Synergistic analysis of lignin degrading bacterial consortium and its application in rice straw fiber film.

Fiber film have received widespread attention due to its green friendliness. We can use microorganisms to degrade lignin in straw to obtain cellulose and make fiber films. Herein, a group of high-temperature (50 °C) lignin degrading bacterial consortium (LDH) was enriched and culture conditions for lignin degradation were optimized. Combined with high-throughput sequencing technology, the synergistic effect of LDH-composited bacteria was analyzed. Then LDH was used to treat rice straw for the bio-pulping experiment. The results showed that the lignin of rice straw was degraded 32.4 % by LDH at 50 °C for 10 d, and after the optimization of culture conditions, lignin degradation rate increased by 9.05 % (P < 0.001). The bacteria that compose in LDH can synergistically degrade lignin. Paenibacillus can encode all lignin-degrading enzymes present in the LDH. Preliminary tests of LDH in the pulping industry have been completed. This study is the first to use high temperature lignin degrading bacteria to fabricate fiber film.

Effect of bioaugmentation on gas production and microbial community during anaerobic digestion in a low-temperature fixed-bed reactor.

Low temperature is one of the limiting factors for anaerobic digestion in cold regions. To improve the efficiency of anaerobic digestion for methane production in stationary reactors under low-temperature conditions, and to improve the structure of the microbial community for anaerobic digestion at low temperatures. We investigated the effects of different concentrations of exogenous Methanomicrobium (10, 20, 30%) and different volumes of carbon fiber carriers (0, 10, 20%) on gas production and microbial communities to improve the performance of low-temperature anaerobic digestion systems. The results show that the addition of 30% exogenous microorganisms and a 10% volume of carbon fiber carrier led to the highest daily (128.15 mL/g VS) and cumulative (576.62 mL/g VS) methane production. This treatment effectively reduced the concentrations of COD and organic acid, in addition to stabilizing the pH of the system. High-throughput sequencing analysis revealed that the dominant bacteria under these conditions were Acidobacteria and Firmicutes and the dominant archaea were Candidatus_Udaeobacter and Methanobacterium. While the abundance of microorganisms that metabolize organic acids was reduced, the functional abundance of hydrogenophilic methanogenic microorganisms was increased. Therefore, the synergistic effect of Methanomicrobium bioaugmentation with carbon fiber carriers can significantly improve the performance and efficiency of low-temperature anaerobic fermentation systems.

Optimization of semi-continuous dry anaerobic digestion process and biogas yield of dry yellow corn straw: Based on "gradient anaerobic digestion reactor".

In China, the problem of low biogas yield of traditional biogas projects has become increasingly prominent. This study investigated the effects of different hydraulic retention times (HRTs) on the biogas production efficiency and microbial community under pilot conditions. The results show that the "Gradient anaerobic digestion reactor" can stably carry out semi-continuous dry anaerobic digestion and improve biogas yield. The highest volatile solids (VS) biogas yield (413.73 L/kg VS and 221.61 L CH4/kg VS) and VS degradation rate (48.41%) were observed at an HRT of 25 days. When the HRT was 15 days, the volumetric biogas yield was the highest (2.73 L/L/d, 1.43 L CH4/L/d), but the VS biogas yield and degradation rate were significantly decreased. Microbial analysis showed that HRT significantly affected microbial community. It provides basic data support for the development of a new anaerobic digestion process and the practical application of the straw biogas project in China.

The effects of adding exogenous lignocellulose degrading bacteria during straw incorporation in cold regions on degradation characteristics and soil indigenous bacteria communities.

Low temperature is one of the bottleneck factors that limits the degradation of straw during rice straw incorporation. Determining strategies to promote the efficient degradation of straw in cold regions has become a highly active research area. This study was to investigate the effect of rice straw incorporation by adding exogenous lignocellulose decomposition microbial consortiums at different soil depths in cold regions. The results showed that the lignocellulose was degraded the most efficiently during straw incorporation, which was in deep soil with the full addition of a high-temperature bacterial system. The composite bacterial systems changed the indigenous soil microbial community structure and diminished the effect of straw incorporation on soil pH, it also significantly increased rice yield and effectively enhanced the functional abundance of soil microorganisms. The predominant bacteria SJA-15, Gemmatimonadaceae, and Bradyrhizobium promoted straw degradation. The concentration of bacterial system and the depth of soil had significantly positive correlations on lignocellulose degradation. These results provide new insights and a theoretical basis for the changes in the soil microbial community and the application of lignocellulose-degrading composite microbial systems with straw incorporation in cold regions.

