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Applied Microbiology and Biotechnology (v.96, #6)

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products by Vittorio Capozzi; Pasquale Russo; María Teresa Dueñas; Paloma López; Giuseppe Spano (pp. 1383-1394).

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.

Keywords: Bread; B-group vitamins; Lactobacillus plantarum ; Lactobacillus sanfranciscensis

Microbial degradation of chloroform by M. Cappelletti; D. Frascari; D. Zannoni; S. Fedi (pp. 1395-1409).

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

Keywords: Biodegradation; Chloroform; Cometabolism; Monooxygenase; Trichloromethane

Immobilized metal ion affinity chromatography: a review on its applications by Randy Chi Fai Cheung; Jack Ho Wong; Tzi Bun Ng (pp. 1411-1420).

After 35 years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.

Keywords: Immobilized metal ion affinity chromatography; Proteins; Purification; Review

Monascus pigments by Yanli Feng; Yanchun Shao; Fusheng Chen (pp. 1421-1440).

Monascus pigments (MPs) as natural food colorants have been widely utilized in food industries in the world, especially in China and Japan. Moreover, MPs possess a range of biological activities, such as anti-mutagenic and anticancer properties, antimicrobial activities, potential anti-obesity activities, and so on. So, in the past two decades, more and more attention has been paid to MPs. Up to now, more than 50 MPs have been identified and studied. However, there have been some reviews about red fermented rice and the secondary metabolites produced by Monascus, but no monograph or review of MPs has been published. This review covers the categories and structures, biosynthetic pathway, production, properties, detection methods, functions, and molecular biology of MPs.

Keywords: Monascus ; Secondary metabolite; Pigment; Chemical structure; Molecular biology

Effects of encapsulation of microorganisms on product formation during microbial fermentations by Johan O. Westman; Päivi Ylitervo; Carl Johan Franzén; Mohammad J. Taherzadeh (pp. 1441-1454).

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.

Keywords: Encapsulation; Microbial cell; Whole-cell biocatalyst; Ethanol; Lactic acid

The plant strengthening root endophyte Piriformospora indica: potential application and the biology behind by P. Franken (pp. 1455-1464).

The successful conversion of plant production systems from conventional resource-exhausting to sustainable strategies depends on knowledge-based management of environmental factors. Root-inhabiting fungi came more and more into focus because their hyphae connect in ideal manner resources and challenges of the surrounding with the plant. A paradigm for such root endophytes is presented by the basidiomycete Piriformospora indica. This fungus possesses a broad host spectrum and positively affects different aspects of plant performance. This so far unique combination of attributes makes P. indica and its close relatives among the Sebacinales very interesting tools for cultivation of various crops. This review will outline the different aspects required to apply this root endophyte in agri- and horticulture concerning plant growth, plant nutrition and plant defence or tolerance thereby explaining what is known about the biological basis for the observed effects. Open questions and challenges for successful inoculum production and application will be discussed.

Keywords: Abiotic stress tolerance; Induced resistance; Inoculum formulation; Piriformospora indica ; Plant growth promotion; Sebacinales

Physiological heterogeneity of Pseudomonas taetrolens during lactobionic acid production by Saúl Alonso; Manuel Rendueles; Mario Díaz (pp. 1465-1477).

Physiological heterogeneity constitutes a critical parameter in biotechnological systems since both metabolite yield and productivity are often hampered by the presence of undesired physiological cell subpopulations. In the present study, the physiological status and functionality of Pseudomonas taetrolens cells were monitored by multiparameter flow cytometry during fermentative lactobionic acid production at the shake-flask and bioreactor scale. In shake-flask fermentation, the onset of the lactobionic acid production phase was accompanied by a progressive loss of cellular metabolic activity, membrane polarization, and membrane integrity concomitantly to acidification. In fact, population dynamics has shown the prevalence of damaged and dead subpopulations when submitted to a pH < 4 from 16 h onwards. Furthermore, fluorescence-activated cell sorting revealed that these sublethally injured cells were nonculturable. In contrast, P. taetrolens cells exhibited a robust physiological status during bioreactor cultivations performed with a pH-shifted strategy at 6.5, remaining predominantly healthy and metabolically active (>96 %) as well as maintaining bioconversion efficiency throughout the course of the fermentation. Additionally, an assessment of the seed culture’s physiological robustness was carried out in order to determine the best seed culture age. Results showed that bioreactor culture performance, growth, and lactobionic acid production efficiency were strongly dependent on the physiological heterogeneity displayed by the seed culture. This study provides the most suitable criteria for optimizing lactobionic acid production efficiency through a novel flow cytometric-based approach based on the physiological status of P. taetrolens. It also constitutes a valuable, broad-ranging methodology for the enhancement of microbial bioprocesses involved in the production of secondary metabolites.

