’s genomic DNA was extracted using a FastDNA spin kit for soil (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s instructions. The primer sets Bakt_341F (5′-CCT ACG GGN GGC WGC AG-3′) and Bakt_805R (5′-GAC TAC HVG GGT ATC TAA TCC-3′) were used to amplify the bacterial 16S rRNA gene fragments, whereas Arch349F (5′-GYG CAS CAG KCG MGA AW-3′) and Arch806R (5′-GGA CTA CVS GGG TAT CTA AT-3′) were used to amplify the archaeal 16S rRNA gene fragments. We used Illumina MiSeq deep sequencing with 2 × 300 bp reads. The obtained sequence data were then analyzed with the QIIME pipeline [30]. Operational taxonomic units (OTUs; 97% identity threshold) representing less than 0.01% of the total reads were eliminated from further analysis. Alpha diversity was assessed on an OTU level. Unique and shared microbial OTUs were additionally visualized using the Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed on 15 August 2021)). For the taxonomic assignment of OTUs, the Silva database [31] was used.

3. Results and Discussion

The efficiency of the anaerobic digestion of chicken manure in the mesophilic and thermophilic anaerobic batch reactors was evaluated by the degradation of volatile solids with the production of biomethane. Moreover, the volatile organic acids, VOA/TIC ratio, pH, TAN, and FAN levels in all these reactors were investigated. Batch treatments were conducted at 38 °C and 55–50 °C for 30–40 days to investigate the impact of granular activated carbon at concentrations of 5 g L−1 and 10 g L−1 on the efficiency of the anaerobic digestion of chicken wastes. In addition, the structure of several functional guilds of microbes in these reactors was examined by the amplicon sequencing of 16S rRNA genes.

3.1. Process Stability and Methane Production in Mesophilic Experiments

The specific methane production, methane flow rate, and total methane yield from mesophilic (38 °C) anaerobic reactors loaded with chicken manure in the absence of GAC and the presence of different concentrations of GAC (5 g L−1 and 10 g L−1) are presented in Figure 1. After a concise start period (1–2 h), the GAC-free and GAC-containing anaerobic reactors started to produce methane, and their operation was completed within 30 days (Figure 1a). The addition of GAC (5 g L−1 and 10 g L−1) enhanced the methane flow rate. The methane release from GAC-supplemented and GAC-free mesophilic reactors reached a plateau within 25–30 days. Methane was produced faster by reactors in the presence of GAC at concentrations of 5 g L−1 and 10 g L−1 than by GAC-free reactors. The specific methane production reached 190 ± 0.83 mL g−1VS, 198 ± 1.19 mL g−1VS, and 193 ± 0.46 mL g−1VS from M_C, M_GAC_5, and M_GAC_10, respectively (Figure 1a). The maximum methane production rate from the mesophilic reactors increased by 22.1% in the presence of GAC (5 g L−1) compared to the control reactors (p < 0.01). The mean maximum peaks in methane production from reactors M_C, M_GAC_5, and M_GAC_10 were 757 ± 12 mL, 893 ± 19 mL, and 925 ± 14 mL, accordingly, and the corresponding time was day 6 for control systems and day 5 for experimental systems (Figure 1b). Statistically significant differences were detected for the total methane yield between experiments performed in the presence of 5 g L−1 GAC and the absence of GAC (p < 0.05). In total, 8150 ± 36 mL, 8487 ± 51 mL, and 8283 ± 20 mL of CH4 were produced from all three treatments (0 g L−1, 5 g L−1, and 10 g L−1, respectively) (Figure 1c).
On day 3, the average acid capacity in the mesophilic anaerobic reactors M_C, M_GAC_5, and M_GAC_10 reached 6.19 ± 0.11 g L−1, 5.99 ± 0.13 g L−1, and 5.88 ± 0.12 g L−1, respectively. Concentrations of volatile organic acids in GAC-supplemented anaerobic reactors (M_GAC_5 and M_GAC_10) were significantly lower than those detected in GAC-free reactors on days 7–14 (p < 0.05), indicating the more effective initial destruction of short-chain fatty acids in the presence of activated carbon. Most of the produced volatile organic acids in all reactors were effectively converted to methane during the entire experimental process (Figure 2a). The VOA/TIC ratio in all experiments reached values of 0.65–0.73 on day 3 but finally decreased to 0.10–0.14, as illustrated in Figure 2b. GAC-containing treatments’ values were lower than the values observed in the GAC-free experiments on days 3–14. Table 1 demonstrates the TAN and FAN concentrations measured in mesophilic biogas reactors on selected days. TAN concentrations increased during the experimental period and were comparable in various anaerobic reactors (2.35–2.65 g L−1 on day 23; p > 0.05). In the case of FAN values, their concentrations reached 105–129 mg L−1 in different reactors on day 23 (Table 1).
GAC is an inexpensive material that is produced from carbon-containing raw materials through physical and chemical activation and is often used as a biocarrier or adsorbent in various wastewater treatment processes. Studies that investigated the effect of GAC on the anaerobic digestion of various substrates in mesophilic reactors include the following examples. The results obtained by Yang et al. [21] demonstrate that in reactors containing GAC (up to 5 g L−1), the rate of methane formation and reduction of waste-activated sludge increased. The GAC has enriched several groups of microorganisms, resulting in increased substrate consumption and methane production rates. Capson-Tojo et al. [22] showed that the addition of GAC (10 g L−1) promoted biomass acclimatization, improved acetic acid utilization, and enhanced methane production during the anaerobic digestion of food waste. In our recent work [23], the GAC-containing reactors (5–10 g L−1) significantly enhanced the methane production rate during the anaerobic co-digestion of sugar beet pulp and distiller grains. Kang et al. [26] showed that, with a decreasing inoculum concentration, bioreactors with a culture medium and supplemented with 1 g of GAC (mass/volume) were characterized by faster CH4 release rates and a short lag phase. Peng et al. [32] demonstrated that GAC (27 g L−1) enhanced the syntrophic metabolism between distinct bacteria and methanogens during the anaerobic digestion of waste-activated sludge due to its high electrical conductivity and large surface area. The addition of activated carbon during the anaerobic digestion of residual chicken blood also improved the process performance substantially, as was demonstrated by Cuetos et al. [33].

