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Applied Microbiology and Biotechnology (v.92, #3)

Physisporinus vitreus: a versatile white rot fungus for engineering value-added wood products by Francis W. M. R. Schwarze; Mark Schubert (pp. 431-440).

The credo of every scientist working in the field of applied science is to transfer knowledge “from science to market,” a process that combines (1) science (fundamental discoveries and basic research) with (2) technology development (performance assessment and optimization) and (3) technology transfer (industrial application). Over the past 7 years, we have intensively investigated the potential of the white rot fungus, Physisporinus vitreus, for engineering value-added wood products. Because of its exceptional wood degradation pattern, i.e., selective lignification without significant wood strength losses and a preferential degradation of bordered pit membranes, it is possible to use this fungus under controlled conditions to improve the acoustic properties of tonewood (i.e., “mycowood”) as well as to enhance the uptake of preservatives and wood modification substances in refractory wood species (e.g., Norway spruce), a process known as “bioincising.” This minireview summarizes the research that we have performed with P. vitreus and critically discusses the challenges encountered during the development of two distinct processes for engineering value-added wood products. Finally, we peep into the future potential of the bioincising and mycowood processes for additional applications in the forest and wood industry.

Keywords: Physisporinus vitreus ; Bioincising; Mycowood; Wood permeability; Acoustic properties; Tonewood; Value-added wood products

Physisporinus vitreus: a versatile white rot fungus for engineering value-added wood products by Francis W. M. R. Schwarze; Mark Schubert (pp. 431-440).

Keywords: Physisporinus vitreus ; Bioincising; Mycowood; Wood permeability; Acoustic properties; Tonewood; Value-added wood products

Genomic variations of Oenococcus oeni strains and the potential to impact on malolactic fermentation and aroma compounds in wine by Eveline J. Bartowsky; Anthony R. Borneman (pp. 441-447).

Malolactic fermentation (MLF) is the bacterially driven decarboxylation of l-malic acid to l-lactic acid and carbon dioxide, and brings about deacidification, flavour modification and microbial stability of wine. The main objective of MLF is to decrease wine sourness by a small increase in wine pH via the metabolism of l-malic acid. Oenococcus oeni is the main lactic acid bacterium to conduct MLF in virtually all red wine and an increasing number of white and sparkling wine bases. Over the last decade, it is becoming increasingly recognized that O. oeni exhibits a diverse array of secondary metabolic activities during MLF which can modify the sensory properties of wine. These secondary activities include the metabolism of organic acids, carbohydrates, polysaccharides and amino acids, and numerous enzymes such as glycosidases, esterases and proteases, which generate volatile compounds well above their odour detection threshold. Phenotypic variation between O. oeni strains is central for producing different wine styles. Recent studies using array-based comparative genome hybridization and genome sequencing of three O. oeni strains have revealed the large genomic diversity within this species. This review will explore the links between O. oeni metabolism, genomic diversity and wine sensory attributes.

Keywords: Oenococcus oeni ; Malolactic fermentation; Microarrays; Genomics; Wine aroma

Genomic variations of Oenococcus oeni strains and the potential to impact on malolactic fermentation and aroma compounds in wine by Eveline J. Bartowsky; Anthony R. Borneman (pp. 441-447).

Keywords: Oenococcus oeni ; Malolactic fermentation; Microarrays; Genomics; Wine aroma

Perspectives of biotechnological production of l-ribose and its purification by Chao Hu; Liangzhi Li; Yayue Zheng; Lilian Rui; Cuiying Hu (pp. 449-455).

l-Ribose is a non-natural and expensive sugar that can be used as an important intermediate for the synthesis of l-nucleoside analogues, which are used as antiviral drugs. In contrast to chemical production, biotechnological methods can produce l-ribose from biomass under environmentally friendly conditions. In this mini-review, various strategies for synthesizing l-ribose by applying microorganisms and their enzymes are discussed, including microbial biotransformation and biocatalysis by engineering bacteria. Furthermore, subsequent isolation-and-purification techniques, as an integral step in the whole process, are accordingly described, containing the especial introduction of a promising strategy of l-ribose separation. Particularly, further researches and outlook for the improvement of l-ribose preparation was solely stressed. Compared with each method, this mini-review provides a panorama of respective advantages and disadvantages existing in them.

Keywords: l-Ribose; Biotransformation; Ribitol; l-Arabinose; Escherichia coli ; Purification

Perspectives of biotechnological production of l-ribose and its purification by Chao Hu; Liangzhi Li; Yayue Zheng; Lilian Rui; Cuiying Hu (pp. 449-455).

