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Applied Microbiology and Biotechnology (v.74, #3)

Exploitation of Dunaliella for β-carotene production by R. Raja; S. Hemaiswarya; R. Rengasamy (pp. 517-523).

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.

Keywords: Dunaliella ; 4-carotene glycerol; Salinity; Microalga; Carotenogenesis

Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives by Rojan P. John; K. Madhavan Nampoothiri; Ashok Pandey (pp. 524-534).

The concept of utilizing excess biomass or wastes from agricultural and agro-industrial residues to produce energy, feeds or foods, and other useful products is not necessarily new. Recently, fermentation of biomass has gained considerable attention due to the forthcoming scarcity of fossil fuels and also due to the necessity of increasing world food and feed supplies. A cost-effective viable process for lactic acid production has to be developed for which several attempts have been initiated. Fermentation techniques result in the production of either d (−) or l (+) lactic acid, or a racemic mixture of both, depending on the type of organism used. The interest in the fermentative production of lactic acid has increased due to the prospects of environmental friendliness and of using renewable resources instead of petrochemicals. Amylolytic bacteria Lactobacillus amylovorus ATCC 33622 is reported to have the efficiency of full conversion of liquefied cornstarch to lactic acid with a productivity of 20 g l⁻¹ h⁻¹. A maximum of 35 g l⁻¹ h⁻¹ was reported using a high cell density of L. helveticus (27 g l⁻¹) with a complete conversion of 55- to 60-g l⁻¹ lactose present in whey. Simultaneous saccharification and fermentation is proved to be best in the sense of high substrate concentration in lower reactor volume and low fermentation cost. In this review, a survey has been made to see how effectively the fermentation technology explored and exploited the cheaply available source materials for value addition with special emphasis on lactic acid production.

Keywords: Lactic acid; Renewable resource; Agro-industrial residue; Solid-state fermentation

Bioconversion of butyronitrile to butyramide using whole cells of Rhodococcus rhodochrous PA-34 by J. Raj; A. Seth; S. Prasad; T. C. Bhalla (pp. 535-539).

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.

Keywords: Rhodococcus rhodochrous PA-34; Nitrile hydratase; Butyronitrile; Butyramide

Batch fermentations on synthetic mixed sugar and starch medium with amylolytic lactic acid bacteria by Mette Hedegaard Thomsen; Jean Pierre Guyot; Pauli Kiel (pp. 540-546).

The green crop drying industry in Denmark uses Italian rye grass, clover, and alfalfa as raw materials for the production of green pellets. The green crop drying industry solves its energy economical problems by heating and pressing of the green crop before drying. The produced sidestream is called brown juice. Brown juice was shown to be an excellent medium for lactic acid fermentation. The aim of this study was to investigate the utilisation of brown juice in the production of polylactic acid, where wheat starch would be added to increase the lactic acid yield and, thus, the feasibility of the process. A number of amylolytic lactic acid bacteria have been identified, and in this work, six different strains were tested for their ability to produce α-amylase and to utilise all sugars with high lactic acid yield in a medium with a complex composition of free sugars (brown juice) and starch. Lactobacillus plantarum A6 was the only strain that showed both a good lactic acid production and utilisation of starch in this medium. The growth rate of this strain was approximately 0.4 h⁻¹ and the lactic acid yield was 0.7.

Keywords: Amylolytic lactic acid bacteria; Brown juice; Starch; Polylactic acid

Growth and laccase production kinetics of Trametes versicolor in a stirred tank reactor by A. T. Thiruchelvam; Juliana A. Ramsay (pp. 547-554).

White rot fungi are a promising option to treat recalcitrant organic molecules, such as lignin, polycyclic aromatic hydrocarbons, and textile dyes, because of the lignin-modifying enzymes (LMEs) they secrete. Because knowledge of the kinetic parameters is important to better design and operate bioreactors to cultivate these fungi for degradation and/or to produce LME(s), these parameters were determined using Trametes versicolor ATCC 20869 (ATCC, American Type Culture Collection) in a magnetic stir bar reactor. A complete set of kinetic data has not been previously published for this culture. Higher than previously reported growth rates with high laccase production of up to 1,385 U l⁻¹ occurred during growth without $${ ext{NH}}^{{ ext{ + }}}_{{ ext{4}}} $$ or glucose limitation. The maximum specific growth rate averaged 0.94 ± 0.23 day⁻¹, whereas the maximum specific substrate consumption rates for glucose and ammonium were 3.37 ± 1.16 and 0.15 ± 0.04 day⁻¹, respectively. The maximum specific oxygen consumption rate was 1.63 ± 0.36 day⁻¹.

