Evaluation of Biogas Production Performance and Dynamics of the Microbial Community in Different Straws.

The development and utilization of crop straw biogas resources can effectively alleviate the shortage of energy, environmental pollution, and other issues. This study performed a continuous batch test at 35°C to assess the methane production potential and volatile organic acid contents using the modified Gompertz equation. Illumina MiSeq platform sequencing, which is a sequencing method based on sequencing-by-synthesis, was used to compare the archaeal community diversity, and denaturing gradient gel electrophoresis (DGGE) was used to analyze the bacterial community diversity in rice straw, dry maize straw, silage maize straw, and tobacco straw. The results showed that cumulative gas production values for silage maize straw, rice straw, dry maize straw, and tobacco straw were 4,870, 4,032.5, 3,907.5, and 3,628.3 ml/g ·VS , respectively, after 24 days. Maximum daily gas production values of silage maize straw and rice straw were 1,025 and 904.17 ml/g ·VS, respectively, followed by tobacco straw and dry maize straw. The methane content of all four kinds of straws was > 60%, particularly that of silage maize straw, which peaked at 67.3%. Biogas production from the four kinds of straw was in the order silage maize straw > rice straw > dry maize straw > tobacco straw, and the values were 1,166.7, 1,048.4, 890, and 637.4 ml/g ·VS, respectively. The microbial community analysis showed that metabolism was mainly carried out by acetate-utilizing methanogens, and that Methanosarcina was the dominant archaeal genus in the four kinds of straw, and the DGGE bands belonged to the phyla Firmicutes, Bacteroidetes, and Chloroflexi. Silage maize is useful for biogas production because it contains four kinds of straw.

Material and microbial changes during corn stalk silage and their effects on methane fermentation.

Silage efficiency is crucial for corn stalk storage in methane production. This study investigated characteristics of dynamic changes in materials and microbes during the silage process of corn stalks from the initial to stable state. We conducted laboratory-scale study of different silage corn stalks, and optimized silage time (0, 2, 5, 10, 20, and 30days) for methane production and the endogenous microbial community. The volatile fatty acid concentration increased to 3.00g/L on Day 10 from 0.42g/L on Day 0, and the pH remained below 4.20 from 5.80. The lactic acid concentration (44%) on Day 10 lowered the pH and inhibited the methane yield, which gradually decreased from 229mL/g TS at the initial state (Day 0, 2) to 207mL/g TS at the stable state (Day 10, 20, 30). Methanosaeta was the predominant archaea in both fresh and silage stalks; however, richness decreased from 14.11% to 4.75%.

[Biodiversity of mesophilic microbial community BYND-8 capability of lignocellulose degradation and its effect on biogas production].

The biodiversity of a mesophilic microbial community BYND-8 capable of degrading lignocellulose at 30 degrees C was detected using denaturing gradient gel electrophoresis (DGGE) and the isolation of pure cultures, and the effect of the liquid of rice straw degradation by BYND-8 on biogas production was measured. Six bacterial strains were isolated using peptone cellulose solution medium, and the highest similarities of their 16S rDNA gene sequences to Serratia sp. PSGB 13, S. marcescens strain UFLA-25LS, S. marcescens strain DAP33, Alcaligenes sp. YcX-20, Stenotrophomonas maltophilia strain C6, Bacillus cereus isolate BRL02-71 were 99%, 100%, 96%, 100%, 100% and 99%, respectively. In addition, one band was detected besides six bands of cultured isolates on the DGGE gel, and it showed 100% sequence similarity to uncultured bacterium clone ATB-KS-1446. The cumulative biogas and methane productions of biogas fermentation system added with the liquid of rice straw degraded by BYND-8 were 13 167 mL and 7 248 mL, 44.5% and 95.3% higher than those of the control, respectively, in the early 15 days of fermentation. The results showed that the biodiversity of microbial community BYND-8 was very high, and the time of producing biogas was put forward and biogas production was increased with application of microbial community for rice straw pretreatment during the biogas fermentation.

