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Applied Microbiology and Biotechnology (v.85, #3)

Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits by Kenji Okano; Tsutomu Tanaka; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 413-423).

Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

Keywords: Lactic acid fermentation; Lactic acid bacteria; Yeast; Optically pure lactic acid

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment by Pilar Junier; Verónica Molina; Cristina Dorador; Ora Hadas; Ok-Sun Kim; Thomas Junier; Karl-Paul Witzel; Johannes F. Imhoff (pp. 425-440).

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

Keywords: AOB; AOA; Anammox; Functional marker; Phylogenetic marker

Carbamoylases: characteristics and applications in biotechnological processes by Sergio Martínez-Rodríguez; Ana Isabel Martínez-Gómez; Felipe Rodríguez-Vico; Josefa María Clemente-Jiménez; Francisco Javier Las Heras-Vázquez (pp. 441-458).

Enzymatic kinetic resolution is a widely used biotechnological tool for the production of enantiomerically pure/enriched compounds. This technique takes advantage of the enantioselectivity or enantiospecificity of an enzyme for one of the enantiomers of a racemic substrate to isolate the desired isomer. N-Carbamoyl-d- and l-amino acid amidohydrolases (d- and l-carbamoylases) are model enzymes for this procedure due to their strict enantiospecificity. Carbamoylase-based kinetic resolution of amino acids has been applied for the last three decades, allowing the production of optically pure d- or l-amino acids. Furthermore, this enzyme has become crucial in the industrially used multienzymatic system known as “Hydantoinase Process,” where the kinetic resolution produced by coupling an enantioselective hydantoinase and the enantiospecific carbamoylase is enhanced by the enzymatic/chemical dynamic kinetic resolution of the low-rate hydrolyzed substrate. This review outlines the properties of d- and l-carbamoylases, emphasizing their biochemical/structural characteristics and their biotechnological applications. It also pinpoints new applications for the exploitation of carbamoylases over the forthcoming years.

Keywords: l-Carbamoylase; N-Carbamoyl-l-amino acid amidohydrolase; d-Carbamoylase; N-Carbamoyl-d-amino acid amidohydrolase; Hydantoinase Process; Ureidopropionase; Peptidase family M20/M25/M40; Nitrilase family; Kinetic resolution

Silkworm expression system as a platform technology in life science by Tatsuya Kato; Mizuho Kajikawa; Katsumi Maenaka; Enoch Y. Park (pp. 459-470).

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.

Keywords: Silkworm; Bombyx mori nucleopolyhedrovirus (BmNPV); Bacmid; Protein expression; Baculovirus display

Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook by Toru Jojima; Crispinus A. Omumasaba; Masayuki Inui; Hideaki Yukawa (pp. 471-480).

There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.

Keywords: Sugar transporter; Lignocellulose; Mixed-sugar utilization; Carbon catabolite repression

Bioprospecting for microbial products that affect ice crystal formation and growth by Brent C. Christner (pp. 481-489).

At low temperatures, some organisms produce proteins that affect ice nucleation, ice crystal structure, and/or the process of recrystallization. Based on their ice-interacting properties, these proteins provide an advantage to species that commonly experience the phase change from water to ice or rarely experience temperatures above the melting point. Substances that bind, inhibit or enhance, and control the size, shape, and growth of ice crystals could offer new possibilities for a number of agricultural, biomedical, and industrial applications. Since their discovery more than 40 years ago, ice nucleating and structuring proteins have been used in cryopreservation, frozen food preparation, transgenic crops, and even weather modification. Ice-interacting proteins have demonstrated commercial value in industrial applications; however, the full biotechnological potential of these products has yet to be fully realized. The Earth’s cold biosphere contains an almost endless diversity of microorganisms to bioprospect for microbial compounds with novel ice-interacting properties. Microorganisms are the most appropriate biochemical factories to cost effectively produce ice nucleating and structuring proteins on large commercial scales.

Keywords: Ice-nucleating proteins; Antifreeze proteins; Ice-structuring proteins; Cryopreservation; Bioprospecting; Cryosphere

Fermentative production of branched chain amino acids: a focus on metabolic engineering by Jin Hwan Park; Sang Yup Lee (pp. 491-506).