Keywords: Growth; Kinetics; Laccase; Stirred tank reactor; Trametes versicolor

Online recovery of nisin during fermentation and its effect on nisin production in biofilm reactor by Thunyarat Pongtharangku; Ali Demirci (pp. 555-562).

An online removal of nisin by silicic acid coupled with a micro-filter module was proposed as an alternative to reduce detrimental effects caused by adsorption of nisin onto producer, enzymatic degradation by protease, and product inhibition during fermentation. In this study, silicic acid was successfully used to recover nisin from the fermentation broth of Lactococcus lactis subsp. lactis NIZO 22186. The effect of pH (at 6.8 and 3.0) during adsorption process and several eluents (deionized water, 20% ethanol, 1 M NaCl, and 1 M NaCl + 20% ethanol) for desorption were evaluated in a small batch scale. Higher nisin adsorption onto silicic acid was achieved when the adsorption was carried out at pH 6.8 (67% adsorption) than at pH 3.0 (54% adsorption). The maximum recovery was achieved (47% of nisin was harvested) when the adsorption was carried out at pH 6.8 and 1 M NaCl + 20% ethanol was used as an eluent for desorption. Most importantly, nisin production was significantly enhanced (7,445 IU/ml) when compared with the batch fermentation without the online recovery (1,897 IU/ml). This may possibly be attributed to preventing the loss of nisin due the detrimental effects and a higher biomass density achieved during online recovery process, which stimulated production of nisin during fermentation.

Keywords: Nisin production; Biofilm reactor; Online recovery; Silicic acid

Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production by Jinshan Li; Cuiqing Ma; Yanhe Ma; Yan Li; Wei Zhou; Ping Xu (pp. 563-571).

An optimization strategy based on desirability function approach (DFA) together with response surface methodology (RSM) has been used to optimize production medium in L-glutamine fermentation. Fermentation problems often force to reach a compromise between different experimental variables in order to achieve the most suitable strategy applying in industrial production. The importance of the use of multi-objective optimization methods lies in the ability to cope with this kind of problems. A sequential RSM with different combinations of glucose and (NH₄)₂SO₄ was performed to attain the optimal medium (OM-1) in glutamine production. Based on the result of RSM and the evaluation of production cost, a more economical optimal medium (OM-2) was obtained with the aid of DFA. In DFA study, glutamate, the main by-product in glutamine fermentation as another response was considered. Compared with OM-1 in validated experiment, similar amounts of glutamine were obtained in OM-2 while the concentration of glutamate and the production cost decreased by 53.6 and 7.1%, respectively.

Keywords: Response surface methodology; Desirability function; Glutamine production; Medium optimization

Enhancement of polysaccharides production in Ganoderma lucidum by the addition of ethyl acetate extracts from Eupolyphaga sinensis and Catharsius molossus by Gao-Qiang Liu; Ke-Chang Zhang (pp. 572-577).

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.

Keywords: Extracellular polysaccharides; Ganoderma lucidum ; Intracellular polysaccharides; Medicinal insects; Submerged fermentation

The inhibition of aggregation of recombinant human consensus interferon-α mutant during Pichia pastoris fermentation by Yuyou Hao; Ju Chu; Yonghong Wang; Yingping Zhuang; Siliang Zhang (pp. 578-584).

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-α mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20°C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20°C was 4.04 times as high as that at 30°C. There was no obvious effect on the cell growth at 20°C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l⁻¹ Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20°C was 19.9-fold higher than that without Tween-20 at 30°C.

Keywords: Aggregation; Consensus interferon-α; Pichia pastoris ; Temperature; Tween-20

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima by Satoshi Kakugawa; Shinya Fushinobu; Takayoshi Wakagi; Hirofumi Shoun (pp. 585-591).

The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH₂-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni²⁺ affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K _m and k _cat values of 3.1 μM and 10.8 s⁻¹, respectively, at 60°C and pH 7.5. The addition of O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca²⁺ ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.

Keywords: Esterase; Carboxylesterase; Hyperthermophilic enzyme; Lipase; Alpha/beta-Hydrolase; Lipolytic enzyme

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the Pleurotus eryngii complex by S. Urbanelli; V. Della Rosa; F. Punelli; D. Porretta; M. Reverberi; A. A. Fabbri; C. Fanelli (pp. 592-600).