Biogas Residues Improved Microbial Diversity and Disease Suppression Function under Extent Indigenous Soil Microbial Biomass.

Indigenous soil microbial biomass (ISMB) plays a key role in maintaining essential functions and biodiversity of soil health. One of the critical unknowns is how the indigenous microorganisms respond to different fertilizers which is directly related to agricultural production. Therefore, we used Mi-Seq sequencing and network analyses to compare the response of ISMB to biogas residue and chemical fertilizers. The results showed that crop production was profoundly influenced by levels of ISMB present and is further dependent on the strategy of fertilizer application. Higher ISMB primarily manifests through retention of richer microbial abundance, a balanced community structure, and tightened co-occurrence within a certain proportion of Nitrospirae, Rhizophlyctidaceae, and Gemmatimonadetes. Compared to chemical fertilizer, biogas residue resulted in higher production with more strongly linked nodes such as Actinobacteria, Chloroflexi and Gemmatimonadetes. Under the same level of ISMB, the microbial diversity was richer and co-occurrence was tighter when biogas residues were applied compared with chemical fertilizer. In addition, the higher level of ISMB with biogas residue applied had a lower abundance of potential fungal pathogens in both bulk and rhizosphere soil compared with chemical fertilizer. This study provides critical data to understand the influence of ISMB and biogas residue on soil ecological system.

Effect of temperature and storage methods on liquid digestate: Focusing on the stability, phytotoxicity, and microbial community.

Identifying the stability and phytotoxicity of liquid digestate (LD) is necessary for safe agricultural utilization. Storage temperature, method, and time are critical factors that affect the stability and phytotoxicity of LD. This study therefore aimed to explore the dynamics of stability, phytotoxicity, and microbial community of LD in cattle farms under different storage conditions. The results showed that the contents of solids, organic matter, nitrogen, and phosphorous decreased during storage and exhibited temperature dependency. Conversely, the seed germination index increased, which was negatively correlated with dissolved organic carbon and ammonium nitrogen and positively correlated with certain bacteria (Thermovirga and Fastidiosipila). Open storage and/or higher temperature were found to contribute to the stabilization efficiency and phytotoxicity disappearance of LD. Open storage of LD at 30 °C for 60 days and 20 °C for 90 days was safe for its agricultural utilization, while hermetic storage of LD at 30 °C for 120 days and 20 °C for 150 days was safe. However, for storage at 10 °C for 180 days, additional post-treatment is required.

Obtainment of lignocellulose degradation microbial community: the effect of acid-base combination after restrictive enrichment.

Microbial communities for bioconversion of lignocellulose have received widespread attention. Many cellulose-degrading microbial communities have been enriched from different sources. Combining two microbial communities with acidic and basic properties (acid-base combination) is a technique used alongside restricted enrichment culturing. Understanding how changes to microbial communities result in community's structure and function is important for mechanistic reconstruction of microbiomes. In this study, we analyzed changes in microbial community structure to elucidate determination of the mechanisms of acid-base combination. We found that after restricted enrichment, the bacteria that were primarily retaining included not only those that decompose and utilize lignocellulose, such as Clostridium and Pseudomonas, but also synergistic microbiota such as Alkalobacillaceae. When the proportion of these two types of bacteria was unbalanced, the degradative ability of the microbial community was low, or pH changes of it did not compound regular changes, which may lead to the failure of restricted enrichment. Microbial communities were re-constituted by acid-base combination, whereby the degrading and synergistic strains were adjusted to a more appropriate proportion. The acid-base combination fixed the instability of microbial communities caused by the randomness of restrictive screening enrichment; it provided an effective method for obtaining high-quality lignocellulose-degrading microbial community.