The branched chain amino acids (BCAAs), l-valine, l-leucine, and l-isoleucine, have recently been attracting much attention as their potential to be applied in various fields, including animal feed additive, cosmetics, and pharmaceuticals, increased. Strategies for developing microbial strains efficiently producing BCAAs are now in transition toward systems metabolic engineering from random mutagenesis. The metabolism and regulatory circuits of BCAA biosynthesis need to be thoroughly understood for designing system-wide metabolic engineering strategies. Here we review the current knowledge on BCAAs including their biosynthetic pathways, regulations, and export and transport systems. Recent advances in the development of BCAA production strains are also reviewed with a particular focus on l-valine production strain. At the end, the general strategies for developing BCAA overproducers by systems metabolic engineering are suggested.

Keywords: Branched chain amino acids (BCAAs); l-Valine; l-Leucine; l-Isoleucine; Systems metabolic engineering; Fermentation

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida) by Ayako Hirao; Ralf-Udo Ehlers (pp. 507-515).

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 × 10³ DJ per milliliter for S. carpocapsae and 1 to 8 × 10³ DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 × 10³ DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3–6 × 10³ DJs per milliliter in order to obtain >10³ parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 × 10³ DJs per milliliter to reach >2 × 10³ parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 × 10³ and 105 × 10³ per mililiter for S. feltiae.

Keywords: Biocontrol nematodes; Liquid culture; Inoculum density; Steinernema ; Xenorhabdus ; Dauer juvenile yields

Investigation of extractive microbial transformation in nonionic surfactant micelle aqueous solution using response surface methodology by Yingying Xue; Chen Qian; Zhilong Wang; Jian-He Xu; Rude Yang; Hanshi Qi (pp. 517-524).

Extractive microbial transformation of L-phenylacetylcarbinol (L-PAC) in nonionic surfactant Triton X-100 micelle aqueous solution was investigated by response surface methodology. Based on the Box–Behnken design, a mathematical model was developed for the predication of mutual interactions between benzaldehyde, Triton X-100, and glucose on L-PAC production. It indicated that the negative or positive effect of nonionic surfactant strongly depended on the substrate concentration. The model predicted that the optimal concentration of benzaldehyde, Triton X-100, and glucose was 1.2 ml, 15 g, and 2.76 g per 100 ml, respectively. Under the optimal condition, the maximum L-PAC production was 27.6 mM, which was verified by a time course of extractive microbial transformation. A discrete fed-batch process for verification of cell activity was also presented.

Keywords: Nonionic surfactant; Micelle; Response surface methodology; Extractive microbial transformation; L-phenylacetylcarbinol

Development of an attached microalgal growth system for biofuel production by Michael B. Johnson; Zhiyou Wen (pp. 525-534).

Algal biofuel production has gained a renewed interest in recent years but is still not economically feasible due to several limitations related to algal culture. The objective of this study is to explore a novel attached culture system for growing the alga Chlorella sp. as biodiesel feedstock, with dairy manure wastewater being used as growth medium. Among supporting materials tested for algal attachment, polystyrene foam led to a firm attachment, high biomass yield (25.65 g/m², dry basis), and high fatty acid yield (2.31 g/m²). The biomass attached on the supporting material surface was harvested by scraping; the residual colonies left on the surface served as inoculum for regrowth. The algae regrowth on the colony-established surface resulted in a higher biomass yield than that from the initial growth on fresh surface due to the downtime saved for initial algal attachment. The 10-day regrowth culture resulted in a high biodiesel production potential with a fatty acid methyl esters yield of 2.59 g/m² and a productivity of 0.26 g/m⁻² day⁻¹. The attached algal culture also removed 61–79% total nitrogen and 62–93% total phosphorus from dairy manure wastewater, depending on different culture conditions. The biomass harvested from the attached growth system (through scraping) had a water content of 93.75%, similar to that harvested from suspended culture system (through centrifugation). Collectively, the attached algal culture system with polystyrene foam as a supporting material demonstrated a good performance in terms of biomass yield, biodiesel production potential, ease to harvest biomass, and physical robustness for reuse.

Keywords: Algal biofuel; Attached growth; Chlorella; Lipids; Microalgae mass culture; Waste water treatment

Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells by Wook-Dong Kim; Miwako Tokunaga; Hiroyuki Ozaki; Takuya Ishibashi; Kohsuke Honda; Hiroyuki Kajiura; Kazuhito Fujiyama; Ryutaro Asano; Izumi Kumagai; Takeshi Omasa; Hisao Ohtake (pp. 535-542).