3.2. Process Stability and Methane Production in Thermophilic Experiments

The SMP, methane flow rate, and total methane production from thermophilic reactors converting chicken manure are demonstrated in Figure 3. Methane was released faster from GAC-containing reactors than from GAC-free reactors. However, when exposed to higher temperatures (55 °C), the initial methane flow rate decreased compared to the treatments with lower temperatures (38 °C) (Figure 1 and Figure 3). After a 10-day experimental period, the inhibition of the process was observed, and on day 19 the operating temperature in reactors T_C and T_GAC_5 was changed to 50 °C at 1 °C day−1 (the experiments with T_GAC_10 reactors were stopped). The control and T_GAC_5 reactors were then partially recovered, and their operation was continued until day 40. The specific methane production from T_C and T_GAC_5 achieved 156 ± 2.09 mL g−1VS and 170 ± 2.27 mL g−1VS, respectively (Figure 3a). The maximum CH4 production rate from the thermophilic reactors increased by 13.3% in the presence of GAC (10 g L−1) compared to the control reactors (p < 0.05). These values are significantly lower compared with the values observed at mesophilic temperatures (p < 0.05). The mean maximum peaks in CH4 production from all reactors T_C, T_GAC_5, and T_GAC_10 were 654 ± 23 mL, 705 ± 22 mL, and 744 ± 22 mL, respectively, and the appropriate time was day 6 for T_C and T_GAC_5, and day 5 for T_GAC_10 (Figure 3b). Statistically significant differences were observed for total methane production between experiments conducted in the presence of GAC (5 g L−1) and in the absence of GAC (p < 0.05). Finally, 6661 ± 89 mL and 7269 ± 97 mL of CH4 were generated from two thermophilic group reactors (0 g L−1 and 5 g L−1, respectively) (Figure 3c).
On day 3, the mean acid capacity in reactors T_C, T_GAC_5, and T_GAC_10 achieved 5.76 ± 0.15 g L−1, 5.67 ± 0.14 g L−1, and 5.56 ± 0.16 g L−1, respectively (Figure 4). The concentrations of organic acids in GAC-added reactors were significantly lower than the concentrations of acids detected in GAC-free reactors on day 7 (p < 0.05). This means a more effective initial conversion of short-chain fatty acids in the presence of GAC, also at thermophilic temperatures. However, compared to mesophilic experiments, the produced acids in all thermophilic reactors were not effectively converted to methane during the whole experimental period. The process at thermophilic temperatures was possibly inhibited due to a higher level of free ammonia in these experiments. Table 2 shows TAN and FAN concentrations measured in the thermophilic reactors on selected days. Therefore, the operation temperature in T_C and T_GAC_5 was changed from 55 °C to 50 °C on day 19. This resulted in a decrease of free ammonia levels (data not shown) and a recovery of the process. However, most of the organic acids have not yet been utilized by microorganisms. As seen in Table 1 and Table 2, the average TAN levels in the mesophilic and thermophilic reactors were comparable, but the FAN level was 3–6 times higher in the thermophilic reactors. This led to an instability of the anaerobic digestion of chicken manure at thermophilic temperatures.
Investigations of the effect of GAC, as well as other conductive materials, on the thermophilic anaerobic digestion are very limited, which to a certain extent limits the further development of these technologies. So, among a small list of these studies, we note a work of Jan et al. [34] in which GAC (10 g L−1), as well as carbon nanotubes (1 g L−1), were added to anaerobic reactors and tested at a thermophilic temperature (55 °C). The results showed a reduced start-up period of the thermophilic process, a doubled maximum methane-producing rate, and a lower accumulation of volatile fatty acids in reactors supplemented with conductive materials. The influence of biochar, another type of conductive material, on the anaerobic digestion of dry dairy manure was investigated by Jang et al. [35], where various concentrations of biochar were tested under different temperature conditions (20 °C; 35 °C; 55 °C). Researchers demonstrated that the addition of biochar (10 g L−1) enhanced the methane production rate and shortened the lag period in all tested conditions. The results of the above works and the present study demonstrate that carbon-based materials can improve the thermophilic process performance.
Research aimed at elucidating the mechanisms and participants of direct interspecies electron transfer in microbial consortia will help us improve the anaerobic digestion process [21,22,23,24,27]. The addition of conductive materials to the tested anaerobic systems may have allowed the electrons generated in the bacterial stages of biomass conversion to be directly consumed by methanogens, thus increasing the rate of methanogenesis. Considering that certain microorganisms (bacteria and archaea) can adhere to the surface of conductive materials and exchange electrons through them [36,37,38], we can assume that the improvement in the methane flow rate in our anaerobic systems supplemented with GAC, as a relatively large conductive material, can be explained by providing a large surface area by GAC to which some electroactive microorganisms can attach. However, we also cannot exclude the possibility of an enhanced mediated interspecies electron transfer (MIET), because of the formation of biofilms on the surface of GAC, which made it possible to reduce the distance between microorganisms and improve the kinetics of the anaerobic process. In addition, GAC adsorbs various inhibitory components. The results of this study also demonstrated that the mesophilic and thermophilic GAC-containing reactors had a higher rate of conversion of organic acids to methane, but the thermophilic reactors supplemented with the carbon-based conductive material had high levels of free ammonia level, as did the control reactors (Table 2). Considering the instability of the thermophilic anaerobic digestion process at high FAN levels, further studies are required to elucidate the mechanisms of interspecies electron transfer in the presence of conductive materials of various compositions, especially under thermophilic conditions and during the conversion of complex substrates.