[Characteristics and diversity of a microbial community decomposing wheat straw under aerobic conditions].

A microbial community with the stable ability of effectively decomposing wheat stalks was constructed at room temperature under aerobic conditions by the method of restricted cultivation. The degradation ability of the microbial community shows that when performed in 100 mL improved CMC liquid medium, the depth and diamerer of the fluid level were 2 cm and 9 cm, it kept higher degradation efficiency with capability of decomposing straws of 66.1% during the initial six days. By the tenth day of decomposition, the degradation ratio was slowly up to 77.0%. Analysis of the components of wheat stalk (1.86 g) showed that the cellulose lost 0.78 g, hemicellulose lost 0.16 g and lignin lost 0.24 g. The composition diversity of the microbial community shows that 13 genera of microorganisms were identified by the technologies of clone library and isolation of pure strain. The results indicated that the dominate genera were Hydrogenophaga, Pseudomonas, Bacteroides and Clostridium taking up 78% in one hundred of positive clones. Phylogenetic dendrogram indicates that the relations among clones and isolated strains and their closest relatives presented certain distances; both Isolated 7 (FJ439527) and Clone 86 (EU834839) were closely related to Pseudomonas xiamenensis.

[Screening and characteristics of normal temperature lignocellulose-degradation microbial community].

A normal temperature lignocellulose-degrading microflora has been constructed by our laboratory. We researched the degradation activity and compose of the community in 28 degrees C fermentation condition. The results showed that the microbial community could degrade 39.6% of rice stew gross weight within five days.The volatile products were detected using CP-Chirasil-Dex CB capillary column by GC-MS, propionic acid, ethanol, isopropyl alcohol, 4-amino-1-butanol, butanoic acid, diethoxydimethyl-silane, lactic acid, ethanol,2,2'-oxybis-, diethyl phthalate and glycerin,more than 10 kinds of volatile products were detected. The state volatile products of changed largely along with the process of decompose, the productions gradually increase, and the content changes much with the process of decompose. Denaturing gradient gel electrophoresis (DGGE) detected the dynamic change of bacterium compose, the bacterium changes much in different period,the result of Blast from 16S rDNA sequence was found that the closest relative in community belong to Clostridium sp., Brevibacillus sp., Rhizobium sp., Bacterium sp. four genera.

[Composition diversity of the multifunctional bacterium community NSC-7].

The NSC-7 microbial community could decompose cellulose and lindan with high efficiency. In order to determine the bacterial composition of the community, 11 isolate strains were detected by plate isolation, while a community reset by the 11 isolate strains lost the capacity of degrading cellulose. The capacity of degrading of the filter paper in double deck plate and monolayer plate were determined, only the filter paper in double deck plate were degraded, that means the main or key microbe are anaerobic. The denaturing gradient gel electrophoresis (DGGE) and construction of 16S rDNA clone library were used to identify the composition diversity of NSC-7 community. 195 clones and 25 strains were detected in clone library, and about 60% closest relative among them was known the detailed information which were belonged to Clostridium, Petrobacter, Bacteria, Paenibacillus, Proteobacterium. Furthermore, there were 40% closest relative belonged to uncultured bacterium clone.

[Degradation of cyanide and maturity in cassava processing wastes composting].

An investigation was carried out to approach the degradation of cyanide and maturity during the cassava processing wastes composting process. Mixtures of cassava hull, cassava residues and pig manure were used in the experiment. Parameters like temperature, pH, cyanide, cellulose, hemicellulose, lignin and C/N ratio were assessed during the composting process, the effect of composting process on the degradation of cyanide and maturity were evaluated. The results reveal that the content of cyanide decreases sharply and declines to 2.08 mg/kg (30 days of composting), the degradation rate of cyanide is 94.16% and is in accord with food safety standard. After 15 days of the composting process, degradation of composting materials containing carbon (starch, cellulose, hemicellulose) and cyanide are quick and the degradation rates of them are more than 80%, properties tend towards stability basically. During 30 days of the composting process, the composting temperature drops to normal temperature and tends to stability, pH remains stable at 7.2. Parameters like C/N ratio, nitrate-nitrogen (NO3(-)-N) and ammonia nitrogen (NH4(+)-N) as maturity evaluation index were measured, and the results indicate that physical and chemical properties keep stability after 15 days of cassava processing wastes composting process. At the end of fermentation, C/N ratio is 17.55, the content of nitrate-nitrogen and ammonia nitrogen reach 2.5g/kg and 10 mg/kg respectively, NO3(-)-N/NH4(+)-N ratio is 250. The changes of these above mentioned parameters meet with maturity evaluation standard. Proving that cassava processing wastes during 30 days of composting treatment can achieve stability and security state.