Bioaugmentation with methanogens cultured in a micro-aerobic microbial community for overloaded anaerobic digestion recovery.

Anaerobic digestion (AD) is widely used for conversion of waste materials into biogas, but inhibition of methane production caused by overloading can be a major problem. The micro-aerobic microbial community MC1 was used to successfully culture methanogens, Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC 101360. The maximum 16S rRNA gene concentrations of Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC101360 were 1.06 × 106 and 1.35 × 103 copies/mL, respectively. The five key bacteria in MC1 were quantified to assess the effect of inoculation on the abundances of the bacteria in the mixed culture. The original MC1 total 16S rRNA gene concentration was 1.93 × 108 copies/mL, and the total 16S rRNA gene concentration had increased to 4.79 × 109 copies/mL on day 9 (p < 0.05). The proportions of the key strains in MC1+MST had changed by day 9. Cells were harvested and used to bioaugment and increase the pH values of the high- and medium-temperature anaerobic systems. After bioaugmentation, thermophilic AD recovered well. The cumulative amounts of gas produced were 44.78% and 28.28% higher in the MC1+MST and MC1 groups, respectively, than the sterilized control. The MC1+MST group gave better results than the chemical addition control group (CaCO3). There was no clear effect of bioaugmentation in mesophilic AD. When compared with traditional pure culture of methanogens as inoculants, methanogen cultivation in MC1 was simple and there was no need to separate and purify the target strains. This simplified methanogenic bioaugmentation agent was useful to study the mechanism of bioaugmentation for the recovery from low pH inhibition, showing the potential for practical application.

An innovative strategy to enhance the ensiling quality and methane production of excessively wilted wheat straw: Using acetic acid or hetero-fermentative lactic acid bacterial community as additives.

Ensiling is an effective storage strategy for agricultural biomass, especially for energy crops (mainly energy grasses and maize). However, the ensiling of excessively wilted crop straw is limited due to material characteristics, such as a high lignocellulosic content and low water-soluble carbohydrate and moisture contents. In this study, acetic acid or hetero-fermentative lactic acid bacterial community (hetero-fermentative LAB) were employed as silage additives to improve the ensiling process of excessively wilted wheat straw (EWS). The results showed that the additives inhibited the growth of Enterobacteriaceae and Clostridium_sensu_stricto_12, whose abundances decreased from 55.8% to 0.03-0.2%, respectively. The growth of Lactobacillus was accelerated, and the abundances increased from 1.3% to 80.1-98.4% during the ensiling process. Lactic acid fermentation was the dominant metabolic pathway in the no additive treatment. The additives increased acetic acid fermentation and preserved the hemicellulose and cellulose contents, increasing the methane yield by 17.7-23.9%. This study shows that ensiling with acetic acid or hetero-fermentative LAB is an effective preservation and storage strategy for efficient methane production from EWS.

Degradation of lignocelluloses in straw using AC-1, a thermophilic composite microbial system.

In composting, the degradation of lignocellulose in straw is problematic due to its complex structures such as lignin. A common solution to this problem is the addition of exogenous inoculants. AC-1, a stable thermophilic microbial composite, was isolated from high temperature compost samples that can decompose lignocellulose at 50-70 °C. AC-1 had a best degradation efficiency of rice straw at 60 °C (78.92%), of hemicellulose, cellulose and lignin were 82.49%, 97.20% and 20.12%, respectively. It showed degrad-ability on both simple (filter paper, absorbent cotton) and complex (rice straw) cellulose materials. It produced acetic and formic acid during decomposition process and the pH had a trend of first downward then upward. High throughput sequencing revealed the main bacterial components of AC-1 were Tepidimicrobium, Haloplasma, norank-f-Limnochordaceae, Ruminiclostridium and Rhodothermus which provides major theoretical basis for further application of AC-1.

Metagenomic Insight into Lignocellulose Degradation of the Thermophilic Microbial Consortium TMC7.