3.3. Description of the Bacterial Community Structure

An increasing number of works have proved that individual microorganisms are capable of efficient direct electron transfer within associations through conductive carbon materials, including GAC and biochar, as well as iron-bearing minerals, such as magnetite [37,38,39,40]. This makes the study and discovery of new electroactive microbes a topical issue in biology and biotechnology. In this work, the impact of granular activated carbon on the mesophilic and thermophilic anaerobic digestion of chicken manure as a nitrogen-rich substrate was investigated. The bacterial communities of chicken manure, inocula for mesophilic and thermophilic treatments, and samples obtained on day 7 from GAC-free reactors (M1_C and T1_C) and GAC-supplemented reactors (M4_GAC_5 and T4_GAC_5) were further characterized by the amplicon sequencing of bacterial 16S rRNA genes.
After the quality-filtering steps, ~569 thousand high-quality 16S rRNA gene sequences were obtained with an average of 81,256 per sample (from 57,280 to 130,476). Alpha diversity indices were further calculated on the OTU level to estimate the diversity and richness of the bacterial community of each sample, and these values are summarized in Table 3 (abundance > 0.01%). It should be noted that the mesophilic and thermophilic systems in the presence and absence of GAC differed in their alpha diversity indices. Relatively low values of the Shannon index (the index values increase as the number of species in the sample increases and reaches the maximum when all species in the sample have the same abundance) and the Simpson index (which indicates species dominance) were observed in chicken manure and samples from the thermophilic reactors. The Chao1 index (which estimates the total species richness) values were noted as high for all mesophilic samples. The instability of the anaerobic digestion of nitrogen-rich chicken manure at thermophilic temperatures might be due to the scarcity of the diversity of bacterial communities in these systems (Table 3).
The Venn diagram was constructed to visualize the shared and unique OTUs between samples (Figure 5). The number of shared OTUs over the four mesophilic and thermophilic samples was 220, the number of shared OTUs over the two mesophilic samples was 188, and the number of shared OTUs over the two thermophilic samples was lower and reached 100. While samples M1_C and M4_GAC_5 comprised 2 and 4 unique OTUs, respectively, the number of unique OTUs in T1_C and T4_GAC_5 comprised 3 and 2 unique OTUs, accordingly.
OTUs present in chicken manure, in mesophilic and thermophilic inocula, in two samples from the mesophilic reactors (M1_C as a GAC-free control reactor and M4_GAC_5 as an experimental reactor), and in two samples from the thermophilic reactors (T1_C as a GAC-free control reactor and T4_GAC_5 as an experimental reactor) were distributed within 24 phyla and 38 classes. Chicken manure contained a high level of members of the phyla Firmicutes and Actinobacteria. Firmicutes was the substantial bacterial phylum in all samples with complete dominance in the thermophilic anaerobic reactors (up to 94% of the total 16S rRNA gene sequences). Bacteroidetes was the major bacterial phylum in the mesophilic anaerobic reactors (up to 46%) and the second main phylum in the mesophilic inoculum, with an average relative abundance of 28%. Compared to the mesophilic inoculum, the thermophilic inoculum had a higher relative abundance of Firmicutes (92%) and a lower relative abundance of Bacteroidetes (2.3%). Other phyla representing more than 1% of the sequences in at least one of the samples were Chloroflexi, Cloacimonetes, Fibrobacteres, Patescibacteria, Planctomycetes, Proteobacteria, Spirochaetia, Synergistia, and Tenericutes (data not shown).
Members of the class Clostridia were detected at high levels in all reactors, showing a higher abundance in the thermophilic reactors (up to 90% of the total 16S rRNA gene sequences; Figure 6). This class was also the main one for both mesophilic and thermophilic inocula (48% and 90%, respectively). In chicken manure, these Gram-stain-positive, anaerobic, spore-forming bacterial cells accounted for no more than 3% of all bacterial 16S rRNA gene sequences. It should be noted that a high proportion of bacteria of the class Clostridia is characteristic of various animal feces and suggests the presence of a large proportion of pathogenic bacteria in substrates that require proper disposal [41]. However, the observed importance of the class Clostridia has been previously reported during the mesophilic and thermophilic anaerobic digestion of various agricultural wastes, including manure [42,43,44]. The importance of the representatives of Clostridia is reflected in their participation in the stages of hydrolysis, acidogenesis, and acetogenesis [45,46]. It is important to note that several studies demonstrated the syntrophic acetate-oxidizing (SAO) activity within representatives of the phylum Firmicutes, including representatives of the class Clostridia [47,48]. Thus, an increase in the proportion of microorganisms of Clostridia in our thermophilic anaerobic systems treating chicken manure as substrate containing high concentrations of nitrogen may also indicate their participation in syntrophic acetate oxidization [49,50].
