[DNA assembly by multi-fragment digestion/ligation and homologous recombination].

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Characterization of the effective cellulose degrading strain CTL-6.

An efficient cellulose degrading bacteria exists in the thermophilic wheat straw-degrading community, WDC2. However, this strain cannot be isolated and cultured using conventional separation techniques under strict anaerobic conditions. We successfully isolated a strain of effective cellulose degrading bacteria CTL-6 using a wash, heat shock, and solid-liquid alternating process. Analysis of its properties revealed that, although the community containing the strain CTL-6 grew under aerobic conditions, the purified strain CTL-6 only grew under anaerobic culture conditions. The strain CTL-6 had a striking capability of degrading cellulose (80.9% weight loss after 9 days of culture). The highest efficiency value of the endocellulase (CMCase activity) was 0.404 micromol/(min mL), cellulose degradation efficiency by CTL-6 was remarkably high at 50-65 degrees C with the highest degradation efficiency observed at 60 degrees C. The 16S rRNA gene sequence analysis indicated that the closest relative to strain CTL-6 belonged to the genus Clostridium thermocellum. Strain CTL-6 was capable of utilizing cellulose, cellobiose, and glucose. Strain CTL-6 also grew with Sorbitol as the sole carbon source, whereas C. thermocellum is unable to do so.

Functional characteristics and diversity of a novel lignocelluloses degrading composite microbial system with high xylanase activity.

To obtain an efficient natural lignocellulolytic complex enzyme, we screened an efficient lignocellulose degrading composite microbial system (XDC-2) from composted agricultural and animal wastes amended soil following a long-term directed acclimation. The XDC-2 could not only degrade natural lignocelluloses, but could also secret extracellular xylanase efficiently in liquid culture under static conditions at room temperature. The XDC-2 degraded rice straw by 60.3% after fermentation for 15 days. Hemicelluloses were decomposed effectively, while the extracellular xylanase activity was dominant with an activity of 8.357 U ml-1 on day 6 of the fermentation period. The extracellular crude enzyme noticeably hydrolyzed natural lignocelluloses. The optimum temperature and pH for the xylanase activity were 40 degrees and 6.0. However, the xylanase was activated in a wide pH range of 3.0-10.0, and retained more than 80% of its activity at 25-35 degrees and pH 5.0-8.0 after three days of incubation in liquid culture under static conditions. PCR-DGGE analysis of successive subculture indicated that the XDC-2 was structurally stable over long-term restricted and directed cultivation. Analysis of 16S rRNA gene clone library showed that the XDC-2 was mainly composed of mesophilic bacteria related to the genera Clostridium, Bacteroides, Alcaligenes, Pseudomonas, etc. Our results offer a new approach to exploring efficient lignocellulolytic enzymes by constructing a high-performance composite microbial system with synergistic complex enzymes.

Characterization of Microbial Communities in Chinese Rice Wine Collected at Yichang City and Suzhou City in China.

Two typical microbial communities from Chinese rice wine fermentation collected in Yichang city and Suzhou city in China were investigated. Both communities could ferment glutinous rice to rice wine in 2 days. The sugar and ethanol contents were 198.67 and 14.47 mg/g, respectively, for rice wine from Yichang city, and 292.50 and 12.31 mg/g, respectively, for rice wine from Suzhou city. Acetic acid and lactic acid were the most abundant organic acids. Abundant fungi and bacteria were detected in both communities by high-throughput sequencing. Saccharomycopsis fibuligera and Rhizopus oryzae were the dominant fungi in rice wine from Suzhou city, compared with R. oryzae, Wickerhamomyces anomalus, Saccharomyces cerevisiae, Mucor indicus, and Rhizopus microsporus in rice wine from Yichang city. Bacterial diversity was greater than fungal diversity in both communities. Citrobacter was the most abundant genus. Furthermore, Exiguobacterium, Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were highly abundant in both communities.

Dynamic changes in the composite microbial system MC1 during and following its rapid degradation of lignocellulose.

To monitor the dynamics of the composite microbial system MC1 during its degradation of lignocellulose and to improve our understanding of the microbial communities involved in this biomass conversion, MC1 was characterized at eight time points over an 18-day, thermophilic, aerobic, static cultivation. We found the microbial communities to be dynamic, rhythmic consortia capable of changing in response to lignocellulose degradation. The growth curve over 18 days was M-shaped. Based on the quantitative changes in five major components of MC1 (Clostridium straminisolvens CSK-1, Clostridium sp. FG4, Pseudoxanthomonas sp. M1-3, Brevibacillus sp. M1-5, and Bordetella sp. M1-6), reduction in rice straw weight, cellulase (CMCase) activity, xylanase activity, and changes in medium pH, we found that the process comprised two identifiable phases. Rapid degradation occurred from day 0 to day 9, while the post-rapid degradation phase included days 10 to 18. Day 3 and day 12 were two key time points in the rapid degradation phase and post-rapid degradation phase, respectively. Two anaerobes, C. straminisolvens CSK-1 and Clostridium sp. FG4, dominated the MC1 system from day 0 to day 18.

Enhancing the cellulose-degrading activity of cellulolytic bacteria CTL-6 (Clostridium thermocellum) by co-culture with non-cellulolytic bacteria W2-10 (Geobacillus sp.).

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml(-1) and 45.1 µg ml(-1) (DNA content) up to 0.47 U ml(-1) and 112.2 µg ml(-1), respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml(-1). This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.

The effect of temperature on the structure and function of a cellulose-degrading microbial community.

The purpose of this study was to investigate the effect of temperature on the structure and straw degradation capability of a microbial community grown from wheat straw compost. Two cellulolytic microbial communities, WDC1 and WDC2, were obtained from compost. The communities had been cultured under 50 and 60 °C by continuous enrichment, respectively. The wheat straw degradation capabilities were 45.69 % (WDC1) and 59.5 % (WDC2). By changing the culture temperatures, two new stable communities were obtained: WDC1-6N (WDC1, cultivated at 60 °C for eight generations) and WDC2-5N (WDC2, cultivated at 50 °C for eight generations). The wheat straw degradation capabilities for the new communities were 59.75 and 52.60 %, respectively. The results showed that compared to 50 °C, the wheat straw degradation capability of the communities cultured at 60 °C was stronger. Sequencing of selected denaturing gradient gel electrophoresis (DGGE) bands and analysis of DGGE profiles indicated that the WDC2 structure was significantly different from the structure of WDC1. This was so even though the two communities were enriched from the same compost. With the change of culture temperature, the community structures underwent significant transitions. Included communities were thermophilic, anaerobic bacteria, and any cellulolytic bacteria (e.g., Clostridium thermocellum) that were active and abundant at conditions under 60 °C. These results have the potential to significantly aid in the enrichment of a cellulose-degrading community from the environment and to enhance the community capability to conduct straw biotransformation.

[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.

[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.

[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.

[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.