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Applied Microbiology and Biotechnology (v.74, #5)

Towards industrial pentose-fermenting yeast strains by Bärbel Hahn-Hägerdal; Kaisa Karhumaa; César Fonseca; Isabel Spencer-Martins; Marie F. Gorwa-Grauslund (pp. 937-953).

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

Modulation of volatile sulfur compounds by wine yeast by J. H. Swiegers; I. S. Pretorius (pp. 954-960).

Sulfur compounds in wine can be a ‘double-edged sword’. On the one hand, certain sulfur-containing volatile compounds such as hydrogen sulfide, imparting a rotten egg-like aroma, can have a negative impact on the perceived quality of the wine, and on the other hand, some sulfur compounds such as 3-mercaptohexanol, imparting fruitiness, can have a positive impact on wine flavor and aroma. Furthermore, these compounds can become less or more attractive or repulsive depending on their absolute and relative concentrations. This presents an interesting challenge to the winemaker to modulate the concentrations of these quality-determining compounds in wine in accordance with consumer preferences. The wine yeast Saccharomyces cerevisiae plays a central role in the production of volatile sulfur compounds. Through the sulfate reduction sequence pathway, the HS^- is formed, which can lead to the formation of hydrogen sulfide and various mercaptan compounds. Therefore, limiting the formation of the HS^- ion is an important target in metabolic engineering of wine yeast. The wine yeast is also responsible for the transformation of non-volatile sulfur precursors, present in the grape, to volatile, flavor-active thiol compounds. In particular, 4-mercapto-4-methylpentan-2-one, 3-mercaptohexanol, and 3-mercaptohexyl acetate are the most important volatile thiols adding fruitiness to wine. This paper briefly reviews the metabolic processes involved in the production of important volatile sulfur compounds and the latest strategies in the pursuit of developing wine yeast strains as tools to adjust wine aroma to market specifications.

Keywords: Aroma; Flavor; Fermented beverages; Thiols; Wine; Yeast

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems by Hermann J. Heipieper; Grit Neumann; Sjef Cornelissen; Friedhelm Meinhardt (pp. 961-973).

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.

Optimization of culture conditions and scale-up to pilot and plant scales for coenzyme Q₁₀ production by Agrobacterium tumefaciens by Suk-Jin Ha; Sang-Yong Kim; Jin-Ho Seo; Deok-Kun Oh; Jung-Kul Lee (pp. 974-980).

This report describes the optimization of culture conditions for coenzyme Q₁₀ (CoQ₁₀) production by Agrobacterium tumefaciens KCCM 10413, an identified high-CoQ₁₀-producing strain (Kim et al., Korean patent. 10-0458818, 2002b). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ₁₀ production. When the pH and DO levels were controlled at 7.0 and 0–10%, respectively, a dry cell weight (DCW) of 48.4 g l⁻¹ and a CoQ₁₀ production of 320 mg l⁻¹ were obtained after 96 h of batch culture, corresponding to a specific CoQ₁₀ content of 6.61 mg g-DCW⁻¹. In a fed-batch culture of sucrose, the DCW, specific CoQ₁₀ content, and CoQ₁₀ production increased to 53.6 g l⁻¹, 8.54 mg g-DCW⁻¹, and 458 mg l⁻¹, respectively. CoQ₁₀ production was scaled up from a laboratory scale (5-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V _tip) as a scale-up parameter. CoQ₁₀ production at the laboratory scale was similar to those at the pilot and plant scales. This is the first report of pilot- and plant-scale productions of CoQ₁₀ in A. tumefaciens.

Keywords: Agrobacterium tumefaciens ; Coenzyme Q₁₀ ; Optimization of culture conditions; DO level; Fed-batch culture; Scale-up; Impeller tip velocity

Optimizing conditions for poly(β-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source by Jorge Quillaguamán; Marlene Muñoz; Bo Mattiasson; Rajni Hatti-Kaul (pp. 981-986).

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.

Keywords: Halomonas boliviensis ; Moderate halophile; Poly(β-hydroxybutyrate) (PHB) production

Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of Haematococcus pluvialis: C/N ratio and light intensity by C. D. Kang; J. S. Lee; T. H. Park; S. J. Sim (pp. 987-994).

