Zongjun Cui

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

ABSTRACT A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

Characterization of a microbial consortium capable of degrading lignocellulose

A microbial consortium, designated WCS-6, was established by successive subcultivation in the presence of rice straw under static conditions. The degradation efficiencies of WSC-6 for 0.5 g filter paper, cotton and rice straw after 3 days of cultivation were 99.0±0.7%, 76.9±1.5% and 81.3±0.8%, respectively as determined by gravimetrical methods. Nine bacterial isolates were obtained from WCS-6 plated under aerobic conditions, and sequencing of their 16S rDNA indicated that these bacteria were related to Bacillus thermoamylovorans BTa, Paenibacillus barengoltzii SAFN-016, Proteobacterium S072, Pseudoxanthomonas taiwanensis CB-226, Rhizobiaceae str. M100, Bacillus sp. E53-10, Beta proteobacterium HMD444, Petrobacter succinimandens 4BON, and Tepidiphilus margaritifer N2-214. DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rDNA sequences amplified from total consortium DNA revealed the presence of sequences related to those of Ureibacillus thermosphaericus, uncultured bacterium clone GC3, uncultured Clostridium sp. clone A1-3, Clostridium thermobutyricum, and Clostridium thermosuccinogenes in addition to the sequences identified from the cultured bacteria. The microbial community identified herein is a potential candidate consortium for the degradation of waste lignocellulosic biomass.

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.

Effects of different pretreatment strategies on corn stalk acidogenic fermentation using a microbial consortium

The effects of sulfuric acid, acetic acid, aqueous ammonia, sodium hydroxide, and steam explosion pretreatments of corn stalk on organic acid production by a microbial consortium, MC1, were determined. Steam explosion resulted in a substrate that was most favorable for microbial growth and organic acid productions. The total amounts of organic acids produced by MC1 on steam exploded, sodium hydroxide, sulfuric acid, acetic acid, and aqueous ammonia pretreated corn stalk were 2.99, 2.74, 1.96, 1.45, and 2.21g/l, respectively after 3days of fermentation at 50°C. The most prominent organic products during fermentation of steam-exploded corn stalks were formic (0.86g/l), acetic (0.59g/l), propanoic (0.27g/l), butanoic (0.62g/l), and lactic acid (0.64g/l) after 3days of fermentation; ethanol (0.18g/l), ethanediol (0.68g/l), and glycerin (3.06g/l) were also produced. These compounds would be suitable substrates for conversion to methane by anaerobic digestion.

Enhancing the anaerobic digestion of lignocellulose of municipal solid waste using a microbial pretreatment method

The use of biological pretreatment in anaerobic digestion systems has some potential; however, to date, these methods have not been able to effectively increase methane production of lignocellulose of municipal solid waste (LMSW). In this study a thermophilic microbial consortium (MC1) was used as a pretreatment method in order to enhance biogas and methane production yields. The results indicated that sCOD concentration increased significantly in the early stages of pretreatment. Ethanol, acetic acid, propionic acid, and butyric acid were the predominant volatile organic products in the MC1 hydrolysate. Biogas and methane production yields of LMSW significantly increased following MC1 pretreatment. In addition, the methane production rate of the treated LMSW was greater than that observed from the untreated sample.

Bioreactor performance and methanogenic population dynamics in a low-temperature (5-18°C) anaerobic fixed-bed reactor.

The effect of temperature on the functionality of microbial community structure in a low temperature, anaerobic fixed-bed reactor was studied by decreasing the operating temperature from 18 °C to 5 °C. The reactor was productive within 20 days and produced stable methane content in biogas (above 77%) throughout the trial period. At 17 °C and 15 °C, chemical oxygen demand (COD) removal efficiency and biogas production of reactor were significantly reduced. These might be temperature thresholds when fixed-bed reactors are operated under low temperatures. The methanogen community composition was analyzed using 16S rRNA gene clone library screening and quantitative PCR. At low ambient temperatures, Methanomicrobiales were dominant methanogens, and they preferentially adhered to the carbon fiber carrier. The results indicated that 16S rRNA levels of Methanomicrobiales and Methanosaetaceae in adhering sludge were higher than in deposited sludge, and they all contributed to the efficient performance of the fixed-bed reactor at low operating temperatures.

Selection and characteristics of a switchgrass-colonizing microbial community to produce extracellular cellulases and xylanases.

