INDISIM-Denitrification, an individual-based model for study the denitrification process Abstract Denitrification is one of the key processes of the global nitrogen (N) cycle driven by bacteria. It has been widely known for more than 100 years as a process by which the biogeochemical N-cycle is balanced. To study this process, we develop an individual-based model called INDISIM-Denitrification. The model embeds a thermodynamic model for bacterial yield prediction inside the individual-based model INDISIM and is designed to simulate in aerobic and anaerobic conditions the cell growth kinetics of denitrifying bacteria. INDISIM-Denitrification simulates a bioreactor that contains a culture medium with succinate as a carbon source, ammonium as nitrogen source and various electron acceptors. To implement INDISIM-Denitrification, the individual-based model INDISIM was used to give sub-models for nutrient uptake, stirring and reproduction cycle. Using a thermodynamic approach, the denitrification pathway, cellular maintenance and individual mass degradation were modeled using microbial metabolic reactions. These equations are the basis of the sub-models for metabolic maintenance, individual mass synthesis and reducing internal cytotoxic products. The model was implemented in the open-access platform NetLogo. INDISIM-Denitrification is validated using a set of experimental data of two denitrifying bacteria in two different experimental conditions. This provides an interactive tool to study the denitrification process carried out by any denitrifying bacterium since INDISIM-Denitrification allows changes in the microbial empirical formula and in the energy-transfer-efficiency used to represent the metabolic pathways involved in the denitrification process. The simulator can be obtained from the authors on request.

Identification of a cell-surface protein involved in glucose assimilation and disruption of the crystalline region of cellulose by Cytophaga hutchinsonii Abstract The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557, which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii. Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.

Microbial production of 2,3-butanediol for industrial applications Abstract 2,3-Butanediol (2,3-BD) has great potential for diverse industries, including chemical, cosmetics, agriculture, and pharmaceutical areas. However, its industrial production and usage are limited by the fairly high cost of its petro-based production. Several bio-based 2,3-BD production processes have been developed and their economic advantages over petro-based production process have been reported. In particular, many 2,3-BD-producing microorganisms including bacteria and yeast have been isolated and metabolically engineered for efficient production of 2,3-BD. In addition, several fermentation processes have been tested using feedstocks such as starch, sugar, glycerol, and even lignocellulose as raw materials. Since separation and purification of 2,3-BD from fermentation broth account for the majority of its production cost, cost-effective processes have been simultaneously developed. The construction of a demonstration plant that can annually produce around 300 tons of 2,3-BD is scheduled to be mechanically completed in Korea in 2019. In this paper, core technologies for bio-based 2,3-BD production are reviewed and their potentials for use in the commercial sector are discussed.

Increasing l -threonine production in Escherichia coli by overexpressing the gene cluster phaCAB Abstract l-Threonine is an important branched-chain amino acid and could be applied in feed, drugs, and food. In this study, l-threonine production in an l-threonine-producing Escherichia coli strain TWF001 was significantly increased by overexpressing the gene cluster phaCAB from Ralstonia eutropha. TWF001/pFW01-phaCAB could produce 96.4-g/L l-threonine in 3-L fermenter and 133.5-g/L l-threonine in 10-L fermenter, respectively. In addition, TWF001/pFW01-phaCAB produced 216% more acetyl-CoA, 43% more malate, and much less acetate than the vector control TWF001/pFW01, and meanwhile, TWF001/pFW01-phaCAB produced poly-3-hydroxybutyrate, while TWF001/pFW01 did not. Transcription analysis showed that the key genes in the l-threonine biosynthetic pathway were up-regulated, the genes relevant to the acetate formation were down-regulated, and the gene acs encoding the enzyme which converts acetate to acetyl-CoA was up-regulated. The results suggested that overexpression of the gene cluster phaCAB in E. coli benefits the enhancement of l-threonine production.

Promoting microbial utilization of phenolic substrates from bio-oil Abstract The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween® 20 and 30 wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Affinity-binding immobilization of d -amino acid oxidase on mesoporous silica by a silica-specific peptide Abstract Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits the recovery ability. Here, affinity-binding immobilization of enzymes was explored using a silica-specific affinity peptide (SAP) as a fusion tag to intensify the binding force between the enzyme and mesoporous silica (MPS) carrier. d-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO–SAP–MPS was superior comparing with DAAO–2SAP–MPS and DAAO–3SAP–MPS; meanwhile DAAO–SAP–MPS shows 36% higher activity than that of DAAO–MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO–SAP–MPS compared to that of DAAO–MPS at 50 °C for 3 h. Moreover, the activity recovery of immobilized DAAO–SAP–MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO–MPS.