In the mesophilic anaerobic systems, the most important class was Bacteroidia (up to 46%); its share in thermophilic systems did not exceed 0.4%. The representatives of the class Bacteroidia belong to a large group of bacteria with a wide range of abilities. They are involved in the hydrolysis of polysaccharides and proteins, as well as in the fermentation of sugars with the production of various organic acids, which emphasizes their importance for the final stage of anaerobic digestion, methanogenesis [51]. Regarding the differences in the composition of the bacterial communities of the studied anaerobic systems at different temperature regimens, the abundance of Bacteroidia under thermophilic conditions sharply decreased, with a clear predominance of Clostridia (Figure 6). Our data on the competitive advantage of bacteria within Firmicutes over the representatives of Bacteroidetes during the thermophilic anaerobic conversion process are consistent with the results of previous studies [44,52,53]. Bacteroidia and Clostridia were the key groups of microorganisms during the mesophilic anaerobic digestion of beet pulp and distillers grains with solubles in the reactors supplied with GAC [23] and magnetite [25] used as agents to improve biogas production. As for the substrate for methane production, the most important bacterial classes detected in poultry manure were Bacilli, with an average relative abundance of 65%, and Actinobacteria, with an average relative abundance of 21%, while the percentage of representatives of the classes Clostridia and Bacteroidia was 3% and 6%, respectively (Figure 6).
On a genus taxonomic scale, there were many OTUs that showed differences between samples. Figure 7 demonstrates a heat map of the relative abundances of the most common genera detected in different samples, including the substrate itself, inocula, and samples from the mesophilic and thermophilic treatments with and without GAC.
The bacterial community of chicken manure was mainly represented by the genera Virgibacillus, Lactobacillus (Bacilli), and Brevibacterium (Actinobacteria), with average relative abundances of 32%, 15%, and 13%, respectively. Although similarities in the composition of the microbial communities were found between M1_C and M4_GAC_5 on the phylum and class levels, differences in the composition of the bacterial communities were observed on a genus level. Thus, the relative abundances of OTUs assigned to an uncultured bacterium within Bacteroidales UCG-001, Proteiniphilum, DMER64 (Rikenellaceae), LNR A2-18 (Cloacimonadaceae), Sedimentibacter, uncultured Syntrophomonadaceae, and some others were higher in M4_GAC_5 (Figure 7). The Syntrophomonadaceae family (Clostridia), which includes the genera Syntrophomonas, Pelospora, Syntrophothermus, and Thermosyntropha, is involved in the degradation of fatty acids in close association with hydrogen- or formate-utilizing partner organisms [54]. Species affiliated with the genus Sedimentibacter are strictly anaerobic and likely to produce acetic, propionic, and butyric acids, but only from several substrates [55,56]. Members of the genera DMER64 (Rikenellaceae) [57] and Syntrophomonas [58] have been proposed as bacteria that can potentially create magnetite-mediated DIET with several methanogenic archaea. The slight predominance of these and some other microorganisms in our GAC-containing systems allows them to be considered as microorganisms capable of GAC-mediated DIET.
A substantial difference in the structure of bacterial communities was observed between the mesophilic and thermophilic treatments. Ruminiclostridium 1 and uncultured bacteria within the MBA03 of Clostridia were more abundant in the T1_C and T4_GAC_5 reactors, with a slight margin in the experimental reactor (Figure 7). The genus Ruminiclostridium unites anaerobic bacteria which have strategies for the depolymerization of cellulose and the related plant cell wall polysaccharides, which make it possible to classify them as promising prokaryotes for various biotechnologies [59,60]. The mesophilic and moderately thermophilic isolate R. herbifermentans sp. nov. was isolated from a lab-scale anaerobic digester fed with maize silage. It ferments arabinose, cellobiose, crystalline, and amorphous cellulose, ribose, and xylan, and produces mainly acetate and ethanol [61]. Our results showed that the addition of GAC could also increase the abundance of Ruminiclostridium 1 and MBA03 within Clostridia under thermophilic conditions. Furthermore, the level of Herbinix and Pseudomonas members with different metabolic abilities were higher in the thermophilic systems (Figure 7).