Keywords: Flow cytometry; Physiological status; Lactobionic acid; Whey; Pseudomonas taetrolens ; Secondary metabolite

Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved by Yuanjing Li; Chunjie Tian; Hua Tian; Jiliang Zhang; Xin He; Wenxiang Ping; Hong Lei (pp. 1479-1487).

Nowadays, bacterial cellulose has played more and more important role as new biological material for food industry and medical and industrial products based on its unique properties. However, it is still a difficult task to improve the production of bacterial cellulose, especially a large number of byproducts are produced in the metabolic biosynthesis processes. To improve bacterial cellulose production, ethanol and sodium citrate are added into the medium during the fermentation, and the activities of key enzymes and concentration of extracellular metabolites are measured to assess the changes of the metabolic flux of the hexose monophosphate pathway (HMP), the Embden–Meyerhof–Parnas pathway (EMP), and the tricarboxylic acid cycle (TCA). Our results indicate that ethanol functions as energy source for ATP generation at the early stage of the fermentation in the HMP pathway and the supplementation of ethanol significantly reduces glycerol generation (a major byproduct). While in the EMP pathway, sodium citrate plays a key role, and its supplementation results in the byproducts (mainly acetic acid and pyruvic acid) entering the gluconeogenesis pathway for cellulose synthesis. Furthermore, by adding ethanol and sodium citrate, the main byproduct citric acid in the TCA cycle is also reduced significantly. It is concluded that bacterial cellulose production can be improved by increasing energy metabolism and reducing the formation of metabolic byproducts through the metabolic regulations of the bypasses.

Keywords: Bacterial cellulose; Ethanol; Sodium citrate; Metabolite; Bypass

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications by Fabien Durand; Sébastien Gounel; Christian H. Kjaergaard; Edward I. Solomon; Nicolas Mano (pp. 1489-1498).

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100 mg for 2 L culture, twice that of Myrothecium verrucaria. The k _cat/K _m ratio for conjugated bilirubin (1,513 mM⁻¹ s⁻¹) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980 mM⁻¹ s⁻¹), with the lowest K _m measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300 min at 37 °C, high stability at pH 7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.

Keywords: Bilirubin oxidase; Pichia pastoris ; Oxygen reduction; Biofuel cells; Magnaporthe oryzae ; Decolorization of dyes; Bilirubin detection

Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice by Jingjie Li; Wen Zhang; Chuan Wang; Qian Yu; Ruirui Dai; Xiaofang Pei (pp. 1499-1506).

The endogenous β-galactosidase expressed in intestinal microbes is demonstrated to help humans in lactose usage, and treatment associated with the promotion of beneficial microorganism in the gut is correlated with lactose tolerance. From this point, a kind of recombinant live β-galactosidase delivery system using food-grade protein expression techniques and selected probiotics as vehicle was promoted by us for the purpose of application in lactose intolerance subjects. Previously, a recombinant Lactococcus lactis MG1363 strain expressing food-grade β-galactosidase, the L. lactis MG1363/FGZW, was successfully constructed and evaluated in vitro. This study was conducted to in vivo evaluate its efficacy on alleviating lactose intolerance symptoms in post-weaning Balb/c mice, which were orally administered with 1 × 10⁶ CFU or 1 × 10⁸ CFU of L. lactis MG1363/FGZW daily for 4 weeks before lactose challenge. In comparison with naïve mice, the mice administered with L. lactis MG1363/FGZW showed significant alleviation of diarrhea symptoms in less total feces weight within 6 h post-challenge and suppressed intestinal motility after lactose challenge, although there was no significant increase of β-galactosidase activity in small intestine. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora. Therefore, this recombinant L. lactis strain effectively alleviated diarrhea symptom induced by lactose uptake in lactose intolerance model mice with the probable mechanism of promotion of lactic acid bacteria to differentiate and predominantly colonize in gut microbial community, thus making it a promising probiotic for lactose intolerance subjects.