Biodegradation is the key process involved in natural lignocellulose biotransformation and utilization. Microbial consortia represent promising candidates for applications in lignocellulose conversion strategies for biofuel production; however, cooperation among the enzymes and the labor division of microbes in the microbial consortia remains unclear. In this study, metagenomic analysis was performed to reveal the community structure and extremozyme systems of a lignocellulolytic microbial consortium, TMC7. The taxonomic affiliation of TMC7 metagenome included members of the genera Ruminiclostridium (42.85%), Thermoanaerobacterium (18.41%), Geobacillus (10.44%), unclassified_f__Bacillaceae (7.48%), Aeribacillus (2.65%), Symbiobacterium (2.47%), Desulfotomaculum (2.33%), Caldibacillus (1.56%), Clostridium (1.26%), and others (10.55%). The carbohydrate-active enzyme annotation revealed that TMC7 encoded a broad array of enzymes responsible for cellulose and hemicellulose degradation. Ten glycoside hydrolases (GHs) endoglucanase, 4 GHs exoglucanase, and 6 GHs ß-glucosidase were identified for cellulose degradation; 6 GHs endo-ß-1,4-xylanase, 9 GHs ß-xylosidase, and 3 GHs ß-mannanase were identified for degradation of the hemicellulose main chain; 6 GHs arabinofuranosidase, 2 GHs α-mannosidase, 11 GHs galactosidase, 3 GHs α-rhamnosidase, and 4 GHs α-fucosidase were identified as xylan debranching enzymes. Furthermore, by introducing a factor named as the contribution coefficient, we found that Ruminiclostridium and Thermoanaerobacterium may be the dominant contributors, whereas Symbiobacterium and Desulfotomaculum may serve as "sugar cheaters" in lignocellulose degradation by TMC7. Our findings provide mechanistic profiles of an array of enzymes that degrade complex lignocellulosic biomass in the microbial consortium TMC7 and provide a promising approach for studying the potential contribution of microbes in microbial consortia.

The effects of C/N (10-25) on the relationship of substrates, metabolites, and microorganisms in "inhibited steady-state" of anaerobic digestion.

The carbon/nitrogen ratio (C/N) is a key parameter that affects the performance of anaerobic digestion (AD). Recent AD research has focused on optimizing the C/N of feedstock. The so-called "inhibited steady-state" refers to a special state of ammonia inhibition of AD that often occurs at low-C/N (below 25) when degradable nitrogen-rich substrates, such as livestock manure, are used as feedstock. However, the mechanism behind the "inhibited steady-state" is still unknown. In the current study, co-digestion and recirculation were used to create a C/N gradient in the influent to explore the relationship between substrates, metabolites, and microorganisms in the "inhibited steady-state." Data were collected at the macro, microbial, and genetic levels. Three CSTRs were successfully made run into the "inhibited steady-state" using influent C/Ns of 10-12. Digestion performance levels of R10-R12 were low and stable, transitioning from an aceticlastic methane-producing pathway to a hydrogenotrophic pathway as the C/N gradually decreased. As the abundance of the hydrogenophilic methanogens increased, the abundance of syntrophic acetate-oxidizing bacteria (SAOB) also increased. The succession between populations of Methanosaeta and Methanosarcina may be used as a microbiological indicator of ammonia inhibition. Under high-C/Ns, cooperation among bacteria was high, while under low-C/Ns, competition among bacteria was high. These results clarify the processes underlying the "inhibited steady-state," which is a condition often faced in actual large-scale biogas facilities that use degradable nitrogen-rich substrates. Moreover, practical guidelines for evaluating ammonia inhibition are provided, and strategies to alleviate ammonia suppression are developed.

A comparison and evaluation of the effects of biochar on the anaerobic digestion of excess and anaerobic sludge.