3.4. Description of the Archaeal Community Structure

Since the presence of certain groups of methanogenic archaea and their abundance in anaerobic systems directly affect the rate and efficiency of methanogenesis, the structure of the archaeal communities was assessed in both the mesophilic and thermophilic reactors operating without (M1_C and T1_C) and with the addition of granular activated carbon (M4_GAC_5 and T4_GAC_5). More than 438 thousand high-quality 16S rRNA gene sequences were obtained after processing six samples (two inocula and four reactors’ samples) with an average of 72,951 per sample (from 47,641 to 105,104). The alpha diversity indices calculated on the OTU level are shown in Table 3 (abundance > 0.01%). The number of archaeal OTUs in effluent samples from the mesophilic treatments was higher than those observed in the thermophilic reactors; the diversity indices were comparable in the systems with and without granular activated carbon.
The Venn diagram was constructed to visualize the shared and unique OTUs between samples (Figure 8). The number of shared archaeal OTUs over the four mesophilic and thermophilic samples was 29, the number of shared OTUs over the two mesophilic samples was 10, and the number of shared OTUs over the two thermophilic samples was lower and reached 4.
A total of 95–99% of archaeal 16S rRNA gene sequences amplified from the mesophilic and thermophilic GAC-free reactors and GAC-containing reactors belonged to the classes Methanomicrobia, Methanobacteria, and Thermoplasmata, within the phylum Euryarchaeota, while only one class Bathyarchaeia within the phylum Crenarchaeota was detected in all anaerobic systems. Communities that functioned in the absence and presence of GAC showed a relative similarity with a variation in the proportion of individual representatives. Methanomicrobia was a major group in mesophilic systems, accounting for 76–81% of the archaeal communities, and in the thermophilic reactors, reaching 65–72%. Methanobacteria was the second most important group within the studied anaerobic systems, with a preponderance in the thermophilic reactors (up to 32%) (data not shown).
The reactor-specific profile of the archaeal communities (the genus level) in the mesophilic and thermophilic reactors is demonstrated in Figure 9. On the genus level, Methanosarcina (Methanomicrobia) was the dominant group in the mesophilic GAC-free and GAC-containing reactors, accounting for 64–69% of the archaeal communities. In the thermophilic reactors, Methanosarcina remained abundant at the level of 46–58% of the total 16S rRNA gene sequences, but it was slightly pushed by strict hydrogenotrophic competitors (Figure 9). Representatives of the genus Methanosarcina have been repeatedly mentioned as the main methanogens in various anaerobic digesters [25,62,63]. It should be noted that this group of archaea in the mesophilic and thermophilic inocula accounted for 35% and 39% of all archaeal 16S rRNA gene sequences, respectively. Previous studies have also shown the direct involvement of these microorganisms in DIET [38,39,40]. The archaeal communities of thermophilic samples had lower OTU numbers than the mesophilic samples, which can be explained by the sensitivity of some methanogenic species to high temperatures and high ammonia levels [64]. In the thermophilic reactors, the second important group of archaea (after Methanosarcina) was the genus Methanothermobacter (Methanobacteria), which is characteristic of thermophilic sources [65]. Representatives of the chemolithoautotrophic thermophilic genus Methanothermobacter are the objects of active research to clarify the biochemistry of methane formation from H2 and CO2 [66].
The relative abundance of Methanothrix, Methanobacterium, Methanobrevibacter, Methanomassiliicoccus, and Methanomethylophilaceae species in the mesophilic system with the addition of GAC was slightly higher compared to control treatments. Methanogenic microbes, including the Methanothermobacter, Methanoculleus, Methanobrevibacter, and Methanomassiliicoccus species, were slightly higher in the thermophilic system with GAC (Figure 9). An increase in the level of hydrogenotrophic methanogens (e.g., Methanothermobacter, Methanoculleus) and the complete absence of Methanosaetaceae members in the thermophilic reactors may suggest a potential shift in the acetate conversion, namely, the creation of conditions for the SAO pathway [49]. The representatives of the Methanosarcinaceae are acetoclastic, methylotrophic, and mixotrophic methanogens, but they have also been shown to participate in SAO as hydrogenotrophic partners [67].
Thus, during the anaerobic digestion of chicken manure, the methanogens of the genus Methanosarcina dominated in the mesophilic and thermophilic reactors, while strict hydrogenotrophic methanogens also played an important role in the thermophilic systems, with a greater prevalence in the GAC-system. The results of the kinetics of methanogenesis and the data on metabolites indicate that the addition of conductive carbon material allowed methanogenic archaea to generate methane more efficiently. Overall, the findings of this study are that the decomposition of the produced organic acids and methane formation in the mesophilic and thermophilic batch tests were faster in the presence of GAC than in the absence of GAC. New data about anaerobic microbial communities will help us better understand the phenomena of direct interspecies electron transfer using conductive materials for technological advances in ammonia-stressed anaerobic digestion systems.

