Jinzhu Song

Developing a mesophilic co-culture for direct conversion of cellulose to butanol in consolidated bioprocess

BackgroundConsolidated bioprocessing (CBP) of butanol production from cellulosic biomass is a promising strategy for cost saving compared to other processes featuring dedicated cellulase production. CBP requires microbial strains capable of hydrolyzing biomass with enzymes produced on its own with high rate and high conversion and simultaneously produce a desired product at high yield. However, current reported butanol-producing candidates are unable to utilize cellulose as a sole carbon source and energy source. Consequently, developing a co-culture system using different microorganisms by taking advantage of their specific metabolic capacities to produce butanol directly from cellulose in consolidated bioprocess is of great interest.ResultsThis study was mainly undertaken to find complementary organisms to the butanol producer that allow simultaneous saccharification and fermentation of cellulose to butanol in their co-culture under mesophilic condition. Accordingly, a highly efficient and stable consortium N3 on cellulose degradation was first developed by multiple subcultures. Subsequently, the functional microorganisms with 16S rRNA sequences identical to the denaturing gradient gel electrophoresis (DGGE) profile were isolated from consortium N3. The isolate Clostridium celevecrescens N3-2 exhibited higher cellulose-degrading capability was thus chosen as the partner strain for butanol production with Clostridium acetobutylicum ATCC824. Meanwhile, the established stable consortium N3 was also investigated to produce butanol by co-culturing with C. acetobutylicum ATCC824. Butanol was produced from cellulose when C. acetobutylicum ATCC824 was co-cultured with either consortium N3 or C. celevecrescens N3-2. Co-culturing C. acetobutylicum ATCC824 with the stable consortium N3 resulted in a relatively higher butanol concentration, 3.73 g/L, and higher production yield, 0.145 g/g of glucose equivalent.ConclusionsThe newly isolated microbial consortium N3 and strain C. celevecrescens N3-2 displayed effective degradation of cellulose and produced considerable amounts of butanol when they were co-cultured with C. acetobutylicum ATCC824. This is the first report of application of co-culture to produce butanol directly from cellulose under mesophilic condition. Our results indicated that co-culture of mesophilic cellulolytic microbe and butanol-producing clostridia provides a technically feasible and more simplified way for producing butanol directly from cellulose.

Haloterrigena daqingensis sp. nov., an extremely haloalkaliphilic archaeon isolated from a saline-alkaline soil

A haloalkaliphilic archaeon, strain JX313(T), was isolated from a saline-alkaline soil from Daqing, Heilongjiang Province, China. Its morphological, physiological and biochemical features and 16S rRNA gene sequence were determined. Colonies of the strain were orange-red and cells were non-motile cocci and Gram-stain-variable. The strain required at least 1.7 M NaCl for growth, with optimal growth occurring in 2.0-2.5 M NaCl. Growth was observed at 20-50°C and pH 8.0-10.5, with optimal growth at 35°C and pH 10.0. The G+C content of its genomic DNA was 59.3 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain JX313(T) is associated with the genera Haloterrigena and Natrinema and is most closely related to Haloterrigena salina XH-65(T) (96.2  % sequence similarity) and Haloterrigena hispanica FP1(T) (96.2 %). DNA-DNA hybridization experiments revealed that the relatedness of strain JX313(T) to type strains of related species of the genus Haloterrigena or Natrinema was less than 50 %. Furthermore, the cellular polar lipids of strain JX313(T), identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and mannose-2,6-disulfate (1→2)-glucose glycerol diether (S₂-DGD), were consistent with the polar lipid characteristics of the genus Haloterrigena. Therefore, phylogenetic analysis, phenotypic assessment and chemotaxonomic data showed that JX313(T) represents a novel species within the genus Haloterrigena, for which the name Haloterrigena daqingensis sp. nov. is proposed. The type strain is JX313(T) (=CGMCC 1.8909(T) =NBRC 105739(T)).

A new serine protease gene from Trichoderma harzianum is expressed in Saccharomyces cerevisiae

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to biocontrol processes. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these proteases for industrial application. We report here a successful expression of the serine protease SL41 from a biocontrol fungus Trichoderma harzianum in Saccharomyces cerevisiae. A new serine proteases gene SL41 has been cloned from T. harzianum. The full-length cDNA was isolated by 5′ and 3′ rapid amplification of cDNA ends. The isolated cDNA of SL41 was then sequenced. The results showed that the open reading frame of SL41 was 1.617 bp long, encoding 538 amino acids. The cloning vector pMD18-T and an E. coli DH5α host were used to yield clones as E. coli DH5α/SL41. The SL41 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/SL41. Serine protease expressed by pYES2/SL41 was induced by galactose (maximal activity 16.5 units ml−1 at 40°C, pH 8.0) and was produced in fermentation liquid cultured for 60 h. Northern blot analysis indicated that SL41 transcripts are differentially accumulated at different culture time.

Cloning, annotation and expression analysis of mycoparasitism-related genes in Trichoderma harzianum 88.

