Zhiyong Zheng

A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol.

The effect of osmotic pressure on erythritol and mannitol production by an osmophilic yeast strain of Yarrowia lipolytica CICC 1675 using glycerol as the sole carbon source was investigated. Appropriately high osmotic pressure was found to enhance erythritol production and inhibit mannitol formation. A novel two-stage osmotic pressure control fed-batch strategy based on the kinetic analysis was developed for higher erythritol yield and productivity. During the first 96 h, the osmotic pressure was maintained at 4.25 osmol/kg by feeding glycerol to reduce the inhibition of cell growth. After 132 h, the osmotic pressure was controlled at 4.94 osmol/kg to maintain a high dp(ery)/dt. Maximum erythritol yield of 194.3g/L was obtained with 0.95 g/L/h productivity, which were 25.7% and 2.2%, respectively, improvement over the best results in one-stage fed-batch fermentation. This is the first report that a novel osmotic pressure control fed-batch strategy significantly enhanced erythritol production.

Enhanced production of curdlan by Alcaligenes faecalis by selective feeding with ammonia water during the cell growth phase of fermentation

Curdlan is a water insoluble exopolysaccharide produced by Alcaligenes faecalis under nitrogen-limiting conditions. After excretion, the polysaccharide is attached the cell wall. Thus enhancement of biomass production during the cell growth phase is important to curdlan production. A strategy of increasing nitrogen source to improve biomass production was adopted for curdlan production by Alcaligenes faecalis (ATCC 31749). In the batch fermentation of curdlan, a relatively higher NH4Cl level of 3.6 g/L with continuous glucose feeding increased the cell density leading to improvement of curdlan production. However, excessive NH4Cl would inhibit curdlan production and biomass production was not improved significantly. In addition, feeding of ammonia water at the initial phase replaced NaOH solution to control pH at 7.0. Subsequently, feeding of NaOH solution was resumed to control pH at 5.6 for curdlan production after ammonia was consumed. As a result, biomass production and curdlan yield were both enhanced remarkably. Feeding of ammonia water during the first 24 h led to biomass production of 18.8 g/L. However, higher cell density did not lead to increase in curdlan production. The maximum curdlan production (72 g/L) was obtained by feeding ammonia water for the first 14 h, during which the cell density was about 11.9 g/L.

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Curdlan β-1,3-glucooligosaccharides induce the defense responses against Phytophthora infestans infection of potato (Solanum tuberosum L. cv. McCain G1) leaf cells.

Activation of the innate immune system before the invasion of pathogens is a promising way to improve the resistance of plant against infection while reducing the use of agricultural chemicals. Although several elicitors were used to induce the resistance of potato plant to microbial pathogen infection, the role of curdlan oligosaccharide (CurdO) has not been established. In the current study, the defense responses were investigated at biochemical and proteomic levels to elucidate the elicitation effect of CurdOs in foliar tissues of potato (Solanum tuberosum L. cv. McCain G1). The results indicate that the CurdOs exhibit activation effect on the early- and late-defense responses in potato leaves. In addition, glucopentaose was proved to be the shortest active curdlan molecule based on the accumulation of H2O2 and salicylic acid and the activities of phenylalanine amino-lyase, β-1,3-glucanase and chitinase. The 2D-PAGE analysis reveals that CurdOs activate the integrated response reactions in potato cells, as a number of proteins with various functions are up-regulated including disease/defense, metabolism, transcription, and cell structure. The pathogenesis assay shows that the ratio of lesion area of potato leaf decreased from 15.82%±5.44% to 7.79%±3.03% when the plants were treated with CurdOs 1 day before the infection of Phytophthora infestans. Furthermore, the results on potato yield and induction reactions indicate that the defense responses induced by CurdOs lasted for short period of time but disappeared gradually.

Changes in gene transcription and protein expression involved in the response of Agrobacterium sp. ATCC 31749 to nitrogen availability during curdlan production.