We investigated the effect of carbon/nitrogen (C/N) ratio on astaxanthin synthesis in Haematococcus pluvialis during photoautotrophic induction by continuous input of both CO₂–air mixture and intense light. When H. pluvialis was induced by constant irradiance induction at 200 μmol photon m⁻² s⁻¹, there was a positive correlation with astaxanthin content and C/N ratio, which was similar to the case for heterotrophic induction. Lower C/N ratios did not retard Haematococcus encystment, but did increase culture biomass, resulting in a decrease in astaxanthin production because of light limitation. However, induction using variable irradiance showed that reduction of astaxanthin production at low C/N ratios was successfully overcome by simply increasing the light intensity from 200 to 300 μmol photon m⁻² s⁻¹ to overcome the light limitation. This resulted in a greatly enhanced astaxanthin synthesis in proportion to cell density in cultures with low C/N ratios. Our results indicate that light intensity is more critical than C/N ratio in astaxanthin production by H. pluvialis during photoautotrophic induction.

Enhanced hydroxylation of imidacloprid by Stenotrophomonas maltophilia upon addition of sucrose by Yi-jun Dai; Ting Chen; Feng Ge; Yu Huan; Sheng Yuan; Fei-fei Zhu (pp. 995-1000).

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.

Keywords: Imidacloprid; Microbial transformation; Sucrose; NADH; P450

Substrate and product hydrolysis specificity in family 11 glycoside hydrolases: an analysis of Penicillium funiculosum and Penicillium griseofulvum xylanases by Jean-Guy Berrin; El Hassan Ajandouz; Jacques Georis; Filip Arnaut; Nathalie Juge (pp. 1001-1010).

Two genes encoding family 11 endo-(1,4)-β-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.

Keywords: Glycoside hydrolase family 11; Heterologous expression; Kinetic parameters; Wheat arabinoxylan; Xylo-oligosaccharides; Xylanase inhibitor

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity by K. Ravi-Kumar; K. S. Venkatesh; S. Umesh-Kumar (pp. 1011-1015).

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent α-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of α-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Keywords: Aspergillus niger ; Proteolytic processing; Starch-hydrolyzing enzyme; Acarbose; Taka-amylase; α-anomeric configuration

Characterization of the new β-glucuronidase from Streptococcus equi subsp. zooepidemicus by Ján Krahulec; Jana Krahulcová (pp. 1016-1022).

Recently, a new gene encoding β-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37°C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52°C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of β-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of β-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified β-glucuronidase from SEZ glcHis was four times as low as β-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal β-glucuronidase from E. coli with E. coli β-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.

Keywords: Glucuronidase; Streptococcus; Activity; Recombinant; Mutant

Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis by Jingang Wang; Qing Zhang; He Huang; Zhongyi Yuan; Dafu Ding; Sheng Yang; Weihong Jiang (pp. 1023-1030).

Site-directed mutagenesis based on predicted modeled structure of pencillin G acylase from Bacillus megaterium (BmPGA) was followed to increase its performance in the kinetically controlled synthesis of cephalexin with high reactant concentrations of 133 mM 7-amino-desaceto-xycephalosporanic acid (7-ADCA) and 267 mM d-phenylglycine amide (D-PGA). We directed changes in amino acid residues to positions close to the active site that were expected to affect the catalytic performance of penicillin acylase: alpha Y144, alpha F145, and beta V24. Alpha F145 was mutated into tyrosine, alanine, and leucine. Alpha Y144 and beta V24 were mutated into arginine and phenylalanine, respectively. The S/H ratios of three mutants, BmPGAα144R, BmPGAβ24F, and BmPGAβ24F+α144R, were up to 1.3–3.0 times higher values. Compared to the wild-type BmPGA, BmPGAβ24F+α144R showed superior potential of the synthetic performance, allowing the accumulation of up to twofold more cephalexin at significantly higher conversion rates.

Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing Escherichia coli strains by Monica M. Olson; Lori J. Templeton; Wonchul Suh; Philip Youderian; F. Sima Sariaslani; Anthony A. Gatenby; Tina K. Van Dyk (pp. 1031-1040).

Escherichia coli K12 strains producing l-phenylalanine were converted to l-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked ΔpheLA and Ptrc-tyrA::Kan^R genetic modifications were moved into l-phenylalanine producing strains by generalized transduction to convert l-phenylalanine-producing strains to l-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled l-tyrosine-production from sucrose.

Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases by Anu Saloheimo; Jenita Rauta; Oleh V. Stasyk; Andrei A. Sibirny; Merja Penttilä; Laura Ruohonen (pp. 1041-1052).