4. Conclusions

In summary, this research demonstrates that methane is produced faster under GAC-supplemented conditions. These results show that GAC addition efficiently enhances the degradation of organic acids in the mesophilic and thermophilic batch reactors loaded with chicken manure as substrate. Bacteroidia and Clostridia were the two main bacterial classes in the mesophilic reactors, while Clostridia had a competitive advantage over other bacterial groups in the thermophilic reactors. The methanogenesis in the mesophilic reactors was mainly performed by representatives of the genera Methanosarcina, Methanobacterium, and Methanotrix, while in the thermophilic systems methane production occurred mostly by members of the genera Methanosarcina, Methanoculleus, and Methanothermobacter. GAC could provide large attachment surfaces for microorganisms, as well as special conditions for electron exchange, which contributed to the improved methane production in both the mesophilic and thermophilic reactors. In addition, GAC can adsorb various inhibitory components. Due to an increase in the proportion of syntrophic bacteria and hydrogenotrophic methanogens involved in SAO, the presence of GAC might facilitate SAO during the thermophilic anaerobic digestion of chicken manure. However, further research is needed to achieve a sustained efficacy of the anaerobic conversion of poultry manure in the presence of GAC on an industrial scale.

Author Contributions

Conceptualization, E.E.Z. and A.M.Z.; methodology, E.E.Z. and A.M.Z.; software, E.E.Z., S.S.B. and A.M.Z.; validation, E.E.Z., S.S.B. and A.M.Z.; formal analysis, E.E.Z., S.S.B. and A.M.Z.; investigation, E.E.Z., S.S.B. and A.M.Z.; resources, A.M.Z.; data curation, E.E.Z. and A.M.Z.; writing—original draft preparation, E.E.Z.; writing—review and editing, S.S.B. and A.M.Z.; visualization, E.E.Z. and S.S.B.; supervision, A.M.Z.; project administration, A.M.Z.; funding acquisition, A.M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The reported study was funded by the Russian Foundation for Basic Research [Grant no. 18-29-25058]. The work was carried out within the framework of the Kazan Federal University Strategic Academic Leadership Program.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the technicians of the Department of Microbiology (KFU) for their support during the implementation of the project.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Impact of GAC on specific methane production (SMP) (a), methane flow rate (b), and total methane production (c) during the mesophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (M1_C/M2_C, M3_GAC_5/M4_GAC_5, and M5_GAC_10/M6_GAC_10, respectively). Values sharing the same letter indicate no significant difference (p values < 0.05 were considered to indicate statistical significance) (c).
Figure 1. Impact of GAC on specific methane production (SMP) (a), methane flow rate (b), and total methane production (c) during the mesophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (M1_C/M2_C, M3_GAC_5/M4_GAC_5, and M5_GAC_10/M6_GAC_10, respectively). Values sharing the same letter indicate no significant difference (p values < 0.05 were considered to indicate statistical significance) (c).
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Figure 2. Impact of GAC addition on VOA concentrations (a) and VOA/TIC ratio (b) during the mesophilic anaerobic digestion of chicken manure. Different amounts of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to reactors (M1_C/M2_C, M3_GAC_5/M4_GAC_5, and M5_GAC_10/M6_GAC_10, respectively). The error bars show the standard deviation on technical variability (n = 3).