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.

Keywords: DNA fingerprinting; AFLP; Laccase gene; Manganese Peroxidase gene; Pleurotus eryngii complex

Translation elongation factor 1-α gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter by Jungoh Ahn; Jiyeon Hong; Hyeokweon Lee; Myongsoo Park; Eungyo Lee; Chunsuk Kim; Euisung Choi; Joonki Jung; Hongweon Lee (pp. 601-608).

The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P TEF1) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP) by using a bacterial lipase gene as a reporter gene. P TEF1 demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P GAP. The sequence of the gene was deposited in GenBank under accession no. EF014948.

Keywords: Pichia pastoris ; Translation elongation factor; Constitutive promoter

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4 by Hwa-Yong An; Harutoshi Tsuda; Taku Miyamoto (pp. 609-616).

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.

Keywords: Recombinant vector; Citrate permease; Lactobacillus casei ; Diacetyl/acetoin

Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose by Karin Nijkamp; R. G. Maaike Westerhof; Hendrik Ballerstedt; Jan A. M. de Bont; Jan Wery (pp. 617-624).

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp. by Luan Tao; Natalia Sedkova; Henry Yao; Rick W. Ye; Pamela L. Sharpe; Qiong Cheng (pp. 625-633).

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1–crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.

Keywords: Astaxanthin production; Oxygen limitation; Bacterial hemoglobin; β-Carotene ketolase; β-Carotene hydroxylase; Methanotroph

High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis by Markus A. Durban; Jörg Silbersack; Thomas Schweder; Frieder Schauer; Uwe T. Bornscheuer (pp. 634-639).

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.

Keywords: Phospholipase C; Bacillus cereus ; Bacillus subtilis ; Expression; Cloning

Transfer of nisin gene cluster from Lactococcus lactis ATCC 11454 into the chromosome of Bacillus subtilis 168 by Sahru Yuksel; J. Norman Hansen (pp. 640-649).

Nisin is an antimicrobial peptide produced by certain strains of Lactococcus lactis. It is a gene-encoded peptide that contains unusual amino acid residues. These novel residues are introduced by posttranslational modification machinery and confer unique chemical and physical properties that are not attainable by regular amino acid residues. To study the modification mechanisms and to create structural analogs with superior properties, it would be advantageous to insert the nisin genes into a bacterial strain that is amenable to genetic manipulation. In this study, we report the cloning and integration of the complete and intact nisin gene cluster into the Bacillus subtilis 168 chromosome. Furthermore, we demonstrate that the nisin genes are transcriptionally active. These results should greatly facilitate the studies of the genes and proteins involved in nisin expression, as well as provide a standard system for the manipulation and expression of genes involved in other members of the lantibiotic family of antimicrobial peptides.

Keywords: Nisin; Lantibiotic; Nisin biosynthesis; Antimicrobial peptide; Genetic engineering

Expressed sequence tags-based identification of genes in the biocontrol agent Chaetomium cupreum by HaiYan Zhang; Qian Yang (pp. 650-658).

Chaetomium cupreum has a potential as biocontrol agent against a range of plant pathogens on the basis of production of antifungal metabolites, mycoparasitism, competition for space and nutrients, or various combinations of these. To explore genes expressed in C. cupreum, a cDNA library was constructed from mycelium and 3,066 expressed sequence tags (ESTs) were generated. Clusters analysis enabled the identification of 1,471 unigenes with 392 contigs and 1,079 singleton sequences. Putative functions were assigned to 874 unigenes that exhibited strong similarity to genes/ESTs in public databases putatively containing genes involved in cellular component, molecular function, and biological process. Other 597 ESTs representing novel genes showed no significant similarity to public database resource of NCBI. A proportion of genes was identified related to degradation of pathogen cell wall, antifungal metabolite production, as was estimated in the biocontrol fungus. The paper described is a first step towards the knowledge of the C. cupreum genome. The results present the useful application of EST analysis on C. cupreum and provide a preliminary indication of gene expression putatively involved in biocontrol.

Keywords: Chaetomium cupreum; Biocontrol; Expressed sequence tags

Biotransformation of gallic acid by Beauveria sulfurescens ATCC 7159 by Feng-Lin Hsu; Li-Ming Yang; Shwu-Fen Chang; Li-Hsuan Wang; Chung-Yi Hsu; Pan-Chun Liu; Shwu-Jiuan Lin (pp. 659-666).