Keywords: l-Ribose; Biotransformation; Ribitol; l-Arabinose; Escherichia coli ; Purification

Recent advances on biological difructose anhydride III production using inulase II from inulin by Hua Hang; Wanmeng Mu; Bo Jiang; Meng Zhao; Liuming Leon Zhou; Tao Zhang; Ming Miao (pp. 457-465).

Difructose anhydride III (DFA III), the smallest cyclic disaccharide, consists of two fructose residues. DFA III is a hydrolysate of inulin and is rarely found in nature. Industrial interest in DFA III as a low-calorie sugar substitute is increasing. The present review describes the properties and physiological functions of DFA III as well as its commercial importance. Focus is also given on the biological production of DFA III from inulin, which contains enzyme resources, inulase II properties, and the capacity for mass DFA III production. Inulase II as an industrial enzyme and its molecular evolution are discussed as well. The aim is to better understand commercial-scale DFA III production as a food product.

Keywords: DFA III; Inulase II; Inulin; Properties; Functions; Production

Recent advances on biological difructose anhydride III production using inulase II from inulin by Hua Hang; Wanmeng Mu; Bo Jiang; Meng Zhao; Liuming Leon Zhou; Tao Zhang; Ming Miao (pp. 457-465).

Keywords: DFA III; Inulase II; Inulin; Properties; Functions; Production

Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs) by Roger A. Sheldon (pp. 467-477).

Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes.

Keywords: Immobilization; Hydrolases; Oxidoreductases; Lyases; Magnetic; Sustainable

Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs) by Roger A. Sheldon (pp. 467-477).

Keywords: Immobilization; Hydrolases; Oxidoreductases; Lyases; Magnetic; Sustainable

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives by Frédéric Collin; Shantanu Karkare; Anthony Maxwell (pp. 479-497).

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

Keywords: DNA gyrase; DNA topoisomerase; Supercoiling; Quinolones; Aminocoumarins; NBTIs

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives by Frédéric Collin; Shantanu Karkare; Anthony Maxwell (pp. 479-497).

Keywords: DNA gyrase; DNA topoisomerase; Supercoiling; Quinolones; Aminocoumarins; NBTIs

Effect of culture operating conditions on succinate production in a multiphase fed-batch bioreactor using an engineered Escherichia coli strain by Jiangfeng Zhu; Chandresh Thakker; Ka-Yiu San; George Bennett (pp. 499-508).

A metabolically engineered Escherichia coli strain SBS550MG (pHL413) was used in this study to investigate the impact of various culture operating conditions for improving the specific succinate production rate for better final titer while maintaining the theoretical succinate yield on glucose in multiphase fed-batch cultures. Previously, we reported that changes in the level of aeration during the cell growth phase significantly modified gene expression profiles and metabolic fluxes in this system (Martinez et al. 2010). Based on these observations, the examination of culture conditions was mainly focused on the aerobic growth phase. It was found that 2–5 h of low dissolved oxygen culture during the aerobic phase improves cell productivity, but pH control during the aerobic phase was not favorable for the system. Cell viability has been identified as a major limiting factor for succinate production. Supplementing LB medium and betaine, an anti-osmotic stress reagent, did not improve cell activity. A higher succinate titer (537.8 mM) using the current metabolic engineering E. coli strain was achieved, which can potentially be improved further by increasing cell viability.

Keywords: Succinate production; Escherichia coli ; Culture conditions; Multiphase fermentation

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha by Xuerong Han; Yasuharu Satoh; Toshifumi Satoh; Ken’ichiro Matsumoto; Toyoji Kakuchi; Seiichi Taguchi; Tohru Dairi; Masanobu Munekata; Kenji Tajima (pp. 509-517).

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10⁵, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T _g, T _m, and T _d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.

Keywords: Polyhydroxyalkanoate (PHA); Improved two-phase reaction system (iTPRS); 2-Hydroxybutyrate (2HB); Ralstonia eutropha ; Class I PHA synthase

1,3-Propanediol production in a two-step process fermentation from renewable feedstock by Filipa Soares Mendes; Maria González-Pajuelo; Hélène Cordier; Jean M. François; Isabel Vasconcelos (pp. 519-527).

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.

Keywords: Molasses; Glycerol; 1,3-Propanediol; Two-step fermentation; Saccharomyces cerevisiae ; Clostridium acetobutylicum

High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase by Xiaojuan Liu; Mingjie Wen; Jing Li; Fangli Zhai; Jing Ruan; Liqing Zhang; Shentao Li (pp. 529-537).