Efficient production of levan using a recombinant yeast Saccharomyces cerevisiae hypersecreting a bacterial levansucrase Abstract Levan is a fructose polymer with diverse applications in the food and medical industries. In this study, levansucrase from Rahnella aquatilis (RaLsrA) was hyper-secreted using a Saccharomyces cerevisiae protein secretion system. An optimal secretion signal, a translation fusion partner (TFP) containing an N-terminal 98 amino acid domain from a mitochondrial inner membrane protein, UTH1, was employed to secrete approximately 50 U/mL of bioactive RaLsrA into culture media with 63% secretion efficiency by fed-batch fermentation. Although the purified RaLsrA was useful for enzymatic conversion of high-molecular-weight levan of approximately 3.75 × 106 Da, recombinant yeast secreting RaLsrA could produce levan more efficiently by microbial fermentation. In a 50-L scale fermenter, 76-g/L levan was directly converted from 191-g/L sucrose by recombinant yeast cells, attaining an 80% conversion yield and 3.17-g/L/h productivity. Thus, we developed a cost-effective and industrially applicable production system for food-grade levan.

Metabolic adaptability shifts of cell membrane fatty acids of Komagataeibacter hansenii HDM1-3 improve acid stress resistance and survival in acidic environments Abstract Komagataeibacter hansenii HDM1-3 (K. hansenii HDM1-3) has been widely applied for producing bacterial cellulose (BC). The yield of BC has been frequently limited by the acidification during sugar metabolism, due to the generation of organic acids such as acetic acid. In this study, the acid resistance mechanism of K. hansenii HDM1-3 has been investigated from the aspect of metabolic adaptability of cell membrane fatty acids. Firstly, we observed that the survival rate of K. hansenii HDM1-3 was decreased with lowered pH values (adjusted with acetic acids), accompanied by increased leakage rate. Secondly, the cell membrane adaptability in response to acid stress was evaluated, including the variations of cell membrane fluidity and fatty acid composition. The proportion of unsaturated fatty acids was increased (especially, C18-1w9c and C19-Cyc), unsaturation degree and chain length of fatty acids were also increased. Thirdly, the potential molecular regulation mechanism was further elucidated. Under acid stress, the fatty acid synthesis pathway was involved in the structure and composition variations of fatty acids, which was proved by the activation of both fatty acid dehydrogenase (des) and cyclopropane fatty acid synthase (cfa) genes, as well as the addition of exogenous fatty acids. The fatty acid synthesis of K. hansenii HDM1-3 may be mediated by the activation of two-component sensor signaling pathways in response to the acid stress. The acid resistance mechanism of K. hansenii HDM1-3 adds to our knowledge of the acid stress adaptation, which may facilitate the development of new strategies for improving the industrial performance of this species under acid stress.

Evaluating changes to Ralstonia pickettii in high-purity water to guide selection of potential calibration materials for online water bioburden analyzers Abstract Online water bioburden analyzers (OWBAs) can provide real-time feedback on viable bacteria in high-purity water (HPW) systems for pharmaceutical manufacturers. To calibrate and validate OWBAs, which detect bacteria using scattered light and bacterial autofluorescence, standards are needed that mimic the characteristics of bacteria in HPW. To guide selection of potential standards, e.g., fluorescent microspheres, a relevant bacterial contaminant, Ralstonia pickettii, was characterized for size, count, viability, and autofluorescence after exposure for 24 h to HPW or a nutrient environment. The cells exposed to HPW showed smaller sizes, with lower counts and autofluorescence intensities, but similar spectral features. The cell characteristics are discussed in comparison with a set of fluorescent microspheres, considering factors relevant to OWBAs. These studies suggest that fluorescent microspheres should be relatively small (< 1 µm diameter) and dim, while covering a broad emission range from ≈ (420 to 600) nm to best mimic the representative R. pickettii.

Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri Abstract Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.