Preparative-scale fermentation of gallic acid (3,4,5-trihydroxybenzoic acid) (1) with Beauveria sulfurescens ATCC 7159 gave two new glucosidated compounds, 4-(3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yloxy)-3-hydroxy-5-methoxy-benzoic acid (4), 3-hydroxy-4,5-dimethoxy-benzoic acid 3,4-dihydroxy-6-hydroxymethyl-5-methoxy-tetrahydro-pyran-2-yl ester (7), along with four known compounds, 3-O-methylgallic acid (2), 4-O-methylgallic acid (3), 3,4-O-dimethylgallic acid (5), and 3,5-O-dimethylgallic acid (6). The new metabolite genistein 7-O-β-D-4″-O-methyl-glucopyranoside (8) was also obtained as a byproduct due to the use of soybean meal in the fermentation medium. The structural elucidation of the metabolites was based primarily on 1D-, 2D-NMR, and HRFABMS analyses. Among these compounds, 2, 3, and 5 are metabolites of gallic acid in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, B. sulfurescens might be a useful tool for generating mammalian metabolites of related analogs of gallic acid (1) for complete structural identification and for further use in investigating pharmacological and toxicological properties in this series of compounds. In addition, a GRE (glucocorticoid response element)-mediated luciferase reporter gene assay was used to initially screen for the biological activity of the 6 compounds, 2–6 and 8, along with 1 and its chemical O-methylated derivatives 9–13. Among the 12 compounds tested, 11–13 were found to be significant, but less active than the reference compounds of methylprednisolone and dexamethasone.

Keywords: Biotransformation; Gallic-acid; Beauveria sulfurescens

Fusion of the genes for AHL-lactonase and S-layer protein in Bacillus thuringiensis increases its ability to inhibit soft rot caused by Erwinia carotovora by Lei Zhang; Lifang Ruan; Chaohua Hu; Huaiguang Wu; Shouwen Chen; Ziniu Yu; Ming Sun (pp. 667-675).

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA 4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.

Keywords: N-acyl-homoserine lactonase; Fusion protein; AiiA_4Q7 protein; Bacillus thuringiensis ; Surface layer; Soft rot disease

Degradation of environmental endocrine disruptor di-2-ethylhexyl phthalate by a newly discovered bacterium, Microbacterium sp. strain CQ0110Y by Ji-an Chen; Xiang Li; Jun Li; Jia Cao; Zhiqun Qiu; Qing Zhao; Chuan Xu; Weiqun Shu (pp. 676-682).

In this study di-2-ethylhexyl phthalate (DEHP)-degradation strain CQ0110Y was isolated from activated sludge. According to the biophysical/biochemical characteristics and analysis of 16S rDNA, the strain was identified as Microbacterium sp. The results of this study showed the optimal pH value and optimal temperature which influenced the degradation rate in wastewater: pH 6.5–7.5, 25–35°C. Kinetics of degradation reaction had been performed at different initial concentrations and different time. Analyzed with SPSS10.0 software, the DEHP degradation can be described as the same exponential model when the initial DEHP concentration was lower than 1,350 mg/l. The kinetics equation was ln C = −0.4087t + A, with the degradation half life of DEHP in wastewater (1.59 days). To the best of our knowledge, this is the first reported case of DEHP degradation by Microbacterium sp. strain.

Keywords: Di-2-Ethylhexyl phthalate; Degradation; Microbacterium ; Catechol-dioxygenase

Response of Saccharomyces cerevisiae to lead ion stress by Can Chen; Jianlong Wang (pp. 683-687).

The response of Saccharomyces cerevisiae to different concentrations of Pb²⁺ was investigated. The results demonstrated that the growth of S. cerevisiae in the presence of Pb²⁺ showed a lag phase much longer than that in the absence of Pb²⁺. The inhibition was dependent upon Pb²⁺ concentrations. The Pb²⁺ at a concentration of 5 µM inhibited the microbial growth by approximately 30% with regard to control, whereas Pb²⁺ at concentration of 2 µM did not have a significant effect on the microbial growth. The existence of Pb²⁺ did not perturb cell-protein synthesis and there was a good correlation between dry cell weights and total protein content (R ² = 0.98). The RNA/DNA ratio in the microbial cells varied with Pb²⁺ concentration and there was a significant positive correlation between Pb²⁺ concentration and the RNA/DNA ratio. The microbial assimilation of ammonium ion was inhibited by the presence of Pb²⁺ in the medium; when Pb²⁺ concentration was 10 µM, the microbial ammonium assimilation was inhibited about 50%, in comparison with the control experiment.