Figure 2. Impact of GAC addition on VOA concentrations (a) and VOA/TIC ratio (b) during the mesophilic anaerobic digestion of chicken manure. Different amounts of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to reactors (M1_C/M2_C, M3_GAC_5/M4_GAC_5, and M5_GAC_10/M6_GAC_10, respectively). The error bars show the standard deviation on technical variability (n = 3).
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Figure 3. Impact of GAC on SMP (a), methane flow rate (b), and total methane production (c) during the thermophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (T1_C/T2_C, T3_GAC_5/T4_GAC_5, and T5_GAC_10/T6_GAC_10, respectively). After a 10-day experimental period, the inhibition of the process was observed, and on day 19 the operating temperature in the reactors T_C and T_GAC_5 was changed from 55 °C to 50 °C at 1 °C day−1. Values sharing the same letter indicate no significant difference (p values < 0.05 were considered to indicate statistical significance) (c).
Figure 3. Impact of GAC on SMP (a), methane flow rate (b), and total methane production (c) during the thermophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (T1_C/T2_C, T3_GAC_5/T4_GAC_5, and T5_GAC_10/T6_GAC_10, respectively). After a 10-day experimental period, the inhibition of the process was observed, and on day 19 the operating temperature in the reactors T_C and T_GAC_5 was changed from 55 °C to 50 °C at 1 °C day−1. Values sharing the same letter indicate no significant difference (p values < 0.05 were considered to indicate statistical significance) (c).
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Figure 4. Impact of GAC addition on VOA concentrations during the thermophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (T1_C/T2_C, T3_GAC_5/T4_GAC_5, and T5_GAC_10/T6_GAC_10, respectively). The error bars show the standard deviation on technical variability (n = 3).
Figure 4. Impact of GAC addition on VOA concentrations during the thermophilic anaerobic digestion of chicken manure. Different concentrations of GAC (0 g L−1, 5 g L−1, and 10 g L−1) were added to the reactors (T1_C/T2_C, T3_GAC_5/T4_GAC_5, and T5_GAC_10/T6_GAC_10, respectively). The error bars show the standard deviation on technical variability (n = 3).
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Figure 5. The Venn diagram showing the number of unique and shared OTUs comparing the bacterial communities of the reactors M1_C, M4_GAC_5, T1_C, and T4_GAC_5 (sampled on day 7).
Figure 5. The Venn diagram showing the number of unique and shared OTUs comparing the bacterial communities of the reactors M1_C, M4_GAC_5, T1_C, and T4_GAC_5 (sampled on day 7).
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Figure 6. Taxonomic composition of bacterial communities in chicken manure, inocula, and anaerobic reactors (sampled on day 7). Bacterial community composition according to the amplicon sequencing of the bacterial 16S rRNA gene is shown on the class level. Classes with maximum abundances below 1% are summarized as “other”.
Figure 6. Taxonomic composition of bacterial communities in chicken manure, inocula, and anaerobic reactors (sampled on day 7). Bacterial community composition according to the amplicon sequencing of the bacterial 16S rRNA gene is shown on the class level. Classes with maximum abundances below 1% are summarized as “other”.
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Figure 7. Heatmap illustrating the relative abundances of bacterial taxa in chicken manure, inocula, and anaerobic reactors (sampled on day 7; genus level). Only taxa comprising at least 1% relative abundance in at least one sample are presented.
Figure 7. Heatmap illustrating the relative abundances of bacterial taxa in chicken manure, inocula, and anaerobic reactors (sampled on day 7; genus level). Only taxa comprising at least 1% relative abundance in at least one sample are presented.