Keywords: Food grade; β-Galactosidase; Lactococcus lactis ; Lactose intolerance

Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon by Rama Krishna Gudiminchi; Charlene Randall; Diederik J. Opperman; Oluwafemi A. Olaofe; Susan T. L. Harrison; Jacobus Albertyn; Martha S. Smit (pp. 1507-1516).

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6–0.8 μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5–1.0 μmol P450 g _DCW ⁻¹ , for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol L _BRM ⁻¹ was obtained within 24 h (0.34 g L _BRM ⁻¹ h⁻¹) with IPTG-induced cells containing only 0.20 μmol P450 g _DCW ⁻¹ , when glucose (22 g L _BRM ⁻¹ ) was added for cofactor regeneration.

Keywords: CYP153A6; Octane; Alkane hydroxylation; Whole-cell biotransformation; Cofactor regeneration

Purification and characterization of a novel extracellular inulinase from a new yeast species Candida kutaonensis sp. nov. KRF1^T by Bo Yuan; Nan Hu; Juan Sun; Shi-An Wang; Fu-Li Li (pp. 1517-1526).

A novel extracellular exoinulinase was purified and characterized from a new yeast strain KRF1^T, and the gene encoding the enzyme was successfully cloned. The enzyme was stable at low pH between 3.0 and 6.5. The K _m and V _max values of the purified enzyme for inulin were 2.3 mg/mL and 4.8 mg/min, respectively. The optimum temperature of the inulinase was 50 °C, and the enzyme remained 78 % of activity at 60 °C for 2 h. The inulinase showed an amino acid sequence identity of 58 % to its closest homolog in Meyerozyma (Pichia) guilliermondii. In the secondary structure, the domain G (VMEVH) of the enzyme contained three unique residues (V, M, and H). Compared with previously reported inulinases, the enzyme from strain KRF1^T displayed strong acid resistance, notable thermostability, and high affinity for the substrate of inulin. Based on sequence analysis of the 26S rDNA D1/D2 domain and phenotypic characterization, the yeast strain KRF1^T was found to represent a novel anamorphic, ascomycetous yeast species. A complete description of the species is given and the name Candida kutaonensis sp. nov (type strain = KRF1^T = AS 2.4027^T = CBS 11388^T) is proposed.

Keywords: Candida kutaonensis sp. nov.; Conserved motif; Inulinase; Purification; Yeast

Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway by Xiaoxia Yin; Catherine Madzak; Guocheng Du; Jingwen Zhou; Jian Chen (pp. 1527-1537).

In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3 %, and the yields of PA decreased by 51.9 and 69.8 % in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5 g L⁻¹ with an evident decrease in PA yield from 35.2 to 13.5 g L⁻¹.

Keywords: Metabolic engineering; Organic acid production; Pyruvate carboxylation; Yarrowia lipolytica

Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide by Yu-Fei Sun; Ying Lin; Jun-Hui Zhang; Sui-Ping Zheng; Yan-Rui Ye; Xing-Xiang Liang; Shuang-Yan Han (pp. 1539-1550).

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3′-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94 % CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64 % CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170 % hydrolytic activity and 155 % synthetic activity compared with strain KCSe1 as well as 144 % hydrolytic activity and 121 % synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124 % hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.

Keywords: 2A peptide; Co-display; CALB; Sed1p; Sag1p; Pichia pastoris

Membrane interaction and antibacterial properties of chensinin-1, an antimicrobial peptide with atypical structural features from the skin of Rana chensinensis by Dejing Shang; Yue Sun; Che Wang; Shi Wei; Lijie Ma; Li Sun (pp. 1551-1560).

Many antimicrobial peptides from amphibian skin have been purified and structurally characterized and may be developed as therapeutic agents. Here we describe the antibacterial properties and membrane interaction of chensinin-1, a cationic arginine/histidine-rich antimicrobial peptide, from the skin secretions of Rana chensinensis. The amino acid composition, sequence, and atypical structure of chensinin-1 differ from other known antimicrobial peptides from amphibian skin. Chensinin-1 exhibited selective antimicrobial activity against Gram-positive bacteria, was inactive against Gram-negative bacteria, and had no hemolytic activity on human erythrocytes. The CD spectra for chensinin-1 indicated that the peptide adopted an aperiodic structure in water and a conformational structure with 20 % β-strands, 8 % α-helices, and the remaining majority of random coils in the trifluoroethanol or SDS solutions. Time-kill kinetics against Gram-positive Bacillus cereus demonstrated that chensinin-1 was rapidly bactericidal at 2× MIC and PAE was found to be >5 h. Chensinin-1 caused rapid and large dye leakage from negatively charged model vesicles. Furthermore, membrane permeation assays on intact B. cereus indicated that chensinin-1 induced membrane depolarization in less than 1 min and followed to damage the integrity of the cytoplasmic membrane and resulted in efflux of molecules from cytoplasma. Hence, the primary target of chensinin-1 action was the cytoplasmic membrane of bacteria. Chensinin-1 was unable to overcome bacterial resistance imposed by the lipopolysaccharide leaflet, the major constituent of the outer membrane of Gram-negative bacteria. Lipopolysaccharide induced oligomerization of chensinin-1, thus preventing its translocation across the outer membrane.