Keywords: Pb²⁺ ; Saccharomyces cerevisiae ; Protein; Heavy metal; RNA/DNA ratio

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates by Z.-W. Chen; Y.-Y. Liu; J.-F. Wu; Q. She; C.-Y. Jiang; S.-J. Liu (pp. 688-698).

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 × 10⁹ and 6.68 × 10⁵, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor _Fx, sor _SA, and sor _SB were identified from metagenomic DNAs of the bioreactors. The sor _Fx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor _SA and sor _SB showed no significant identity to any genes in GenBank databases. The sor _SB was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor _SA and sor _SB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor _SB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.

Keywords: Sulfur oxygenase reductase; Acidithiobacillus ; Sulfur oxidation; Bioleaching; Metagenome

Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge by A. Ramesh; D. J. Lee; J. Y. Lai (pp. 699-707).

This study extracted the soluble microbial products and loosely bound and tightly bound extracellular polymeric substances (EPS) from suspended sludge from a membrane bioreactor, original and aerobically/anaerobically digested, and compared their fouling potentials on a microfiltration membrane. The resistance of cake layer accounts for 95–98% of the total filtration resistances when filtering the whole sludges, with anaerobically digested sludge presenting the highest resistance among the three tested sludges. The tightly bound EPS has the highest potential to foul the membrane; however, the loosely bound EPS contribute most of the filtration resistances of the whole sludges. The foulants corresponding to the irreversible fouling have chemical fingerprints similar to those from loosely bound EPS, which have a greater predilection to proteins and humic substances than to polysaccharides.

Keywords: EPS; SMP; Biofouling; MBR

High-temperature biotrickling filtration of hydrogen sulphide by Indrani Datta; Roberta R. Fulthorpe; Shobha Sharma; D. Grant Allen (pp. 708-716).

Biofiltration of malodorous reduced sulphur compounds such as hydrogen sulphide has been confined to emissions that are at temperatures below 40°C despite the fact that there are many industrial emissions (e.g. in the pulp and paper industry) at temperatures well above 40°C. This paper describes our study on the successful treatment of hydrogen sulphide gas at temperatures of 40, 50, 60 and 70°C using a microbial community obtained from a hot spring. Three biotrickling filter (BTF) systems were set up in parallel for a continuous run of 9 months to operate at three different temperatures, one of which was always at 40°C as a mesophilic control and the other two were for exploring high-temperature operation up to 70°C. The continuous experiment and a series of batch experiments in glass bottles (250 ml) showed that addition of glucose and monosodium glutamate enhanced thermophilic biofiltration of hydrogen sulphide gas and a removal rate of 40 g m⁻³ h⁻¹ was achieved at 70°C. We suggest that the glucose is acting as a carbon source for the existing microbial community in the BTFs, whereas glutamate is acting as a compatible solute. The use of such organic compounds to enhance biodegradation of hydrogen sulphide, particularly at high temperatures, has not been demonstrated to our knowledge and, hence, has opened up a range of possibilities for applying biofiltration to hot gas effluent.

Keywords: Reduced sulphur; Air pollution; Biofiltration; Sulphur oxidisers; Biotreatment; Biological gas cleaning

Use of protein chip mass spectrometry to monitor biotinylation reactions by Levi L. Blazer; Michael D. P. Boyle (pp. 717-722).

Surface-enhanced laser desorption/ionization time-of-flight analysis was used to monitor both the kinetics and heterogeneity of product formation during the biotinylation of a number of model proteins and peptide targets. The selected molecules were the IgG-binding protein, protein A, human serum albumin, and a synthetic peptide corresponding to the N terminus of a streptococcal M1 protein. The extent of biotinylation was determined by kinetic analysis of the shift in molecular mass from the native material. Each residue modified by reaction with N-hydroxysuccinimide biotin resulted in an addition of ∼341 amu to the native protein or polypeptide. The novelty of the method was in the ability to determine the molecular mass shift, without first separating the targeted molecule from the biotinylating reagent. The analysis was rapid, simple, and provided information on the average number of biotin molecules added and the homogeneity of the resulting product.

Keywords: SELDI-TOF; Biotinylation; Protein A

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Applied Microbiology and Biotechnology (v.74, #3)

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Applied Microbiology and Biotechnology (v.74, #3)