The mechanisms and enhancing effects of different biochar loadings on the digesters receiving low and high excess (or anaerobic) sludge loadings were thoroughly examined in the present study. This was done to explore an efficient method for converting excess sludge to anaerobic sludge. Biochar had an obvious effect on the anaerobic digestion of excess sludge but not on the anaerobic sludge. When the amount of biochar added was equivalent to 100% of the sludge TS, the cumulative methane yields of anaerobic digestion inoculated with small and large amounts of excess sludge were respectively 30.2 and 1.7 times that of those without biochar. The number of methanogens in the digesters that received small and large inoculations of excess sludge with 100% biochar, were respectively 105.4% and 20.6% higher than those without biochar. The biochar enhanced the systems performance because it selectively enriched the Trichococcus and Methanomicrobiales tightly attach to it. This enhanced the synergy and overall activity of the system by promoting biofilm development. Ultimately, the integration of 100% biochar and excess sludge can be used as a substitute for anaerobic sludge as an inoculum by giving similar overall performance.

Accelerated biomethane production from lignocellulosic biomass: Pretreated by mixed enzymes secreted by Trichoderma viride and Aspergillus sp.

Biological pretreatment is a promising technology to increase biogas yield. The methane yield and microbial community resulting from anaerobic digestion of maize straw after pretreatment of enzymes [extracted from Trichoderma viride (ETv) and Aspergillus sp. (EAs)] at different mixing ratios [5/0, 4/1, 3/2, 2/3, 1/4, 0/5] were evaluated. The methane yields from mixed enzymes pretreatment were higher than single enzymes pretreatments of ETv and EAs. The optimal enzymes mixing ratio of ETv and EAs was found to be 2/3, with the cumulative methane yield 512.64 mL/g TSadded, which was 31.74% higher than the control. Enzymatic pretreatment promoted an increase in the abundance of bacteria and archaea associated with cellulose decomposition. The majority of bacteria and archaea were assigned to Bacteroidetes, Firmicutes and Methanosaeta.

Methane production from the co-digestion of pig manure and corn stover with the addition of cucumber residue: Role of the total solids content and feedstock-to-inoculum ratio.

This study investigated performance and stability of increasing total solids (TS) content (10-30%) and feedstock-to-inoculum (F/I) ratios (1, 2) on anaerobic co-digestion of agricultural wastes. The cumulative methane yields generally decreased with the increasing TS content except for the TS content of 30% at F/I ratio of 1 and TS content of 10% at F/I ratio of 2. This was consistent with the maximum methane production rate (Rmax) and rate of the hydrolysis (Kh) stage in reactors. The pH, VFAs and NH4+-N content were positively correlated with increasing TS contents and F/I ratios. Economic analysis results indicated the net present value generally increased with increasing TS contents and TS content of 30% at F/I ratio of 1 had the highest net present value (5.7 million US$) and internal rate of return (41.9%). This indicated solid-state anaerobic digestion was financially attractive under analyzed conditions.

Effects of intermittent mixing mode on solid state anaerobic digestion of agricultural wastes.

This study investigated the effects of three different intermittent mixing modes (top, middle and bottom) on the performance of solid state anaerobic digestion (SS-AD) process of pig manure, corn stover and cucumber residues in a stirred tank reactor (STR). Results showed the cumulative methane yields of reactors had similar values (P > 0.05) except for the unmixed reactor (CK), which had a very low methane production. Reactors of top-mixed (T1) had shortest technical digestion time (T80) and more stable physicochemical characteristics than the other treatments. These findings indicated the three mixing modes had almost no effect on the cumulative methane yields, but affected the digestion process. The main bacteria in T1 reactor was Clostridium_sensu_stricto_1. However, Caldicoprobacter accounted for a relatively large proportion of the bacteria in middle-mixed (T2) and bottom-mixed (T3) which was consistent with the later methane production than T1. Methanosarcina was the dominant archaea in T1 reactor. Methanoculleus and Methanosarcina were the main microorganisms in top and bottom area of T2 and T3 reactor. Acidogenic (top area) and methanogenic zones (bottom area) were formed in all reactors respectively, by combining the physicochemical properties and microorganisms. Overall, T1 showed more advantages for methane production during SS-AD.

Full-scale of composting process of biogas residues from corn stover anaerobic digestion: Physical-chemical, biology parameters and maturity indexes during whole process.