Keywords: Arginine/histidine-rich peptide; Circular dichroism; Depolarization; Integrity; Lipopolysaccharide

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns by Irene Aloisio; Cecilia Santini; Bruno Biavati; Giovanni Dinelli; Avrelija Cencič; Walter Chingwaru; Luca Mogna; Diana Di Gioia (pp. 1561-1576).

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.

Keywords: Bifidobacterium ; Probiotics; Newborns; Enteric disorders; Nontumorigenic cell lines

A novel integrated approach to quantitatively evaluate the efficiency of extracellular polymeric substances (EPS) extraction process by Min Sun; Wen-Wei Li; Han-Qing Yu; Hideki Harada (pp. 1577-1585).

A novel integrated approach is developed to quantitatively evaluate the extracellular polymeric substances (EPS) extraction efficiency after taking into account EPS yield, EPS damage, and cell lysis. This approach incorporates grey relational analysis and fuzzy logic analysis, in which the evaluation procedure is established on the basis of grey relational coefficients generation, membership functions construction, and fuzzy rules description. The flocculation activity and DNA content of EPS are chosen as the two evaluation responses. To verify the feasibility and effectiveness of this integrated approach, EPS from Bacillus megaterium TF10 are extracted using five different extraction methods, and their extraction efficiencies are evaluated as one real case study. Based on the evaluation results, the maximal extraction grades and corresponding optimal extraction times of the five extraction methods are ordered as EDTA, 10 h > formaldehyde + NaOH, 60 min > heating, 120 min > ultrasonication, 30 min > H₂SO₄, 30 min > control. The proposed approach here offers an effective tool to select appropriate EPS extraction methods and determine the optimal extraction conditions.

Keywords: Bacillus megaterium ; Evaluation; Extraction efficiency; Extracellular polymeric substances (EPS); Grey-fuzzy logic

Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China by Hongguang Guo; Zhisheng Yu; Ruyin Liu; Hongxun Zhang; Qiding Zhong; Zhenghe Xiong (pp. 1587-1597).

To identify the methanogenic pathways present in a deep coal bed methane (CBM) reservoir associated with Eastern Ordos Basin in China, a series of geochemical and microbiological studies was performed using gas and water samples produced from the Liulin CBM reservoir. The composition and stable isotopic ratios of CBM implied a mixed biogenic and thermogenic origin of the methane. Archaeal 16S rRNA gene analysis revealed the dominance of the methylotrophic methanogen Methanolobus in the water produced. The high potential of methane production by methylotrophic methanogens was found in the enrichments using the water samples amended with methanol and incubated at 25 and 35 °C. Methylotrophic methanogens were the dominant archaea in both enrichments as shown by polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE). Bacterial 16S rRNA gene analysis revealed that fermentative, sulfate-reducing, and nitrate-reducing bacteria inhabiting the water produced were a factor in coal biodegradation to fuel methanogens. These results suggested that past and ongoing biodegradation of coal by methylotrophic methanogens and syntrophic bacteria, as well as thermogenic CBM production, contributed to the Liulin CBM reserves associated with the Eastern Ordos Basin.

Keywords: Coal bed methane; Methanogenesis; Deep subsurface environment; Ordos Basin

Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment by Azeem Khalid; Farzana Kausar; Muhammad Arshad; Tariq Mahmood; Iftikhar Ahmed (pp. 1599-1606).