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2₁2₁2₁ with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = β = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

Keywords: Candida utilis ; Uricase; Prokaryotic expression; Characterization; Crystal structure

The secondary substrate binding site of the Pseudoalteromonas haloplanktis GH8 xylanase is relevant for activity on insoluble but not soluble substrates by Sven Cuyvers; Emmie Dornez; Jan A. Delcour; Christophe M. Courtin (pp. 539-549).

Previously, it has been demonstrated that the glycoside hydrolase family 8 xylanase from the psychrophylic bacterium Pseudoalteromonas haloplanktis (XPH) can bind substrate non-catalytically on the surface of its catalytic module. In the present study, the functional relevance of this secondary binding site (SBS) for the enzyme is investigated by site-directed mutagenesis and evaluation of activity and binding properties of mutant variants on a range of structurally different homoxylan and heteroxylan substrates. The SBS had an impact on the activity on insoluble substrates, whereas the activity on soluble substrates remained unaffected. Unexpectedly, the activity on a soluble oligomeric substrate was also affected for some mutants and results on a chromophoric polymeric model substrate were in contrast with the trends observed on the corresponding natural substrate. All in all, results show that the impact of the SBS on the activity of XPH is in part analogous to the functioning of some carbohydrate-binding modules in modular enzymes.

Keywords: Arabinoxylan; Glucuronoxylan; Homoxylan; Psychrophilic xylanase; Family 8; Non-catalytic binding

Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose by Xinhao Ye; Zhiguang Zhu; Chenming Zhang; Y.-H. Percival Zhang (pp. 551-560).

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

Keywords: Cellodextrin phosphorylase; Carbohydrate-binding module (CBM); Cellulose; Protein engineering

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture by Hiroki Bando; Hiromoto Hisada; Hiroki Ishida; Yoji Hata; Yoshio Katakura; Akihiko Kondo (pp. 561-569).

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

Keywords: Hemolysin-like protein-encoding gene (hlyA); Aspergillus oryzae ; Solid-state culture; Endo-1,4-β-glucanase; Promoter

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from Bacillus thuringiensis by Ahmed Abderrahmani; Arthur Tapi; Farida Nateche; Marlène Chollet; Valérie Leclère; Bernard Wathelet; Hocine Hacene; Philippe Jacques (pp. 571-581).

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.

Keywords: Bacillus thuringiensis ; MALDI-ToF; PCR; Nonribosomal lipopeptides; Kurstakins; Spreading

Transcriptome profiling of degU expression reveals unexpected regulatory patterns in Bacillus megaterium and discloses new targets for optimizing expression by Claudia Borgmeier; Rebekka Biedendieck; Kristina Hoffmann; Dieter Jahn; Friedhelm Meinhardt (pp. 583-596).

The first whole transcriptome assessment of a Bacillus megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.

Keywords: Bacillus megaterium ; Hypersecretion; Extracellular enzymes; DegSU; Transcriptome

Reductive dehalogenation mediated initiation of aerobic degradation of 2-chloro-4-nitrophenol (2C4NP) by Burkholderia sp. strain SJ98 by Janmejay Pandey; Hermann J. Heipieper; Archana Chauhan; Pankaj Kumar Arora; Dhan Prakash; M. Takeo; Rakesh K. Jain (pp. 597-607).

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by “reductive dehalogenation”; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.

Keywords: Chlorinated nitroaromatic compounds; Burkholderia ; Aerobic degradation

Effects of cryoprotectants on viability of Lactobacillus reuteri CICC6226 by Baokun Li; Fengwei Tian; Xiaoming Liu; Jianxin Zhao; Hao Zhang; Wei Chen (pp. 609-616).

Freeze-drying is commonly used to preserve probiotics, but it could cause cell damage and loss of viability. The cryoprotectants play an important role in the conservation of viability during freeze-drying. In this study, we investigated the survival rates of Lactobacillus reuteri CICC6226 in the presence of cryoprotectants such as sucrose, trehalose, and reconstituted skim milk (RSM). In addition, we determined the activities of hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and ATPases immediately following the freeze-drying. The results showed that the differences in HK and PK activities with and without the cryoprotectants during freeze-drying were not significant, but cell viability and activities of LDH and ATPase were significantly different (P<0.01) prior to and after freeze-drying. Meanwhile, the results showed that the maintenance of the membrane integrity and fluidity was improved in the presence of the 10% trehalose or 10% RSM than other treatments during freeze-drying. These results have provided direct biochemical and metabolic evidence of injured cell during freeze-drying. Freeze-drying damaged membrane structure and function of cell and inactivated enzymes (LDH and ATPases). The results imply that LDH and ATPases are key markers and could be used to evaluate the effect of cryoprotectants on viability and metabolic activities of L. reuteri CICC6226 during freeze-drying.