The glycosylation pattern of a humanized anti-EGFR×anti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.

Keywords: Bispecific diabody; Bispecific IgG-like antibody; Glycosylation; Immunoglobulin G; Chinese hamster ovary cells

A step-forward in the characterization and potential applications of solid and liquid oxygen transfer vectors by Guillermo Quijano; Maria Hernandez; Santiago Villaverde; Frederic Thalasso; Raul Muñoz (pp. 543-551).

Silicone oil 20 and 200 cSt, a perfluorocarbon (FC40TM), heptamethylnonane, Kraton, Elvax, and Desmopan were evaluated for their ability to enhance oxygen transfer in stirred tank and airlift reactors (STR and ALR, respectively). None of the vectors tested was either toxic or biodegradable and they exhibited a moderate affinity for oxygen (gas/vector partitioning coefficients $$ K_{{{ ext{g}}/{ ext{v}}}} = C_{ ext{g}} cdot C_{ ext{v}}^{- 1} $$ ranging from 3 to 5.1). FC40 was highly volatile, while KratonTM and ElvaxTM exhibited a low thermal stability, which constitutes a serious handicap for their implementation in fermentations. Silicone oil 20 cSt and Desmopan supported the highest oxygen transfer rates under abiotic conditions in both STR and ALR designs, with enhancement factors of up to 90% and 250%, respectively, compared to control tests (deprived of vector). The fact that these vectors showed the highest K _g/v proved that, besides the classical selection criteria, the in situ hydrodynamic behavior (which affects K L a) must be considered for vector selection. The use of silicone oil 20 cSt and Desmopan in glucose-supplemented Saccharomyces cerevisiae fermentations resulted in a two- and threefold increase in biomass productions, respectively. The better performance of Desmopan in terms of biomass growth enhancement, together with the absence of the operational problems inherent to the use of liquid vectors (such as intensive foaming, high cost, and difficult solvent recovery), make solid vectors a promising and cost-effective alternative in the future developments of two-phase partitioning bioreactors.

Keywords: Oxygen transfer rate; Saccharomyces cerevisiae ; Transfer vector; Vector selection; Two-phase partitioning bioreactors

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli by Zhaofeng Li; Zhengbiao Gu; Miao Wang; Guocheng Du; Jing Wu; Jian Chen (pp. 553-561).

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.

Keywords: Cyclodextrin glycosyltransferase; Paenibacillus macerans ; Extracellular secretion; Glycine; Escherichia coli ; Delayed supplementation

Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration by Mojca Božič; Sina Pricelius; Georg M. Guebitz; Vanja Kokol (pp. 563-571).

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new β-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.

Keywords: Acid Blue 74; Natural Orange 6; Vat Blue 1; Bacillus subtilis ; NADH-dependent reductase; NADH regeneration

Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6 by Patthra Pason; Akihiko Kosugi; Rattiya Waeonukul; Chakrit Tachaapaikoon; Khanok Ratanakhanokchai; Takamitsu Arai; Yoshinori Murata; Jun Nakajima; Yutaka Mori (pp. 573-580).

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C-terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes.

Keywords: Paenibacillus curdlanolyticus ; Xylanase; Cellulase; Multienzyme complex; Hemicellulase

Characterization of novel thermostable dextranase from Thermotoga lettingae TMO by Young-Min Kim; Doman Kim (pp. 581-587).

Biochemical properties of a putative thermostable dextranase gene from Thermotoga lettingae TMO were determined in a recombinant protein (TLDex) expressed in Escherichia coli and purified to sevenfold apparent homogeneity. The 64-kDa protein displayed maximum activity at pH 4.3, and enzyme activity was stable from pH 4.3–10. The optimal temperature was 55–60°C during 15 min incubation, and the half-life of the enzyme was 1.5 h at 65°C. The enzyme showed higher activity against α-(1 → 6) glucan and released isomaltose and isomaltotriose as main products from dextran T2000. An unusual kinetic feature of TLDex was the negative cooperative behavior on the reaction of dextran T2000 cleavage. Enzyme activity was not significantly affected by the presence of metal ions, except for the strong inhibited by 1 mM Fe²⁺ and Ag²⁺. TLDex may prove useful as an enzyme for high temperature sugar milling processes.