Recycling of biogas residues from corn stover anaerobic digestion is crucial for the development of biogas industry. Full-scale composting process is the feasible way to convert biogas residues to fertilizer. The aim of the study was to explore the feasibility of full-scale composting process to dispose biogas residue to fertilizer, and to evaluate the quality of the compost. The results showed the biogas residues could rapidly reach the thermophilic stage and last at least 20 days, NH4+-N, TOC and C/N decreased along with the composting process, while TP, TK and NO3--N showed an opposite trend. Germination index(GI) and seedling growth index showed that raw biogas residues was toxic for plant, but the GI and seedling growth index were increased during the composting process, except for the cooling stage sample. Anaerolineaceae and Limnochordaceae were the main bacteria involved in the composting process, and Chaetomium was the most important fungus.

Microbial Degradation of Zearalenone by a Novel Microbial Consortium, NZDC-6, and Its Application on Contaminated Corncob by Semisolid Fermentation.

A novel microbial consortium (NZDC-6) was screened and characterized to detoxify the estrogenic mycotoxin zearalenone (ZEA), which commonly contaminates maize and is a major threat to food and health security. We found NZDC-6 to be thermophilic and highly effective, with a 90.3% ZEA degradation ratio at an optimum temperature of 60 °C. NZDC-6 was also effective at degrading the more estrogenic ZEA cognates, α-zearalenol (α-ZAL) and ß-zearalenol (ß-ZAL), with >90% degradation ratios. To evaluate a practical application, ZEA-contaminated corncobs were treated with NZDC-6 via semisolid fermentation. Measurements of physicochemical parameters and 16S microbial diversity and redundancy analysis (RDA) indicated that ZEA removal was most efficient at a low corncob solid content (< 5%), as a high solid content overwhelmed the microbial metabolic load, leading to increased dissolved oxygen and lowered pH. Our results demonstrate that the control of environmental variables is crucial for effective ZEA microbial removal in practical applications.

Effect of Substrate to Inoculum Ratio on Biogas Production and Microbial Community During Hemi-Solid-State Batch Anaerobic Co-digestion of Rape Straw and Dairy Manure.

The substrate to inoculum (S/I) ratio is crucial for the rapid start-up of solid-state anaerobic digestion (SS-AD) systems. In this study, the performance of methane production and microbial community structure were evaluated during co-digestion of rape straw (RS) and dairy manure (DM) at different S/I ratios (2:3, 1:1, 2:1, 3:1, and 4:1) in batch hemi-solid-state anaerobic digestion (HSS-AD) tests. The highest methane yield of 209.1 mL/g VSadded and highest volumetric methane production of 0.4 L/(L·d) were achieved at S/I ratios of 2:3 and 2:1, respectively. Lower S/I ratios (1:2, 1:1, and 2:1) steadily produced biogas throughout the AD period, while higher S/I ratios (3:1 and 4:1) failed to produce biogas during the initial stage of AD because of excess accumulation of volatile fatty acids and low pH. The predominant bacteria and archaea in stable digesters were Firmicutes and acetoclastic Methanosaeta, respectively, while Bacteroidetes predominated and the relative abundance of hydrogenotrophic Methanobacterium increased significantly in acidic digesters. Amounts of bacteria and archaea were inhibited in acidic digesters. Our results provide useful information for enhancing efficient methane production and advancing the understanding of the microbiome in HSS-AD of RS and DM at different S/I ratios.

Co-composting of the biogas residues and spent mushroom substrate: Physicochemical properties and maturity assessment.

Recycling of BR and SMS are crucial for the development of biogas industry and commercial mushroom cultivation. The seed germination test is limited to examine the maturity of compost because of lacking the effect of insoluble part on plant growth. The aim of this study was to evaluate the maturity of compost by analysis the relationship between agronomic parameters of plant growth with physicochemical parameters of compost. The thermophilic period (over 50 °C) was lasted 52 days. TOC, C/N, AP and NH4+-N was decreased along with composting process, while TK, TP, AK and NO3--N showed an opposite trend. As for seedling quality, the raw material (T0) showed the worst plant growth but the 100% compost (T1) showed better seedling quality compared with commercial seedlings. According to the analysis of Spearman correlation, the results indicated that TOC, C/N, NH4+-N, NO3--N, AK and lignocellulose can be used to evaluate compost maturity.