Keywords: Lactobacillus reuteri CICC6226; Cryoprotectants; Probiotics; Freeze-drying; Cell membrane fluidity and integrity

Effects of cryoprotectants on viability of Lactobacillus reuteri CICC6226 by Baokun Li; Fengwei Tian; Xiaoming Liu; Jianxin Zhao; Hao Zhang; Wei Chen (pp. 609-616).

Keywords: Lactobacillus reuteri CICC6226; Cryoprotectants; Probiotics; Freeze-drying; Cell membrane fluidity and integrity

Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C₂C₁₂ cells by Amrita Mishra; Suraj Kumar Tripathy; Rizwan Wahab; Song-Hoon Jeong; Inho Hwang; You-Bing Yang; Young-Soon Kim; Hyung-Shik Shin; Soon-Il Yun (pp. 617-630).

Microorganisms, their cell filtrates, and live biomass have been utilized for synthesizing various gold nanoparticles. The shape, size, stability as well as the purity of the bio synthesized nanoparticles become very essential for application purpose. In the present study, gold nanoparticles have been synthesized from the supernatant, live cell filtrate, and biomass of the fungus Penicillium brevicompactum. The fungus has been grown in potato dextrose broth which is also found to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification, following purification and after storing the particles for 3 months under refrigerated condition. Different characterization techniques like X-ray diffraction, Fourier transform infrared spectroscopy, and UV–visible spectroscopy have been used for analysis of the particles. The effect of reaction parameters such as pH and concentration of gold salt have also been monitored to optimize the morphology and dispersity of the synthesized gold nanoparticles. A pH range of 5 to 8 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. Additionally, the cytotoxic nature of prepared nanoparticles has been analyzed using mouse mayo blast cancer C₂C₁₂ cells at different time intervals (24, 48, and 72 h) of incubation period. The cells are cultivated in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum with antibiotics (streptopenicillin) at 37°C in a 5% humidified environment of CO₂. The medium has been replenished every other day, and the cells are subcultured after reaching the confluence. The viability of the cells is analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.

Keywords: Penicillium brevicompactum ; Gold nanoparticles; Live cell filtrate; pH value; Gold ion concentration; Cytotoxicity

Microbial community engineering for biopolymer production from glycerol by Helena Moralejo-Gárate; Emily Mar’atusalihat; Robbert Kleerebezem; Mark C. M. van Loosdrecht (pp. 631-639).

In this work, the potential of using microbial community engineering for production of polyhydroxyalkanoates (PHA) from glycerol was explored. Crude glycerol is a by-product of the biofuel (biodiesel and bioethanol) industry and potentially a good substrate for bioplastic production. A PHA-producing microbial community was enriched based on cultivation in a feast–famine regime as successfully applied before for fatty acids-based biopolymer production. A glycerol-fed sequencing batch reactor operated at a 2-day liquid and biomass residence time and with feast–famine cycles of 24 h was used to enrich a mixed community of PHA producers. In a subsequent fed-batch PHA production step under growth-limiting conditions, the enriched mixed community produced PHA up to a dry weight content of 80 wt.%. The conversion efficiency of substrate to PHA on electron basis was 53%. Since glycerol is entering the metabolic pathways of the cell in the glycolytic pathway, it was anticipated that besides PHA, polyglucose could be formed as storage polymer as well. Indeed, polyglucose was produced in low amounts (∼10 wt.%). The results indicated that the feast–famine-based enrichment strategy was comparably successful to obtain a microbial community compared to fatty acids-based enrichment described before.

Keywords: Glycerol; Biodiesel; Mixed community; Polyhydroxyalkanoate; Bioplastic; Storage polymer

Microbial community engineering for biopolymer production from glycerol by Helena Moralejo-Gárate; Emily Mar’atusalihat; Robbert Kleerebezem; Mark C. M. van Loosdrecht (pp. 631-639).

Keywords: Glycerol; Biodiesel; Mixed community; Polyhydroxyalkanoate; Bioplastic; Storage polymer

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum by Xiongjun Shao; Babu Raman; Mingjun Zhu; Jonathan R. Mielenz; Steven D. Brown; Adam M. Guss; Lee R. Lynd (pp. 641-652).

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

Keywords: Clostridium thermocellum ; Ethanol tolerance; Genome sequencing; Strain adaptation; Mutations

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Applied Microbiology and Biotechnology (v.92, #3)