[Degradation of cassava residue by the cellulose degradation composite microbial system MC1].

The lignocelluloses of cassava residue are good biomass resources. They are mainly used to produce feeds and alcohol. It is a promising approach to utilize them to produce methane. But it is difficult to use cassava residue for producing methane because of its dispersive solid matter and much water. A cellulose degradation composite microbial system MC1 was applied to degrade cassava residue discarded from cassava starch manufactory, and the composition of the lignocelluloses and the soluble ingredients of cassava residue were analyzed. After 18 days' cultivation, the total weight of the cassava residue was reduced by 47.3%, the cellulose, hemi-cellulose and lignin of the cassava residue were reduced by 22.7%, 90.4% and 11.3%, respectively, and 85% of the whole weight relief was made by MC1 within 6 days. The soluble ingredients of the cassava residue were increased from the incipient 18% to 33% in the third day which was the peak value in the process. The total amount of the volatile products, analyzed by GC-MS, came to a maximum in the sixth day. Twelve kinds of volatile products in the fermentation broth were determined, in which ethanol, acetic acid, 1, 2-ethanediol, butanoic acid and glycerine were the major compounds, and they can be utilized by methanogenic organism directly or be changed into compounds that can be utilized by methanogens organism directly. Accordingly, it is very hopeful to use MC1 to degrade cassava residue as a method of prefermentation in methane fermentation.

Development of an enrichment culture growing at low temperature used for ensiling rice straw.

To speed up the conversion of rice straw into feeds in a low-temperature region, a start culture used for ensiling rice straw at low temperature was selected by continuous enrichment cultivation. During the selection, the microbial source for enrichment was rice straw and soil from two places in Northeast China. Lab-scale rice straw fermentation at 10 degrees C verified, compared with the commercial inocculant, that the selected start culture lowered the pH of the fermented rice straw more rapidly and produced more lactic acid. The results from denatured gradient gel eletrophoresis showed that the selected start culture could colonize into the rice straw fermentation system. To analyze the composition of the culture, a 16S clone library was constructed. Sequencing results showed that the culture mainly consisted of two bacterial species. One (A) belonged to Lactobacillus and another (B) belonged to Leuconostoc. To make clear the roles of composition microbes in the fermented system, quantitative PCR was used. For species A, the DNA mass increased continuously until sixteen days of the fermentation, which occupied 65%. For species B, the DNA mass amounted to 5.5% at six days of the fermentation, which was the maximum relative value during the fermentation. To the authors' best knowledge, this is the first report on ensiling rice straw with a selected starter at low temperature and investigation of the fermented characteristics.

[Productions analyses and pH dynamics during rice straw degradation by the lignocellulose degradation bacteria system WSC-6].