In the present study, we modified xylose uptake properties of a recombinant xylose-utilizing yeast Saccharomyces cerevisiae by expression of heterologous and homologous permease-encoding genes. In a mutant yeast strain with the main seven hexose transporter genes deleted, and engineered for xylose utilization, we screened an expression cDNA library of the filamentous fungus Trichoderma reesei (Hypocrea jecorina) for enhanced growth on xylose plates. One cDNA clone with significant homology to fungal sugar transporters was obtained, but when the clone was retransformed into the host, it did not support significant growth on xylose. However, during a long liquid culture of the strain carrying the cDNA clone, adaptive mutations apparently occurred in the host, which led to growth on xylose but not on glucose. The new transporter homologue, Trxlt1 thus appears to code for a protein specific for xylose uptake. In addition, xylose-transporting properties of some homologous hexose transporters were studied. All of them, i.e., Hxt1, Hxt2, Hxt4, and Hxt7 were capable of xylose uptake. Their affinities for xylose varied, K _m values between 130 and 900 mM were observed. The single-Hxt strains showed a biphasic growth mode on xylose, alike the Trxlt1 harboring strain. The initial, slow growth was followed by a long lag and finally by exponential growth.

Keywords: Xylose uptake; Saccharomyces cerevisiae ; Hexose transporters; Trichoderma reesei transporter; Adaptive mutation(s)

Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system by Simone Guglielmetti; Matti Karp; Diego Mora; Isabella Tamagnini; Carlo Parini (pp. 1053-1061).

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.

Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium by Rebekka Biedendieck; Rafael Beine; Martin Gamer; Eva Jordan; Klaus Buchholz; Jürgen Seibel; Lubbert Dijkhuizen; Marco Malten; Dieter Jahn (pp. 1062-1073).

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru.

Keywords: Levansucrase; Bacillus megaterium ; Secretion; Affinity tag; Fructosyloligosaccharide

Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris by Da Teng; Ying Fan; Ya-lin Yang; Zi-gang Tian; Jin Luo; Jian-hua Wang (pp. 1074-1083).

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l⁻¹. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.

Keywords: β-1,3-1,4-Glucanase; Codon optimization; Bacillus licheniformis ; Expression; Pichia pastoris (P. pastoris)

Trehalose synthesis in Saccharomycopsis fibuligera does not respond to stress treatments by L. K. Liang; X. K. Wang; K. L. Zhu; Z. M. Chi (pp. 1084-1091).

Synthesis of trehalose in Saccharomycopsis fibuligera sdu under various stress conditions was investigated. Neither the activation of trehalose-6-phosphate synthase (SfTPS1) nor the change in trehalose content was observed under stress exposure of S. fibuligera sdu cells. The results of reverse transcription polymerase chain reaction, which was performed with the specific primers designed to target the SfTPS1 gene fragment cloned from this strain, also showed that all stress treatments did not increase the expression of SfTPS1 gene. These results demonstrated that synthesis of trehalose in response to stress conditions in S. fibuligera sdu clearly differs from that of Saccharomyces cerevisiae and most other fungi. The phylogenetic analysis of the amino acid sequence deduced from the SfTPS1 gene fragment showed that the SfTPS1 sequence formed a separate family that was far related to S. cerevisiae TPS1. The yeast strain, which can accumulate a large amount of trehalose under normal growth conditions, has many applications and TPS1 gene in such strain may have unique use in transgenic organisms.

Keywords: Trehalose; TPS1 gene; Stress treatment; Saccharomycopsis fibuligera

Biodegradation of methyl tert-butyl ether by cold-adapted mixed and pure bacterial cultures by G. M. Zaitsev; J. S. Uotila; M. M. Häggblom (pp. 1092-1102).

An aerobic mixed bacterial culture (CL-EMC-1) capable of utilizing methyl tert-butyl ether (MTBE) as the sole source of carbon and energy with a growth temperature range of 3 to 30°C and optimum of 18 to 22°C was enriched from activated sludge. Transient accumulation of tert-butanol (TBA) occurred during utilization of MTBE at temperatures from 3°C to 14°C, but TBA did not accumulate above 18°C. The culture utilized MTBE at a concentration of up to 1.5 g l⁻¹ and TBA of up to 7 g l⁻¹. The culture grew on MTBE at a pH range of 5 to 9, with an optimum pH of 6.5 to 7.1. The specific growth rate of the CL-EMC-1 culture on 0.1 g l⁻¹ of MTBE at 22°C and pH 7.1 was 0.012 h⁻¹, and the growth yield was 0.64 g (dry weight) g⁻¹. A new MTBE-utilizing bacterium, Variovorax paradoxus strain CL-8, isolated from the mixed culture utilized MTBE, TBA, 2-hydroxy isobutyrate, lactate, methacrylate, and acetate as sole sources of carbon and energy but not 2-propanol, acetone, methanol, formaldehyde, or formate. Two other isolates, Hyphomicrobium facilis strain CL-2 and Methylobacterium extorquens strain CL-4, isolated from the mixed culture were able to grow on C₁ compounds. The combined consortium could thus utilize all of the carbon of MTBE.