The changes in transcription of genes involved in nitrogen metabolism and curdlan biosynthesis, and total protein expression were firstly analyzed to define the responses of Agrobacterium sp. ATCC 31749 to nitrogen source availability during curdlan fermentation. The transcription of all nitrogen metabolism and regulation genes increased significantly under nitrogen limitation. The genes of carbon (exoC) and nitrogen (ntrB, ntrC, and nifR) metabolism showed distinctive transcriptional responses to nitrogen limitation. Their relative expression level was increased by 14, 9, 7 and 7-fold, respectively. Two-dimentional electrophoresis (2-DE) revealed that the expression of 14 proteins were elevated and 6 proteins were down-regulated significantly under nitrogen starvation. Furthermore, 4 proteins (GroEL, ABC transporter, Atu1730 and enoylacyl carrier protein reductase) in which the expression level changed significantly were identified. The results showed that L sp. regulates its carbon flux and nitrogen assimilation effectively for better survival.

Purification and characterization of a new endo-β-1,3-glucanase exhibiting a high specificity for curdlan for production of β-1,3-glucan oligosaccharides

Endo-β-1,3-glucanase (Endo23) was purified from a Trichoderma reesei GIMCC 3.498 fermentation broth using anion exchange and 2-stage size exclusion chromatography. Purification of 44.5× and a 12% recovery yield of enzyme activity were achieved. The Mw and isoelectric point were estimated to be 24 kDa and 3.85 using SDS-PAGE and IEF, respectively. The highest substrate specificity was observed for water-insoluble curdlan. The optimal conditions for hydrolyzing curdlan were pH 5.0 and 50°C. The main hydrolytic products were glucobiose and glucotriose. Minor amounts of glucose and glucotetraose were detected. Hg2+, Fe2+, Fe3+, and Sn2+ inhibited the hydrolysis activity of Endo23 at 5 and 50 mM. K+ slightly promoted Endo23 activity. Endo23 belongs to the category EC3.2.1.39. The peptide sequences of Endo23 showed identity with conserved sequences that typically exist in β-1,3-glucanases of the glycoside hydrolase family. The Endo23 sequence was partially similar to a hypothetical lignocellulase from Penicillium oxalicum 114-2.

Structure of oligosaccharide F21 derived from exopolysaccharide WL-26 produced by Sphingomonas sp. ATCC 31555

Mild hydrolysis of Sphingomonas sp. ATCC 31555 polysaccharide WL-26 afforded a new oligosaccharide, F21. Structural resolution based on sugar and methylation analyses as well as NMR data revealed the oligosaccharide to have the following structure: [Formula, see text:].

Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb3-type terminal oxidase and cytochrome caa3-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

Full-scale production of VFAs from sewage sludge by anaerobic alkaline fermentation to improve biological nutrients removal in domestic wastewater

A full-scale project of thermal-alkaline pretreatment and alkaline fermentation of sewage sludge was built to produce volatile fatty acids (VFAs) which was then used as external carbon source for improving biological nitrogen and phosphorus removals (BNPR) in wastewater plant. Results showed this project had efficient and stable performances in VFA production, sludge reduce and BNPR. Hydrolysis rate in pretreatment, VFAs yield in fermentation and total VS reduction reached 68.7%, 261.32 mg COD/g VSS and 54.19%, respectively. Moreover, fermentation liquid with VFA presented similar efficiency as acetic acid in enhancing BNPR, obtaining removal efficiencies of nitrogen and phosphorus up to 72.39% and 89.65%, respectively. Finally, the project also presented greater economic advantage than traditional processes, and the net profits for VFAs and biogas productions are 9.12 and 3.71 USD/m3 sludge, respectively. Long-term operation indicated that anaerobic alkaline fermentation for VFAs production is technically and economically feasible for sludge carbon recovery.

Enhanced production of curdlan by coupled fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma harzianum GIM 3.442

A coupled fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma harzianum GIM 3.442 (AT-CFS) with wheat bran as the optimal nitrogen source was established for producing low-molecular-weight curdlan with high production, which can potentially reduce the cost of low-molecular-weight curdlan biosynthesis. The initial inoculate ratio, pH and the fermentation time were optimized. Compared with the curdlan from the single fermentation system of Agrobacterium sp. ATCC 31749 (A-SFS), the molecular weight (Mw) of the curdlan produced from AT-CFS decreased by 34.01% (from 110.85kDa to 73.15kDa), and the curdlan production (47.9g/L) and conversion rate of glucose to curdlan (0.60gg-1) increased by 119.93% and 36.36%, respectively. The results of RT-PCR showed high curdlan production in AT-CFS was highly correlated with aerobic respiration intensity and curdlan synthase activity. The structure of the curdlan from AT-CFS was the same as that from A-SFS.