To detect the metabolic characteristic of rice straw degradation by composite microbial system WSC-6, we cultured WSC-6 in the media used rice straw as the limiting carbon source. The rice straw was added in the style of different quantity once or the same quantity at the different time intervals during 90 days culture. The systems were cultivated under static condition at 50 degrees C. The degradation ratio, absolute degradation quantity,products from degradation and dynamics of pH value of fermentation system were all investigated. The results showed: when 1% rice straw was added once, the pH of fermentation system decreased from initial 7.8 to 6.0 within the first three days inoculation, and after six-day cultivation, it increased to 8.0 and was stable. For dissolved oxygen concentration (DO), the value was maintained at range of 0.01 to 0.12 mg x L(-1) of microaerobic condition. During the rice straw degradation, more than ten kinds of products including ethanol, acetic acid, lactic acid and glycerol and so on were detected using GC-MS. Especially, the highest concentration of lactic acid among all products was 7.381 g x L(-1) at 24 h after inoculation. During 90-day cultivation, for the addition treatments of the different quantity once, the more rice straw added, the quicker and lower the pH decreased, and the longer time intervals returned the pHs were. Especially for 5.0% addition, when 5.0% of rice straw was added once, pH did not increase again after it decreased. Among the addition of the same quantity at the different time intervals, the trend of decrease-increase in pH at 12-day and 15-day intervals could be repeated and high degradation activity well maintained. After 90-day of inoculation, the highest degradation ratio occurred in the treatment at 15-day interval, which was 86.7%. The highest absolute quantity occurred in the treatment at 6-day interval, which was 32.4 g. The trend of pH changes can indicate the activity of lignocellulose degradation and degradation process of the WSC-6.

Network relationships of bacteria in a stable mixed culture.

We investigated the network relationships of bacteria in a structurally stable mixed culture degrading cellulose. The mixed culture consists of four bacterial strains (a cellulose-degrading anaerobe [strain S], a saccharide-utilizing anaerobe [strain F], a peptide- and acetate-utilizing aerobe [strain 3] and a peptide-, glucose-, and ethanol-utilizing aerobe [strain 5]). Interspecies interactions were examined by analyzing the effects of culture filtrates on the growth of the other strains and by comprehensively analyzing population dynamics in the mixed-culture systems with all possible combinations of the four bacterial strains. The persistence of strain S depends on the effects of strain 5. However, strain 5 is a disadvantaged strain because strain 3 has bacteriocidal activity on strain 5. The extinction of strain 5 is indirectly prevented by strain F that suppresses the growth of strain 3. Although strain F directly has suppressive effects on the growth of strain S, strain F is essential for the persistence of strain S, considering the indirect effects (maintaining strain 5, which is essential for the survival of strain S, by inhibiting strain 3). These indirect relationships form a bacterial network in which all the relationships including suppressive effects were well balanced to maintain the structural stability. In addition to direct metabolite interactions, such kind of indirect relationships could have a great impact on microbial community structure in the natural environment.

Effect of adding cellulolytic bacterium on stable cellulose-degrading microbial community.

The introduction of an exogenous cellulolytic bacterium into a microbial community that was degrading rice straw effectively was evaluated. A stable coexistence of the indigenous and exogenous cellulolytic bacteria was achieved by adjusting the cultivation conditions. The obtained community required several subcultures to reach the highest degradation efficiency.

Effects of cultivation conditions on the diversity of microbes involved in the conversion of rice straw to fodder.

To confirm the optimum cultivation conditions for analyzing lactic acid bacterial communities and to provide the cultivation foundation for lactic acid bacterial communities that were used to convert straw into fodder, fermented rice straw was inoculated into 13 different broths. After 48 h of cultivation, pH values, volatile products, and microbial diversity were analyzed. Except for LAB broth, the pH values of the other broths could decrease to approximately 4.5. GC/MS analysis showed that lactic acid in Tomato MRS broth, MRS broth, LAB broth, and Tomato juice broth was higher than that in the other broths. DNA concentration analysis showed that the counts of microbes in Tomato MRS broth were 2.5 times of those in other broths and that tomato juice favored the reproduction of the microbes. Denaturing gradient gel electrophoresis (DGGE) analysis showed that the number of lactic acid bacterial species in HYA broth, Tomato juice broth, and Tomato MRS broth were higher than those in the other broths.

[Effects of a lactic acid bacteria community SFC-2 treated on rice straw].

