Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.74, #4)

Recombinant therapeutic antibodies by Stefan Dübel (pp. 723-729).

Recombinant antibody technology has revolutionized the development of antibody therapeutics. This minireview offers an overview of enabling technologies and future prospects of this rapidly progressing field.

Permeability issues in whole-cell bioprocesses and cellular membrane engineering by Rachel Ruizhen Chen (pp. 730-738).

Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1–2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature’s ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.

Keywords: Permeability; Cellular membrane engineering; Whole-cell; Biocatalysis; Bioremediation; Fermentation

Natural history and experimental evolution of the genetic code by Birgit Wiltschi; Nediljko Budisa (pp. 739-753).

The standard genetic code is a set of rules that relates the 20 canonical amino acids in proteins to groups of three bases in the mRNA. It evolved from a more primitive form and the attempts to reconstruct its natural history are based on its present-day features. Genetic code engineering as a new research field was developed independently in a few laboratories during the last 15 years. The main intention is to re-program protein synthesis by expanding the coding capacities of the genetic code via re-assignment of specific codons to un-natural amino acids. This article focuses on the question as to which extent hypothetical scenarios that led to codon re-assignments during the evolution of the genetic code are relevant for its further evolution in the laboratory. Current attempts to engineer the genetic code are reviewed with reference to theoretical works on its natural history. Integration of the theoretical considerations into experimental concepts will bring us closer to designer cells with target-engineered genetic codes that should open not only tremendous possibilities for the biotechnology of the twenty-first century but will also provide a basis for the design of novel life forms.

Keywords: Amino acid repertoire; Artificial life; Biotechnology; Evolution of codon re-assignment; Genetic code; Protein design and engineering

Efficient induction of formate hydrogen lyase of aerobically grown Escherichia coli in a three-step biohydrogen production process by Akihito Yoshida; Taku Nishimura; Hideo Kawaguchi; Masayuki Inui; Hideaki Yukawa (pp. 754-760).

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA ⁺⁺, ΔhycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 ± 5.0 mmol h⁻¹ g⁻¹ dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 ΔldhA ΔfrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 ± 6.9 mmol h⁻¹ g⁻¹. Furthermore, a maximum hydrogen production rate of 144.2 mmol h⁻¹ g⁻¹ was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h⁻¹ was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.

Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents by Shiho Yamashita; Masafumi Satoi; Yoshihiro Iwasa; Kohsuke Honda; Yuka Sameshima; Takeshi Omasa; Junichi Kato; Hisao Ohtake (pp. 761-767).

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 μg ml⁻¹ by 24 h.

Keywords: Organic solvent-tolerant bacteria; Rhodococcus opacus ; Whole-cell catalyst; Nonaqueous media

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli by Seong-Goo Byun; Myoung-Dong Kim; Won-Heong Lee; Kun-Jae Lee; Nam Soo Han; Jin-Ho Seo (pp. 768-775).

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.

The use of Tetrahymena thermophila mutant cell line for removal of cholesterol from milk by H. G. Gentili; D. G. Noseda; M. L. Nani; A. Nusblat; A. Tiedtke; C. B. Nudel; J. Florin-Christensen (pp. 776-782).

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 × 10 6 cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% ± 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.

Keywords: Cholesterol free milk; Ciliates

Enhanced vanillin production from ferulic acid using adsorbent resin by Dongliang Hua; Cuiqing Ma; Lifu Song; Shan Lin; Zhaobin Zhang; Zixin Deng; Ping Xu (pp. 783-790).

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.

Keywords: Vanillin; Ferulic acid; Streptomyces sp; Biotransformation; Adsorbent resin

Cloning and high-level production of a chitinase from Chromobacterium sp. and the role of conserved or nonconserved residues on its catalytic activity by Seur Kee Park; Chi Wook Kim; Hoon Kim; Jae Sung Jung; G. E. Harman (pp. 791-804).

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K _cat values.

Keywords: Chitinase; Site-directed mutagenesis; Enzyme purification; Enzyme enhancement

Purification, characterization, and potential applications of formate oxidase from Debaryomyces vanrijiae MH201 by H. Uchida; M. Hojyo; Y. Fujii; Y. Maeda; R. Kajimura; H. Yamanaka; A. Sakurai; M. Sakakibara; K. Aisaka (pp. 805-812).

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.

Keywords: Formate oxidase; Debaryomyces vanrijiae ; Formaldehyde; Carbon dioxide; Catalase

Alteration of substrate specificity of fructosyl-amino acid oxidase from Fusarium oxysporum by Maki Fujiwara; Jun-ichi Sumitani; Shinji Koga; Issei Yoshioka; Takuji Kouzuma; Shigeyuki Imamura; Takashi Kawaguchi; Motoo Arai (pp. 813-819).

Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as $$ N^{varepsilon }$$ -fructosyl $$ N^{alpha }$$ -benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.

Keywords: Fructosyl-amino acid oxidase; Fusarium oxysporum ; Diabetes; Diagnosis

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180 by Jun-Tae Kim; Sung Gyun Kang; Jung-Hee Woo; Jung-Hyun Lee; Byeong Chul Jeong; Sang-Jin Kim (pp. 820-828).

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.

Keywords: Cloning; Enantioselective lipase; Marine microorganism; Characterization; Ofloxacin

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms by Erina Kohyama; Mizuho Dohi; Akihiro Yoshimura; Toyokazu Yoshida; Toru Nagasawa (pp. 829-835).

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.

Expression, refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in Escherichia coli by Yuhui Zang; Xu Zhang; Xiaoling Jiang; Haoran Li; Jie Zhu; Chi Zhang; Wei Peng; Junchuan Qin (pp. 836-842).

Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni²⁺ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.

Keywords: TPO/SCF; Megakaryopoiesis; Bacterial expression; Inclusion body; Refolding

Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits by Beatus Schehl; Thomas Senn; Dirk W. Lachenmeier; Rosaura Rodicio; Jürgen J. Heinisch (pp. 843-850).

Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.

Keywords: Fermentation; Saccharomyces ; Yeast; Spirit; Ethyl carbamate

Effect of surfactants on fluoranthene degradation by Pseudomonas alcaligenes PA-10 by Anne Marie Hickey; Linda Gordon; Alan D. W. Dobson; Catherine T. Kelly; Evelyn M. Doyle (pp. 851-856).

Two surfactants, Tween 80 and JBR, were investigated for their effect on fluoranthene degradation by a Pseudomonad. Both surfactants enhanced fluoranthene degradation by Pseudomonas alcaligenes PA-10 in shake flask culture. This bacterium was capable of utilising the synthetic surfactant and the biosurfactant as growth substrates and the critical micelle concentration of neither compound inhibited bacterial growth. The biosurfactant JBR significantly increased polycyclic aromatic hydrocarbon (PAH) desorption from soil. Inoculation of fluoranthene-contaminated soil microcosms with P. alcaligenes PA-10 resulted in the removal of significant amounts (45 ± 5%) of the PAH after 28 days compared to an uninoculated control. Addition of the biosurfactant increased the initial rate of fluoranthene degradation in the inoculated microcosm. The presence of a lower molecular weight PAH, phenanthrene, had a similar effect on the rate of fluoranthene removal.

Keywords: PAHs; Fluoranthene; Biodegradation; Surfactants

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites by Sergio Bordel; Raúl Muñoz; Luis Felipe Díaz; Santiago Villaverde (pp. 857-866).

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO₂, and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO₂ production remained constant at 1.0 ± 0.1 g biomass dry weight (DW) g⁻¹ toluene and 1.91 ± 0.31 g CO₂ g⁻¹ biomass, respectively, under the tested range of concentrations (2–250 mg toluene l⁻¹). The specific growth rate increased up to 70 mg toluene l⁻¹. Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l⁻¹. 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10–100 mg l⁻¹ of the above-mentioned compounds.

Keywords: ATP content; Gas treatment; Metabolites; PpF1 ; Toluene biodegradation; Toxicity

Steroid-1-dehydrogenase of Mycobacterium sp. VKM Ac-1817D strain producing 9α-hydroxy-androst-4-ene-3,17-dione from sitosterol by G. V. Sukhodolskaya; V. M. Nikolayeva; S. M. Khomutov; M. V. Donova (pp. 867-873).

The strain of Mycobacterium sp. VKM Ac-1817D forms 9α-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M _r∼58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.

Keywords: Steroid-1-dehydrogenase; 9α -hydroxy-androst-4-ene-3,17-dione; Androst-4-ene-3,17-dione; Sitosterol; Microbial transformation; Mycobacterium

Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications by L. Boiero; D. Perrig; O. Masciarelli; C. Penna; F. Cassán; V. Luna (pp. 874-880).

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA₃ were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml⁻¹), USDA110 (2.5 μg ml⁻¹), and E109 (0.87 μg ml⁻¹), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml⁻¹). Ethylene was found in all three strains, with highest production rate (18.1 ng ml⁻¹ h⁻¹) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA₃, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.

Determination of copper binding in Pseudomonas putida CZ1 by chemical modifications and X-ray absorption spectroscopy by XinCai Chen; JiYan Shi; YingXu Chen; XiangHua Xu; LiTao Chen; Hui Wang; TianDou Hu (pp. 881-889).

Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.

Keywords: Pseudomonas putida CZ1; Chemical modification; FTIR; XAS; Metal binding

Quantification of host-specific Bacteroides–Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater by Satoshi Okabe; Noriko Okayama; Olga Savichtcheva; Tsukasa Ito (pp. 890-901).

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides–Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9–11 log₁₀ copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides–Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r ² = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50–800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.

Keywords: Fecal pollution; Bacteroides–Prevotella 16S rRNA genetic markers; RTQ-PCR

Degradation of ethanol plant by-products by Exophiala lecanii-corni and Saccharomyces cerevisiae in batch studies by Erica F. Pirnie-Fisker; Jennifer R. Woertz (pp. 902-910).

By-product emissions from ethanol production facilities have become a public health concern. Many of these by-products are classified as hazardous air pollutants by the USEPA and current treatment methods, mainly thermal-oxidation, for these compounds are costly, energy intensive, and may produce other undesirable by-products. Degradation of these by-products by the fungi Exophiala lecanii-corni and Saccharomyces cerevisiae was explored. Ethanol plant by-products, focused on in this study, included formaldehyde, acetaldehyde, ethanol, methanol, glycerol, acetic acid, and lactic acid. Initial batch studies were conducted to determine degradation rates and whether the contaminants would be toxic to the fungi. These batch studies demonstrated that E. lecanii-corni and S. cerevisiae are able to utilize all but methanol and formaldehyde as sole carbon and energy sources for growth; however, both contaminants were utilized as secondary metabolites by cultures initially fed either ethanol or acetic acid. Growth studies also were conducted using two contaminants simultaneously to determine if the presence of one contaminant inhibited the degradation of another. Growth and contaminant utilization was observed in cultures fed two contaminants simultaneously.

Influence of metal ionic characteristics on their biosorption capacity by Saccharomyces cerevisiae by Can Chen; Jianlong Wang (pp. 911-917).

The influence of metal ionic characteristics on their biosorption capacity was analyzed using quantitative structure–activity relationships model. The waste biomass of Saccharomyces cerevisiae was used as biosorbent to adsorb 10 kinds of metal ions, and their maximum biosorption capacity (q _max) was determined by the Langmuir isotherm model. The values of q _max decreased in the following order (in millimole per gram): Pb²⁺ (0.413) > Ag⁺ (0.385) > Cr³⁺ (0.247) > Cu²⁺ (0.161) > Zn²⁺ (0.148) > Cd²⁺ (0.137) > Co²⁺ (0.128) > Sr²⁺ (0.114) > Ni²⁺ (0.108) > Cs⁺ (0.092). Twenty-two parameters of physiochemical characteristics of metal ions were selected and correlated with q _max, i.e., OX, AN, r (Å), ΔIP (eV), ΔE ₀ (V), X _m, |log K _OH|, $$ X^{2}_{{ ext{m}}} r $$ , Z ²/r, AN/ΔIP, $$ sigma _{ ho } $$ , AR, AW, IP, AR/AW, Z/r ², Z/AR², Z/r, Z/AR, Z*²/r·, Z*, N. The linear regression analysis showed that the covalent index $$ X^{2}_{{ ext{m}}} r $$ was correlated well with q _max for all metal ions tested in the following equation: q _max = 0.029 + 0.061 ( $$ X^{2}_{{ ext{m}}} r $$ ) (R ² = 0.70). It suggested that the greater the covalent index value of metal ion was, the greater the potential to form covalent bonds with biological ligands, such as sulphydryl, amino, carboxyl, hydroxyl groups, etc. on the biomass surface, and the higher the metal ion biosorption capacity was. Classification of metal ions, for divalent ion or for soft–hard ion could improve the linear relationship (R ² = 0.89). The equation could be used to predict the biosorption capacity of metal ions.

Keywords: Biosorption; Ionic characteristics; Saccharomyces cerevisiae ; QSAR

Comparison of methods for total community DNA extraction and purification from compost by Zh. H. Yang; Y. Xiao; G. M. Zeng; Zh. Y. Xu; Y. Sh. Liu (pp. 918-925).

The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.

Keywords: Molecular ecology; Vegetable waste compost; 16S rDNA; DNA extraction; ARDRA; DGGE

Specific single-cell isolation and genomic amplification of uncultured microorganisms by Thomas Kvist; Birgitte K. Ahring; Roger S. Lasken; Peter Westermann (pp. 926-935).

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

Keywords: Single cell genomics; Archaea; Multiple displacement amplification

Sections

Personal tools

Applied Microbiology and Biotechnology (v.74, #4)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.74, #4)