Probiotic properties of Lactobacillus and Bifidobacterium strains isolated from porcine gastrointestinal tract by Pyoung Il Kim; Min Young Jung; Young-Hyo Chang; Saehun Kim; Seong-Jae Kim; Yong-Ha Park (pp. 1103-1111).

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.

Keywords: Lactobacillus amylovorus ; Lactobacillus reuteri ; Lactobacillus salivarius ; Bifidobacterium thermacidophilum ; Antagonistic effect; Clostridium perfringens

Antioxidant activity of Haematococcus pluvialis cells grown in continuous culture as a function of their carotenoid and fatty acid content by M. C. Cerón; M. C. García-Malea; J. Rivas; F. G. Acien; J. M. Fernandez; E. Del Río; M. G. Guerrero; E. Molina (pp. 1112-1119).

The influence of culture conditions on the quality of Haematococcus pluvialis biomass is assessed. Continuously grown cells have been characterised with respect to their astaxanthin, fatty acid content, and antioxidant activity and compared with those of non-growing haematocysts. Moderate limitation of nitrate availability (1.7 mM) under continuous growth conditions favoured the production of reddish palmelloid cells whose extracts possessed antioxidant activity equivalent to that of haematocyst extracts, despite the lower astaxanthin content (0.6%d.wt.), which is compensated by a higher fatty acid level (7.6%d.wt.). Green cells produced under nitrate saturation conditions (>4.7 mM) exhibit only 40% antioxidant activity than palmelloid. In addition, the major fatty acid present in palmelloid cells was oleic acid (40%f.a.), whereas, in both green cells and haematocysts, the main fatty acids were myristic, palmitic, and oleic acid (20–30%f.a. each). Biomass extracts were fractionated and analysed. The antioxidant capacity was a function of both the carotenoid and the fatty acid profiles, the antioxidant capacity of astaxanthin diesters fraction being 60% higher than astaxanthin monoesters fraction and twice than free astaxanthin. In such a way, the evaluation of the quality of H. pluvialis biomass must take into account both variables. When considering the production of H. pluvialis biomass for human consumption, special attention should be paid to the one-step continuous system approach for the generation of cells rich in both astaxanthin and fatty acids, as they have high antioxidant activity but without thick hard cell wall.

Keywords: Haematococcus ; Antioxidant; Fatty acid; Carotenoids; Astaxanthin

Growth promoting effect of a transgenic Bacillus mucilaginosus on tobacco planting by Xin Li; Zhiqiang Wu; Weidong Li; Ruixiang Yan; Li Li; Jun Li; Yihang Li; Minggang Li (pp. 1120-1125).

In this study, we have investigated the plant growth promoting effect of Bacillus mucilaginosus strain D₄B₁, a rhizosphere soil organism, and its transgenic strain NKTS-3 on tobacco planting. The transgenic strain contains a phytase expression cassette that can express high active phytase extracellularly and hydrolyze phytate in the soil to liberate inorganic phosphorus for the growth of tobacco plants. Greenhouse study and field experiments showed that both wild-type B. mucilaginosus and the transgenic strain could promote tobacco plant growth. Moreover, the transgenic strain promoted tobacco plant growth (235% more than control in pot experiments and 125% more than control in field experiments) was higher than the wild-type B. mucilaginosus (183% more than control in pot experiments and 108% more than control in field experiments). In addition, the inoculation with transgenic rhizobacteria could significantly improve root acquisition of phosphorus and increase the phosphorus content of the plant.

Keywords: Phytase; Bacillus mucilaginosus ; Phosphorus absorbing; Plant rhizosphere

Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target by Chang Jui Jen; Chia-Hung Chou; Ping-Chi Hsu; Sian-Jhong Yu; Wei-En Chen; Jiunn-Jyi Lay; Chieh-Chen Huang; Fu-Shyan Wen (pp. 1126-1134).

By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.

Keywords: Clostridium ; Hydrogenase; Fluorescence in situ hybridization; Flow cytometry

Leaching and microbial treatment of a soil contaminated by sulphide ore ashes and aromatic hydrocarbons by Alessandro D’Annibale; Vanessa Leonardi; Ermanno Federici; Franco Baldi; Fulvio Zecchini; Maurizio Petruccioli (pp. 1135-1144).

