Hiroshi Habe

Production of Glyceric Acid by Gluconobacter sp. NBRC3259 Using Raw Glycerol

Gluconobacter sp. NBRC3259 converted glycerol to glyceric acid (GA). The enantiomeric composition of the GA produced was a mixture of DL-forms with a 77% enantiomeric excess of D-GA. After culture conditions, such as initial glycerol concentration, types and amounts of nitrogen sources, and initial pH, were optimized, Gluconobacter sp. NBRC3259 produced 54.7 g/l of GA as well as 33.7 g/l of dihydroxyacetone (DHA) from 167 g/l of glycerol during 4 d of incubation in a jar fermentor with pH control. GA production from raw glycerol samples, the main by-product of the transesterification process in the biodiesel production and oleochemical industries, was also evaluated after proper pretreatment of the samples. Using a raw glycerol sample with activated charcoal pretreatment, 45.9 g/l of GA and 28.2 g/l of DHA were produced from 174 g/l of glycerol.

Biotechnological production of d -glyceric acid and its application

Glycerol is currently produced in large amounts as a by-product during fat splitting and biodiesel fuel production. Over the past decade, both chemical and biotechnological processes to convert glycerol to value-added chemicals have been increasingly explored. This mini-review provides recent information about the biotechnological production of a glycerol derivative, d-glyceric acid (d-GA), and its possible applications. Little is known about GA as a bioproduct, but it is naturally found in different kinds of plants as a phytochemical constituent and is reported to have some biological activity. A racemic mixture of dl-GA can be obtained from glycerol via chemical oxidation; however, d-GA is mainly biotechnologically produced with the aid of bacteria. Under aerobic conditions, some acetic acid bacteria transform glycerol into d-GA, and optimization of initial glycerol concentration and aeration rate provided a yield of more than 80xa0g/l d-GA, using a strain of Gluconobacter frateurii.

Biotransformation of glycerol to d-glyceric acid by Acetobacter tropicalis

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3xa0g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20xa0g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7xa0g/l d-GA from 200xa0g/l glycerol during 4xa0days of incubation in a jar fermentor.

Genome and Transcriptome Analysis of the Basidiomycetous Yeast Pseudozyma antarctica Producing Extracellular Glycolipids, Mannosylerythritol Lipids

Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.

Disruption of the Membrane-Bound Alcohol Dehydrogenase-Encoding Gene Improved Glycerol Use and Dihydroxyacetone Productivity in Gluconobacter oxydans

Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant ΔadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. ΔadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting ΔadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on ΔadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

High-resolution phylogenetic analysis of residual bacterial species of fouled membranes after NaOCl cleaning.

Biofouling is one of the major problems during wastewater treatment using membrane bioreactors (MBRs). In this regard, sodium hypochlorite (NaOCl) has been widely used to wash fouled membranes for maintenance and recovery purposes. Advanced chemical and biological characterization was conducted in this work to evaluate the performance of aqueous NaOCl solutions during washing of polyacrylonitrile membranes. Fouled membranes from MBR operations supplemented with artificial wastewater were washed with 0.1% and 0.5% aqueous NaOCl solutions for 5, 10 and 30 min. The changes in organics composition on the membrane surface were directly monitored by an attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectrometer. In addition, high-throughput Illumina sequencing of 16S rRNA genes was applied to detect any residual microorganisms. Results from ATR-FT-IR analysis indicated the complete disappearance of functional groups representing different fouling compounds after at least 30 min of treatment with 0.1% NaOCl. However, the biomolecular survey revealed the presence of residual bacteria even after 30 min of treatment with 0.5% NaOCl solution. Evaluation of microbial diversity of treated samples using Chao1, Shannon and Simpson reciprocal indices showed an increase in evenness while no significant decline in richness was observed. These implied that only the population of dominant species was mainly affected. The high-resolution phylogenetic analysis revealed the presence of numerous operational taxonomic units (OTUs) whose close relatives exhibit halotolerance. Some OTUs related to thermophilic and acid-resistant strains were also identified. Finally, the taxonomic analysis of recycled membranes that were previously washed with NaOCl also showed the presence of numerous halotolerant-related OTUs in the early stage of fouling. This further suggested the possible contribution of such chemical tolerance on their survival against NaOCl washing, which in turn affected their re-fouling potential.

Fine-scale monitoring of shifts in microbial community composition after high organic loading in a pilot-scale membrane bioreactor.

In biological wastewater treatment, municipal wastewater sometimes undergoes unexpected changes in physicochemical parameters, such as organic carbon concentration. The aim of this study was to understand how microbial communities in activated sludge in a membrane bioreactor (MBR) adapt to high organic loading and maintain their degradation ability during reactor operation. A pilot-scale MBR was operated for 19 days. On day 8, the concentration of organic matter in the synthetic wastewater increased from 450 to 900 mg chemical oxygen demand (COD)/L. Even under conditions of high organic loading, COD removal rates were high, ranging from 85.3 to 91.4%. High-throughput sequencing of 16S rRNA genes revealed that microbial communities changed drastically with increased organic loading. After day 8, Aquabacterium- and Azospira-related operational taxonomic units (OTUs) belonging to the class β-proteobacteria became dominant; this potentially enhanced the degradation of organic substances and decreased activated sludge microbial diversity. Due to the use of dissolved oxygen (DO) for degradation of organic substances, DO levels in the reactor decreased. This led to an increase in a subset of OTUs related to not only aerobic but also anaerobic bacteria, e.g., those in the class Clostridia. During this period, anaerobic microorganisms may have contributed to the degradation of organic substances to maintain MBR performance. On the other hand, high-throughput sequencing also made it possible to identify yet-to-be cultured or minor microorganisms affiliated with the candidate phylogenetic division SR1 and ammonia-oxidizing archaea in activated sludge.

Bacterial production of short-chain organic acids and trehalose from levulinic acid: a potential cellulose-derived building block as a feedstock for microbial production.

Levulinic acid (LA) is a platform chemical derived from cellulosic biomass, and the expansion of LA utilization as a feedstock is important for production of a wide variety of chemicals. To investigate the potential of LA as a substrate for microbial conversion to chemicals, we isolated and identified LA-utilizing bacteria. Among the six isolated strains, Pseudomonas sp. LA18T and Rhodococcus hoagie LA6W degraded up to 70 g/L LA in a high-cell-density system. The maximal accumulation of acetic acid by strain LA18T and propionic acid by strain LA6W was 13.6 g/L and 9.1 g/L, respectively, after a 4-day incubation. Another isolate, Burkholderia stabilis LA20W, produced trehalose extracellularly in the presence of 40 g/L LA to approximately 2 g/L. These abilities to produce useful compounds supported the potential of microbial LA conversion for future development and cellulosic biomass utilization.

Application of electrodialysis to glycerate recovery from a glycerol containing model solution and culture broth.

Glyceric acid is produced by the conversion of glycerol via bioprocesses. The glycerate recovery from model solutions and from real culture broth was demonstrated by a desalting electrodialysis (ED) method. The addition of several impurities in glycerate model solutions, such as polypepton or yeast extract, did not have significant adverse effects on the whole ED process, and more than 93% of the glycerol added in the model solutions (50-150 g/l) was excluded. Using culture broth of Acetobacter tropicalis containing 14.6 g/l D-glycerate, the D-glycerate recovery and the energy consumption were 99.4% and 0.24 kWh/kg, respectively.

Use of a Gluconobacter frateurii Mutant to Prevent Dihydroxyacetone Accumulation during Glyceric Acid Production from Glycerol

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.