A microbial community was selected for growth on dried and NaOH-treated switchgrass. During a 14-day liquid cultivation, a 70% loss in dry weight was observed during the first 4 days and after 14 days, the hemicellulose and cellulose in the system were degraded by 73.5% and 67.3%, respectively. The carboxymethyl cellulase (CMCase) and xylanase levels reached 0.21 and 3.75 IU, respectively. The optimal pH for CMCase and xylanase activities was 5 and 6, respectively. The optimal reaction temperature of CMCase and xylanase was 60°C. A library of bacterial and fungal ribosomal gene sequences obtained from the community showed the presence of Achromobacter xylosoxidans and Alcaligenes faecalis and of Fusarium sporotrichioides. To our knowledge, this was the first report on a microbial community selected in the presence of switchgrass to produce extracellular cellulases and xylanases.

Diversity of a mesophilic lignocellulolytic microbial consortium which is useful for enhancement of biogas production

A mesophilic lignocellulolytic microbial consortium BYND-5, established by successive subcultivation, was applied to enhance the biogas production. The degradation efficiency of BYND-5 for rice straw was more than 49.0 ± 1.8% after 7 days of cultivation at 30°C. Various organic compounds, including acetic acid, propionic acid, butyric acid and glycerin were detected during biodegradation. The diversity analysis of BYND-5 was conducted by ARDRA (Amplified ribosomal DNA restriction analysis) of the 16S rDNA clone library. Results indicated that bacterial groups represented in the clone library were the Firmicutes (5.96%), the Bacteroidetes (40.0%), Deferribacteres (8.94%), Proteobacteria (16.17%), Lentisphaerae (2.13%), Fibrobacteraceae (1.7%), and uncultured bacterium (25.1%). Additionally, the enhancement of biogas yield and methane content was directly related to the pretreatment with BYND-5. The microbial community identified herein is potential candidate consortium for the degradation of waste lignocellulose and enhancement of biogas production under the mesophilic temperature conditions.

Effect of pretreatment by a microbial consortium on methane production of waste paper and cardboard

A microbial consortium MC1 was used to pretreat filter paper, office paper, newspaper, and cardboard to enhance methane production. The results of pretreatment indicated that sCOD of hydrolysates of the four substrates increased significantly in the early stage, and peaked on day 7. During pretreatment, ethanol, acetic acid, propionic acid, butyric acid, and glycerol were the predominant volatile organic products in hydrolysates. MC1 had strong degradation ability on the four substrates, and the weight loss of filter paper, office paper, newspaper, and cardboard reached 78.3%, 80.5%, 39.7%, and 49.7%, respectively. The results of anaerobic digestion showed that methane production yields and rates of the four substrates significantly increased after pretreatment. This study is the first attempt to explore the microbial pretreatment method for anaerobic digestion of waste paper and cardboard. Microbial consortium pretreatment could be an effective method for enhancing methane production of waste paper and cardboard into bioenergy.

Degradation of corn stalk by the composite microbial system of MC1

The composite microbial system of MC1 was used to degrade corn stalk in order to determine properties of the degraded products as well as bacterial composition of MC1. Results indicated that the pH of the fermentation broth was typical of lignocellulose degradation by MC1, decreasing in the early phase and increasing in later stages of the degradation. The microbial biomass peaked on the day 3 after degradation. The MC1 efficiently degraded the corn stalk by nearly 70% during which its cellulose content decreased by 71.2%, hemicellulose by 76.5% and lignin by 24.6%. The content of water-soluble carbohydrates (WSC) in the fermentation broth increased progressively during the first three days, and decreased thereafter, suggesting an accumulation of WSC in the early phase of the degradation process. Total levels of various volatile products peaked in the third day after degradation, and 7 types of volatile products were detected in the fermentation broth. These were ethanol, acetic acid, 1,2-ethanediol, propanoic acid, butanoic acid, 3-methyl-butanoic acid and glycerine. Six major compounds were quantitatively analysed and the contents of each compound were ethanol (0.584 g/L), acetic acid (0.735 g/L), 1,2-ethanediol (0.772 g/L), propanoic acid (0.026 g/L), butanoic acid (0.018 g/L) and glycerine (4.203 g/L). Characterization of bacterial cells collected from the culture solution, based on 16S rDNA PCR-DGGE analysis of DNAs, showed that the composition of bacterial community in MC1 coincided basically with observations from previous studies. This indicated that the structure of MC1 is very stable during degradation of different lignocellulose materials.