Presence of huge amount of salts in the wastewater of textile dyeing industry is one of the major limiting factors in the development of an effective biotreatment system for the removal of azo dyes from textile effluents. Bacterial spp. capable of thriving under high salt conditions could be employed for the treatment of saline dye-contaminated textile wastewaters. The present study was aimed at isolating the most efficient bacterial strains capable of decolorizing azo dyes under high saline conditions. Fifty-eight bacterial strains were isolated from seawater, seawater sediment, and saline soil, using mineral salt medium enriched with 100 mg l⁻¹ Reactive Black-5 azo dye and 50 g NaCl l⁻¹ salt concentration. Bacterial strains KS23 (Psychrobacter alimentarius) and KS26 (Staphylococcus equorum) isolated from seawater sediment were able to decolorize three reactive dyes including Reactive Black 5, Reactive Golden Ovifix, and Reactive Blue BRS very efficiently in liquid medium over a wide range of salt concentration (0–100 g NaCl l⁻¹). Time required for complete decolorization of 100 mg dye l⁻¹ varied with the type of dye and salt concentration. In general, there was an inverse linear relationship between the velocity of the decolorization reaction (V) and salt concentration. This study suggested that bacteria isolated from saline conditions such as seawater sediment could be used in designing a bioreactor for the treatment of textile effluent containing high concentration of salts.

Keywords: Bacteria; Decolorization; Reactive dyes; Salinity; Seawater; Sediment

Antibiofilm activity of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 against Pseudomonas aeruginosa by Yong-Guy Kim; Jin-Hyung Lee; Chang-Jin Kim; Jae-Chan Lee; Yoon Jung Ju; Moo Hwan Cho; Jintae Lee (pp. 1607-1617).

Members of the actinomycetes family are a rich source of bioactive compounds including diverse antibiotics. This study sought to identify novel and non-toxic biofilm inhibitors from the actinomycetes library for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. After the screening of 4104 actinomycetes strains, we found that the culture spent medium (1 %, v/v) of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 inhibited P. aeruginosa biofilm formation by 90 % without affecting the growth of planktonic P. aeruginosa cells, while the spent media enhanced the swarming motility of P. aeruginosa. Global transcriptome analyses revealed that the spent medium of Streptomyces sp. BFI 230 induced expression of phenazine, pyoverdine, pyochelin synthesis genes, and iron uptake genes in P. aeruginosa. The addition of exogenous iron restored the biofilm formation and swarming motility of P. aeruginosa in the presence of the spent medium of Streptomyces sp. BFI 230, which suggests that the Streptomyces sp. BFI 230 strain interfered iron acquisition in P. aeruginosa. Experiments on solvent extraction, heat treatment, and proteinase K treatment suggested that hydrophilic compound(s), possibly extracellular peptides or proteins from Streptomyces sp. BFI 230 cause the biofilm reduction of P. aeruginosa. Together, this study indicates that actinomycetes strains have an ability to control the biofilm of P. aeruginosa.

Keywords: Pseudomonas aeruginosa ; Biofilm inhibition; Streptomyces ; Iron acquisition; Swarming motility

Increase of organic solvent tolerance of Escherichia coli by the deletion of two regulator genes, fadR and marR by Hye Yun Oh; Jae Ok Lee; Ok Bin Kim (pp. 1619-1627).

The improvement of bacterial tolerance to organic solvents is a main prerequisite for the microbial production of biofuels which are toxic to cells. For targeted genetic engineering of Escherichia coli to increase organic solvent tolerances (OSTs), we selected and investigated a total of 12 genes that participate in relevant mechanisms to tolerance. In a spot assay of 12 knockout mutants with n-hexane and cyclohexane, the genes fadR and marR were finally selected as the two key genes for engineering. Fatty acid degradation regulon (FadR) regulates the biosynthesis and degradation of fatty acids coordinately, and the multiple antibiotic resistance repressor (MarR) is the repressor of the global regulator MarA for multidrug resistance. In the competitive growth assay, the ΔmarR mutant became dominant when the pooled culture of 11 knockout mutants was cultivated successively in the presence of organic solvent. The increased OSTs in the ΔmarR and ΔfadR mutants were confirmed by a growth experiment and a viability test. The even more highly enhanced OSTs in the ΔfadR ΔmarR double mutant were shown compared with the two single mutants. Cellular fatty acid analysis showed that the high ratio of saturated fatty acids to unsaturated fatty acids plays a crucial role in OSTs. Furthermore, the intracellular accumulation of OST strains was significantly decreased compared with the wild-type strain.

Keywords: Organic solvent tolerances; Biofuel; fadR ; marR ; Saturated fatty acid; MDR

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Applied Microbiology and Biotechnology (v.96, #6)