Aimed to utilize rice straw and lessen the pressure of environment, the rice straw was used as the fermentation material, and a lactic acid bacteria community SFC-2 from my laboratory was inoculated into the rice straw to investigate the inoculation effects. After 30 days fermentation, the inoculated fermented straw smelt acid-fragrant, and the pH value was 3.8, which was lower than the control of 4.1. Furthermore, lactic acid concentration was more than that in the control. Especially L-lactic acid concentration was two times more than in the control, and the crude protein content was 10.16% higher than that in the control, and the crude fiber content was 3.2% lower than that in the control. From the patterns of denaturing gradient gel electrophoresis (DGGE), Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus paracasei rapidly became the advantageous species in the inoculated straws. However, Enterobacter sakazakii, Pantoea agglomerans, Enterobacter endosymbiont, Pantoea ananatis, whichwere predominate in the controls, were not detected in the inoculated straws, and the fermented quality was improved significantly.

[Construction and composition diversity of a lactic acid bacterial community SFC-2].

To obtain the community that could accelerate the fermentation of the air-dried crop straws, a lactic acid bacteria community SFC-2 was constructed from the natural fermented products of the corn straw and rice straw by continuous restricted subcultivation in the MRS-S broth. The SFC-2 could lower the pH of the broth quickly, and produce high amount of lactic acid. The microbial composition of the SFC-2 became stable from the 25th generation according to the results of denaturing gradient gel electrophoresis (DGGE). The pH value of the SFC-2 dropped to 3.8 and the amount of lactic acid reached 10.64 mg x mL(-1) after 12 h cultivation in the broth, which contained 64% L-(+) lactic acid. Using morphological methods, four strains were isolated from the SFC-2 and they all belonged to Lactobacillus. The closest species were L. fermentum, L. plantarum, L. paracasei and L. paracasei sub sp., respectively. At the same time, the composition of the SFC-2 was also analyzed by constructing 16S rDNA clone library. The results of 16S rDNA sequencing showed that the closest species of seven clones were mainly L. fermentum, L. plantarum and L. paracasei. In clone libraries, 76.3% belonged to L. fermentum, 20.3% belonged to L. plantarum and 3.4% belonged to L. paracasei.

[Microbial dynamics during the composting process].

Microbial dynamics of microbial community during the composting process was investigated with the methods of denaturing gradient gel electrophoresis (DGGE) and plating. The results showed that the most of microorganism count was bacterium, the second was actinomycete and the least was fungi. At the same time, the count of the thermophilic microorganism was always less than that of the mesophilic during the composting process. The count of mesophilic microorganism at later stage was less than that at the initial stage. However, the count of thermophilic antinomycete and fungi at later stage were more than that at initial stage, and the count of thermophilic bacterium was stable throughout the composting process. The bands pattern of DGGE and 16S rDNA analyses indicated that bacterial succession was presented during the composting process. The genera of Bdellovibrio, Clostridia bacterium and Bacillus were dominant species at initial stage (before the first 15 days), and Beta proteobacterium, Petrobacter succinimandens, Nitrospirae bacterium and Paenibacillus were dominant species at middle and later stage. Moreover, Clostridium was found throughout the process.

[Composition diversity of lactic acid bacteria (LAB) community Al2 used for alfalfa silage].

Alfalfa is the most important forage grass that is difficult to ensile for good quality. Using silage inoculants are the important way for preservation of alfalfa silage. Through continuous restricted subcultivation, a lactic acid bacteria (LAB) community Al2 was selected from well-fermented alfalfa silage. Plate isolation and Denaturing Gradient Gel Electrophoresis (DGGE), construction of 16S rDNA clone library were used to identify the composition diversity of Al2 community, with 7 strains detected, and they were all belonged to Lactobacillus. The composition ratios of the 7 strains were 55.21%, 19.79%, 14.58%, 3.13%, 3.13%, 3.13%, 1.03% according to 16S rDNA clone library. Al2-1i, Al2-2i, Al2-3i, corresponding to L. plantarum (99.9%), L. kimchii (99.4%), L. farciminis (100%) were detected by plate isolation. Among 3 isolates, Al2-1i had the highest ability of dropping pH and producing lactic acid, and the amount of lactic acid was reach to 18g/L at 24h cultivated in MRS media. The ability of dropping pH and producing lactic acid of Al2-3i was the lowest. From DGGE profiles, the dominant strains in Al2 community were L. plantarum and L. kimchii. L. plantarum was detected during the whole process, and L. kimchii was detected in the later phase.