Keywords: Thermostable dextranase; Thermotoga lettingae ; Negative cooperative behavior; Thermophile; GH family 66

Energy uncoupling inhibits aerobic granulation by Bo Jiang; Yu Liu (pp. 589-595).

Although aerobic granulation has been intensively studied, the possible mechanism of this cell-to-cell self-immobilization phenomenon still remains unclear. Aerobic granulation in the absence and presence of a chemical uncoupler, 3,3′,4′,5-tetrachlorosalicylanilide (TCS), which can dissipate the proton gradient and further disrupt ATP synthesis, was investigated. Upon exposure to TCS, precultivated mature aerobic granules underwent disintegration, indicating that the stability and integrity of aerobic granules would be associated with microbial energy metabolism. It was also shown that the formation of aerobic granules in the presence of TCS was completely inhibited as compared with the control free of TCS. These results, for the first time, reveal that aerobic granulation is energy metabolism dependent, and possible reasons are also discussed.

Keywords: Aerobic granules; ATP; Energy metabolism; TCS; Uncoupler

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli by Bhawna Madan; Prashant Mishra (pp. 597-604).

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 µM and 217 ± 22.5 µmol min⁻¹ mg⁻¹ protein, respectively.

Keywords: Lipase; Co-expression; Foldase; P. aeruginosa ; E. coli

Production of biologically active recombinant annexin B1 with enhanced stability via a tagging system by Yi Zhang; Tao Zheng; Yue Wang; Yingjun Guo; Feixiang Ding; Mingyu Fei; Huixian Cui; Shuhan Sun (pp. 605-614).

Annexin B1 is a novel Ca²⁺-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure–function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.

Keywords: Annexin B1; Hexahistidine tag; Protein stability; Coagulation time; Apoptosis

Sequence analysis and heterologous expression of a new cytochrome P450 monooxygenase from Rhodococcus sp. for asymmetric sulfoxidation by Jian-Dong Zhang; Ai-Tao Li; Yi Yang; Jian-He Xu (pp. 615-624).

In this study, a 3.7-kb DNA fragment was cloned from Rhodococcus sp. ECU0066, and the sequence was analyzed. It was revealed that the largest one (2,361 bp) of this gene fragment encodes a protein consisting of 787 amino acids, with 73% identity to P450RhF (accession number AF45924) from Rhodococcus sp. NCIMB 9784. The gene of this new P450 monooxygenase (named as P450SMO) was successfully expressed in Escherichia coli BL21 (DE3), and the enzyme was also purified and characterized. In the presence of reduced nicotinamide adenine dinucleotide phosphate, the enzyme showed significant sulfoxidation activity towards several sulfides, with (S)-sulfoxides as the predominant product. The p-chlorothioanisole, p-fluorothioanisole, p-tolyl methyl sulfide, and p-methoxythioanisole showed relatively higher activities than the other sulfides, but the stereoselectivity for p-methoxythioanisole was much lower. The optimal activity of the purified enzyme toward p-chlorothioanisole occurred at pH 7.0 and 30°C. The current study is the first to report a recombinant cytochrome P450 enzyme of Rhodococcus sp. which is responsible for the asymmetric oxidation of sulfides. The new enzymatic activity of P450SMO on the above compounds makes it an attractive biocatalyst for asymmetric synthesis of enantiopure sulfoxides.

Keywords: Cytochrome P450 monooxygenase; Rhodococcus sp; Cloning; Expression; Asymmetric sulfoxidation

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac by Jamie E. Prior; Touraj Shokati; Uwe Christians; Ryan T. Gill (pp. 625-633).

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4′-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.

Keywords: Cytochrome P450; Actinoplanes ; Drug metabolism; Diclofenac

Soluble expression, purification, and characterization of Gloydius shedaoensis venom gloshedobin in Escherichia coli by using fusion partners by Xiuping Jiang; Jianqiang Xu; Qing Yang (pp. 635-642).

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40°C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-l-phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.