Trichoderma harzianum 88, a filamentous soil fungus, is an effective biocontrol agent against several plant pathogens. High-throughput sequencing was used here to study the mycoparasitism mechanisms of T. harzianum 88. Plate confrontation tests of T. harzianum 88 against plant pathogens were conducted, and a cDNA library was constructed from T. harzianum 88 mycelia in the presence of plant pathogen cell walls. Randomly selected transcripts from the cDNA library were compared with eukaryotic plant and fungal genomes. Of the 1,386 transcripts sequenced, the most abundant Gene Ontology (GO) classification group was “physiological process”. Differential expression of 19 genes was confirmed by real-time RT-PCR at different mycoparasitism stages against plant pathogens. Gene expression analysis revealed the transcription of various genes involved in mycoparasitism of T. harzianum 88. Our study provides helpful insights into the mechanisms of T. harzianum 88-plant pathogen interactions.

Biobutanol Production from Lignocellulosic Biomass: Prospective and Challenges

Biobutanol production from lignocellulosic biomass is considered promising and economically feasible. This paper provides an updated review on development of lignocellulose-based biobutanol production with a focus on the understanding of the structure of the feedstock, pretreatment technologies, and fermentation processes. To enhance butanol production from lignocellulosic materials, strategies in terms of inhibitors detoxification, strains improvement and process integration and optimization are also addressed. Besides, the reviewer attempts to shed light on the challenges and perspectives for the bioconversion of lignocellulosic biomass-to-butanol.

Halomonas alkalitolerans sp. nov., a novel moderately halophilic bacteriun isolated from soda meadow saline soil in Daqing, China.

A moderately halophilic bacterial strain 15-13T, which was isolated from soda meadow saline soil in Daqing City, Heilongjiang Province, China, was subjected to a polyphasic taxonomic study. The cells of strain 15–13 were found to be Gram-negative, rod-shaped, and motile. The required growth conditions for strain 15–13T were: 1–23% NaCl (optimum, 7%), 10–50°C (optimum, 35°C), and pH 7.0–11.0 (optimum, pH 9.5). The predominant cellular fatty acids were C18:1ω7c (60.48%) and C16:0 (13.96%). The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain 15–13T clustered within a branch comprising species of the genus Halomonas. The closest phylogenetic neighbor of strain 15–13T was Halomonas pantelleriensis DSM 9661T (98.9% 16S rRNA gene sequence similarity). The level of DNA-DNA relatedness between the novel isolated strain and H pantelleriensis DSM 9661T was 33.8%. On the basis of the phenotypic and phylogenetic data, strain 15–13T represents a novel species of the genus Halomonas, for which the name Halomonas alkalitolerans sp. nov. is proposed. The type strain for this novel species is 15–13T (=CGMCC 1.9129T =NBRC 106539T).

Three tubulin genes of Trichoderma harzianum: alpha, beta and gamma

Three tubulin genes of Trichoderma harzianum were cloned followed genomic walking procedure. The tubulins showed high degree of amino acid homology with other fungal tubulins and were homologous with each other with 32 to 38% amino acid identity. Three measures for the degree of codon usage bias indicated the presence of bias in all the sequences, suggesting high expression levels in all the genes. Protein structures were modeled to provide the basis for understanding the tubulins properties and its interactions with microtubule-associated proteins. Potential motifs were also postulated.

New production process of the antifungal chaetoglobosin A using cornstalks

Chaetoglobosin A is an antibacterial compound produced by Chaetomium globosum, with potential application as a biopesticide and cancer treatment drug. The aim of this study was to evaluate the feasibility of utilizing cornstalks to produce chaetoglobosin A by C. globosum W7 in solid-batch fermentation and to determine an optimal method for purification of the products. The output of chaetoglobosin A from the cornstalks was 0.34 mg/g, and its content in the crude extract was 4.80%. Purification conditions were optimized to increase the content of chaetoglobosin A in the crude extract, including the extract solvent, temperature, and pH value. The optimum process conditions were found to be acetone as the extractant, under room temperature, and at a pH value of 13. Under these conditions, a production process of the antifungal chaetoglobosin A was established, and the content reached 19.17%. Through further verification, cornstalks could replace crops for the production of chaetoglobosin A using this new production process. Moreover, the purified products showed great inhibition against Rhizoctonia solani, with chaetoglobosin A confirmed as the main effective constituent (IC50 = 3.88 μg/mL). Collectively, these results demonstrate the feasibility of using cornstalks to synthesize chaetoglobosin A and that the production process established in this study was effective.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Isolation of sequences flanking the Trichoderma asperellum task1 gene using a single specific primer PCR and their use for gene knockout

Gene recombination has been widely used in bacteria, yeast and other lower organisms for gene function research, but its application in filamentous fungi is uncommon because long homologous sequences are required. In this study, first, the task1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was cloned, and then we devised a novel and efficient PCR-based technique to amplify unknown regions adjacent to known genes using a single, specific primer. The feasibility of this technique was demonstrated by isolating sequences flanking the task1 gene of Trichoderma asperellum. The flanking regions obtained were used to construct a binary vector to knock out T. asperellum task1 gene by a Agrobacterium tumefaciens-mediated transformation method.