Contaminated soil from a historical industrial site and containing sulfide ore ashes and aromatic hydrocarbons underwent sequential leaching by 0.5 M citrate and microbial treatments. Heavy metals leaching was with the following efficiency scale: Cu (58.7%) > Pb (55.1%) > Zn (44.5%) > Cd (42.9%) > Cr (26.4%) > Ni (17.7%) > Co (14.0%) > As (12.4%) > Fe (5.3%) > Hg (1.1%) and was accompanied by concomitant removal of organic contaminants (about 13%). Leached metals were concentrated into an iron gel, produced during ferric citrate fermentation by the metal-resistant strain BAS-10 of Klebsiella oxytoca. Concomitantly, the acidic leached soil was bioaugmented with Allescheriella sp. DABAC 1, Stachybotrys sp. DABAC 3, Phlebia sp. DABAC 9, Pleurotus pulmonarius CBS 664.97, and Botryosphaeria rhodina DABAC P82. B. rhodina was most effective, leading to a significant depletion of the most abundant contaminants, including 7-H-benz[DE]anthracene-7-one, 9,10-anthracene dione and dichloroaniline isomers, and to a marked detoxification as assessed by the mortality test with the Collembola Folsomia candida Willem. The overall degradation activities of B. rhodina and P. pulmonarius appeared to be significantly enhanced by the preliminary metal removal.

Keywords: Metal leaching; Bacterial metal precipitation; Aromatic hydrocarbons; Lignin-degrading enzymes; Fungal bioremediation

Kinetics of decolourisation and biotransformation of direct black 38 by C. hominis and P. stutzeri by Amit Bafana; Sivanesan Saravana Devi; Kannan Krishnamurthi; Tapan Chakrabarti (pp. 1145-1152).

In the present study, a consortium of Cardiobacterium hominis and Pseudomonas stutzeri was isolated from an effluent treatment plant of a textile industry, based on its ability to decolourise azo dyes including direct black 38 (DB38), a benzidine-based azo dye. The role of each culture in the decolourisation process was elucidated, and C. hominis was found to decolourise the dye. Although P. stutzeri could not decolourise the dye, it was found to synergistically enhance dye decolourisation activity of C. hominis by scavenging oxygen in the medium and creating an anaerobic condition (oxidation/reduction potential −440 mV), which is known to be necessary for azo dye decolourisation. Together, the cultures could decolourise 90.5% of 100 mg l⁻¹ DB38 within 24 h. Kinetics of DB38 decolourisation was also examined, and P. stutzeri was found to increase V _max and K _m of decolourisation activity of C. hominis by 3.6- and 3-fold, respectively. The study also revealed a pathway of DB38 degradation with the release of benzidine from DB38 and subsequent degradation of benzidine to 4-aminobiphenyl by the cultures.

A highly productive system for cell-free protein synthesis using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis by Takashi Endoh; Tamotsu Kanai; Tadayuki Imanaka (pp. 1153-1161).

We report in this study an improved system for cell-free protein synthesis at high temperatures using the lysate of Thermococcus kodakaraensis. Previous work indicated that cell-free protein synthesis of ChiAΔ4, a derivative of T. kodakaraensis chitinase, was observed within a temperature range of 40–80°C, and the maximum yield of the ChiAΔ4 synthesized was approximately 1.3 μg/ml. To increase productivity of the system, the following approaches were taken. First, the process of lysate preparation was examined, and we found that omitting the preincubation (runoff) step was especially effective to increase the translational activity of lysate. Second, the concentrations of each reaction mixture were optimized. Among them, the requirement of a high concentration of potassium acetate (250 mM) was characteristic to the T. kodakaraensis system. Third, a mutant strain of T. kodakaraensis was constructed in which a heat shock transcriptional regulator gene, phr, was disrupted. By using the lysate made from the mutant, we observed an increase in the optimum reaction temperature by 5°C. Through these modifications to the system, the yield of ChiAΔ4 was dramatically increased to 115.4 μg/ml in a batch reaction at 65°C, which was about 90 times higher than that in the previous study. Moreover, in the optimized system, a high speed of protein synthesis was achieved: over 100 μg/ml of ChiAΔ4 was produced in the first 15 min of reaction. These results indicate that the system for cell-free protein synthesis based on T. kodakaraensis lysate has a high production potential comparable to the Escherichia coli system.

Trehalose synthesis in Saccharomycopsis fibuligera does not respond to stress treatments by L. K. Liang; X. K. Wang; K. L. Zhu; Z. M. Chi (pp. 1162-1162).

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Applied Microbiology and Biotechnology (v.74, #5)

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Applied Microbiology and Biotechnology (v.74, #5)