Keywords: Fibrigenolytic enzyme; GST; NusA; TrxA; Snake venom; Thrombin-like enzyme

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum by Kenji Okano; Qiao Zhang; Shogo Yoshida; Tsutomu Tanaka; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 643-650).

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.

Keywords: CelA; d-lactic acid; Lactobacillus plantarum ; Cellooligosaccharide; β-glucan

Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes by Shota Atsumi; Tung-Yun Wu; Eva-Maria Eckl; Sarah D. Hawkins; Thomas Buelter; James C. Liao (pp. 651-657).

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.

Keywords: Biofuel; Metabolic engineering; Isobutanol; Alcohol dehydrogenase

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae by Jeong-Woo Seo; Mi-Young Seo; Baek-Rock Oh; Sun-Yeon Heo; Jin-Oh Baek; Dina Rairakhwada; Lian Hua Luo; Won-Kyung Hong; Chul Ho Kim (pp. 659-666).

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.

Keywords: Klebsiella pneumoniae ; 1,3-Propanediol; Glycerol metabolism; 1,3-Propanediol oxidoreductase isoenzyme

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway by Alimjan Idiris; Hideki Tohda; Mayumi Sasaki; Katsunori Okada; Hiromichi Kumagai; Yuko Giga-Hama; Kaoru Takegawa (pp. 667-677).

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

Keywords: Fission yeast; Heterologous protein secretion; Protease; Human growth hormone; Vacuolar protein sorting; vps10

Development of siRNA expression vector utilizing rock bream β-actin promoter: a potential therapeutic tool against viral infection in fish by Kosuke Zenke; Yoon Kwon Nam; Ki Hong Kim (pp. 679-690).

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream β-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream β-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream β-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Keywords: RNA interference; β-actin promoter; Short interfering RNA; Fish cell; Rock bream iridovirus

Molecular and kinetic characterization of mixed cultures degrading natural and synthetic estrogens by Mathieu Muller; Dominique Patureau; Jean-Jacques Godon; Jean-Philippe Delgenès; Guillermina Hernandez-Raquet (pp. 691-701).

The biodegradation of estradiol (E2), estrone (E1), and ethinylestradiol (EE2) was investigated using mixed bacterial cultures enriched from activated sludge. Enrichments were carried out on E2 or EE2 in batch conditions with acetonitrile as additional carbon source. Degradation experiments were performed both using hormones as sole carbon source or with an additional source. The hormones were completely degraded by these cultures. Estradiol was rapidly converted to E1 within 24 h. Thereafter, E1 degradation began, displaying a lag phase ranging from 3 to 4 days. Estrone depletion took from 48 h to more than 6 days, depending on the culture conditions. For EE2 degradation, when it was the sole carbon source, the lag phase and the time required for its complete removal (7 and 15 days, respectively) were shorter that in cultures with a supplementary carbon source. The specific degradation rates observed for E2 both with and without an additional carbon source were similar. By contrast, the specific degradation rates for E1 and EE2 were, respectively, seven and 20 times faster when these hormones were supplied as the sole carbon source. The bacterial community structure of each culture was characterized by molecular and cultural methods. The mixed cultures were made up of species belonging to Alcaligenes faecalis, Pusillimonas sp., Denitrobacter sp., and Brevundimonas diminuta or related to uncultured Bacteroidetes. The isolated strain B. diminuta achieved the conversion of E2 to E1.

Keywords: Estrogen; Degradation; Estradiol; Estrone; Ethinylestradiol; Brevundimonas

Radial basis function neural networks for modeling growth rates of the basidiomycetes Physisporinus vitreus and Neolentinus lepideus by Mark Schubert; Safer Mourad; Francis Schwarze (pp. 703-712).

A radial basis function (RBF) neural network was developed and compared against a quadratic response surface (RS) model for predicting the specific growth rates of the biotechnologically important basidiomycetous fungi, Physisporinus vitreus and Neolentinus lepideus, under three environmental conditions: temperature (10–30 °C), water activity (0.950–9.998), and pH (4–6). Both the RBF network and polynomial RS model were mathematically evaluated against experimental data using graphical plots and several statistical indices. The evaluation showed that both models gave reasonably good predictions, but the performance of the RBF neural network was superior to that of the classical statistical method for all three data sets used (training, testing, full). Sensitivity analysis revealed that of the three experimental factors the most influential on the growth rate of P. vitreus was water activity, followed by temperature and pH to a lesser extent. In contrast, temperature in particular and then water activity were the key determinants of the development of N. lepideus. RBF neural networks could be a powerful technique for modeling fungal growth behavior under certain parameters and an alternative to time-consuming, traditional microbiological techniques.