Effects of water-soluble carbohydrate content on silage fermentation of wheat straw.

To determine a suitable initial water-soluble carbohydrate (WSC) content to make wheat straw natural fermentation successful and to study fermentation characteristics, glucose was used to adjust the initial WSC content to 1.4%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, and 10.0% dry matter (DM) in the wheat straw. At 30 d of fermentation, there were three pHs: when the initial WSC content was 1.4%, the pH was 5.5; when the initial WSC contents were 4.0%, 5.0% and 6.0%, the pHs were near 5.1; and when the initial WSC contents were 7.0%, 8.0%, 9.0% and 10.0%, the pHs were near 4.0. The pattern of changes in WSC content during the fermentation was similar to that in pH. At 30 d of fermentation, there existed a dividing line in WSC remnants between the initial 6.0% WSC treatment and the initial 7.0% WSC treatment. When the initial WSC content was more than 7.0%, the remaining WSC content was more than 23.7 g/kg DM. When the initial WSC content was less than 6.0%, the remaining WSC content was less than 13.6 g/kg DM. Particularly for the 1.4% WSC treatment, the remaining WSC content was 2.1 g/kg DM. The results of the microbiological enumeration showed that with the increase in initial WSC content, the numbers of lactic acid bacteria (LAB) and other bacteria generally decreased. Denaturing gradient gel electrophoresis (DGGE) results showed that when the initial WSC content was beyond 7.0%, the LAB of the fermentation system were detected.

Process of rice straw degradation and dynamic trend of pH by the microbial community MC1.

The process of the rice straw degradation in the fermentor with aeration at 290 ml/h was studied. The results of dissolved oxygen (DO) indicated that the optimum DO during cellulose degradation by microbial community MC1 ranged from 0.01 to 0.12 mg/L. The change model of pH values was as follows: irrespective of the initial pH of the medium, pH values decreased rapidly to approximate 6.0 after being inoculated within 48 h when cellulose was strongly degraded, and then increased slowly to 8.0-9.0 until cellulose was degraded completely. During the degradation process, 15 kinds of organic compounds were checked out by GC-MS. Most of them were organic acids. Quantity analysis was carried out, and the maximum content compound was ethyl acetate which reached 13.56 g/L on the day 4. The cellulose degradation quantity and ratio analyses showed that less quantity (under batch fermentation conditions) and longer interval (under semi-fermentation conditions) of rice straw added to fermentation system were contributed to matching the change model of pH, and increasing the quantity and ratio of rice straw degradation during cellulose degrading process. The highest degradation ratio was observed under the condition of rice straw added one time every five days (under semi-fermentation conditions).

[Capability and stability of degrading rice straw of composite microbial system MC1].

The capability of degrading rice straw of lignocellulolytic composite microbial system MC1 was investigated under different methods of preservation and temperatures treatments of 80 degrees C to 95 degrees C, and stability of composite microbial system MC1 was studied through the method of Denaturing Gradient Gel Electrophoresis (DGGE). The results indicate that the rice straw of 2% dry weight of medium can be degraded completely at 50 degrees C within 10 days under static culture. After 9 days inoculating MC1, the dry weight of rice straw, cellulose, hemicellulose and lignin content was degraded by 81% , 99%, 74% and 51%, respectively. Capability of cellulose degrading and stability of composite microbial system MC1 is sustained under 4 years of continuing subculture, 4 years of dry preservation at room temperature, 4 years of preservation at - 20 degrees C, 1 year of liquid preservation at room temperature and at 4 degrees C, and treatment of 90 degrees C for 30 min, respectively. Plate culture results show that composite microbial system MC1 are consisted of bacteria. The main DNA bands are not changed by the method of 16S rDNA PCR-DGGE after culture of six months so that microbial composition of MC1 is very stable.