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Figure 8. The Venn diagram showing the number of unique and shared OTUs comparing the archaeal communities of the reactors M1_C, M4_GAC_5, T1_C, and T4_GAC_5 (sampled on day 7).
Figure 8. The Venn diagram showing the number of unique and shared OTUs comparing the archaeal communities of the reactors M1_C, M4_GAC_5, T1_C, and T4_GAC_5 (sampled on day 7).
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Figure 9. Taxonomic composition of archaeal communities in inocula and anaerobic reactors (sampled on day 7). Archaeal community composition according to the amplicon sequencing of the archaeal 16S rRNA gene is shown on the genus level.
Figure 9. Taxonomic composition of archaeal communities in inocula and anaerobic reactors (sampled on day 7). Archaeal community composition according to the amplicon sequencing of the archaeal 16S rRNA gene is shown on the genus level.
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Table
Table 1. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) concentrations observed in the mesophilic anaerobic reactors.
Table 1. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) concentrations observed in the mesophilic anaerobic reactors.
ReactorTAN (g L−1)FAN (mg L−1)
Day 7Day 23Day 7Day 23
M1_C2.28 ± 0.052.44 ± 0.16137 ± 3.0118 ± 7.8
M2_C2.12 ± 0.062.55 ± 0.07125 ± 3.3129 ± 3.6
M3_GAC_52.07 ± 0.142.35 ± 0.10133 ± 8.8111 ± 4.8
M4_GAC_52.33 ± 0.182.65 ± 0.19156 ± 12.0120 ± 8.7
M5_GAC_102.26 ± 0.162.62 ± 0.12142 ± 10.1106 ± 4.9
M6_GAC_102.18 ± 0.142.47 ± 0.14134 ± 8.7105 ± 6.0
Table
Table 2. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) concentrations detected in the thermophilic anaerobic reactors.
Table 2. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) concentrations detected in the thermophilic anaerobic reactors.
ReactorTAN (g L−1)FAN (mg L−1)
Day 7Day 16Day 32Day 7Day 16Day 32
T1_C2.29 ± 0.092.14 ± 0.062.82 ± 0.03560 ± 21.6455 ± 12.5343 ± 3.8
T2_C2.67 ± 0.042.21 ± 0.072.78 ± 0.11617 ± 14.7404 ± 12.4318 ± 12.2
T3_GAC_52.57 ± 0.072.12 ± 0.072.75 ± 0.14565 ± 14.7359 ± 11.8514 ± 26.5
T4_GAC_52.66 ± 0.042.12 ± 0.062.92 ± 0.11645 ± 3.4426 ± 7.1588 ± 23.0
T5_GAC_102.75 ± 0.062.11 ± 0.04ND638 ± 13.7402 ± 7.6ND
T6_GAC_102.59 ± 0.072.28 ± 0.10ND580 ± 15.8418 ± 18.1ND
ND—not determined.
Table
Table 3. Alpha diversity of microbial communities in chicken manure, inocula, and some anaerobic reactors (sampled on day 7).
Table 3. Alpha diversity of microbial communities in chicken manure, inocula, and some anaerobic reactors (sampled on day 7).
SampleBacteriaArchaea
OTUsChao1ShannonSimpsonOTUsChao1ShannonSimpson
Manure2202815.060.9300--
Inoculum_M5235386.690.9742424.090.91
M1_C5495636.410.9742422.560.65
M4_GAC_55225306.430.9742422.650.67
Inoculum_T4194315.430.9136363.700.89
T1_C4524745.300.9236362.850.77
T4_GAC_53974305.130.9035352.970.80
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Ziganshina, E.E.; Bulynina, S.S.; Ziganshin, A.M. Impact of Granular Activated Carbon on Anaerobic Process and Microbial Community Structure during Mesophilic and Thermophilic Anaerobic Digestion of Chicken Manure. Sustainability 2022, 14, 447. https://doi.org/10.3390/su14010447

AMA Style

Ziganshina EE, Bulynina SS, Ziganshin AM. Impact of Granular Activated Carbon on Anaerobic Process and Microbial Community Structure during Mesophilic and Thermophilic Anaerobic Digestion of Chicken Manure. Sustainability. 2022; 14(1):447. https://doi.org/10.3390/su14010447

Chicago/Turabian Style

Ziganshina, Elvira E., Svetlana S. Bulynina, and Ayrat M. Ziganshin. 2022. "Impact of Granular Activated Carbon on Anaerobic Process and Microbial Community Structure during Mesophilic and Thermophilic Anaerobic Digestion of Chicken Manure" Sustainability 14, no. 1: 447. https://doi.org/10.3390/su14010447

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