Keywords: Radial basis function neural network; Physisporinus vitreus ; Neolentinus lepideus ; Response surface model; Growth rate

Conversion of phenol to glutamate and proline in Corynebacterium glutamicum is regulated by transcriptional regulator ArgR by Soo Youn Lee; Yang-Hoon Kim; Jiho Min (pp. 713-720).

This paper reports a novel integrated metabolic pathway of amino acid production through the utilization of phenol in Corynebacterium glutamicum. In the presence of 8.5 mM phenol, the level of glutamate and proline production increased up to 1.2- and 14.7-fold, respectively, compared to the control condition. In addition, their productivities increased 1.6- and 20-fold in the culture medium using phenol as the sole carbon source with 72 μM FeSO₄ as iron supplementation. Chromatin immunoprecipitation assay showed that the DNA-binding affinity of ArgR as a transcriptional repressor to the upstream of gltB and gdh gene was reduced significantly in the presence of 8.5 mM phenol. In addition, the DNA-binding affinity of ArgR to the upstream of gltB and gdh gene by iron supplementation was severely reduced, more than that under only 8.5 mM phenol. These results are consistent with those showing an increase in the mRNA levels of the two genes in the presence of 8.5 mM phenol and iron supplementation. Overall, iron enhances the biotransformation to glutamate and proline from phenol because of the regulated transcriptional levels. Furthermore, the biotransformation of phenol to glutamate and proline probably occurs through an interaction between ArgR and the gltB and gdh genes.

Keywords: Corynebacterium glutamicum ; Phenol; Glutamate; Proline; ArgR; Transcriptional regulation

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants by Mark L. Thompson; Ray Marriott; Adam Dowle; Gideon Grogan (pp. 721-730).

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.

Keywords: Hops; Enrichment; Rhodococcus ; Biotransformation; Myrcene; Geraniol

Enhancing the flux of D-glucose to the pentose phosphate pathway in Saccharomyces cerevisiae for the production of D-ribose and ribitol by Mervi H. Toivari; Hannu Maaheimo; Merja Penttilä; Laura Ruohonen (pp. 731-739).

Phosphoglucose isomerase-deficient (pgi1) strains of Saccharomyces cerevisiae were studied for the production of D-ribose and ribitol from D-glucose via the intermediates of the pentose phosphate pathway. Overexpression of the genes coding for NAD⁺-specific glutamate dehydrogenase (GDH2) of S. cerevisiae or NADPH-utilising glyceraldehyde-3-phosphate dehydrogenase (gapB) of Bacillus subtilis enabled growth of the pgi1 mutant strains on D-glucose. Overexpression of the gene encoding sugar phosphate phosphatase (DOG1) of S. cerevisiae was needed for the production of D-ribose and ribitol; however, it reduced the growth of the pgi1 strains expressing GDH2 or gapB in the presence of higher D-glucose concentrations. The CEN.PK2-1D laboratory strain expressing both gapB and DOG1 produced approximately 0.4 g l⁻¹ of D-ribose and ribitol when grown on 20 g l⁻¹ (w/v) D-fructose with 4 g l⁻¹ (w/v) D-glucose. Nuclear magnetic resonance measurements of the cells grown with ¹³C-labelled D-glucose showed that about 60% of the D-ribose produced was derived from D-glucose. Strains deficient in both phosphoglucose isomerase and transketolase activities, and expressing DOG1 and GDH2 tolerated only low D-glucose concentrations (≤2 g l⁻¹ (w/v)), but produced 1 g l⁻¹ (w/v) D-ribose and ribitol when grown on 20 g l⁻¹ (w/v) D-fructose with 2 g l⁻¹ (w/v) D-glucose.

Keywords: Sugar alcohols; Pentose sugars; Saccharomyces cerevisiae ; Pentose phosphate pathway; D-ribose; Ribitol; NMR

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases by Watchara Kanchanarach; Gunjana Theeragool; Toshiharu Yakushi; Hirohide Toyama; Osao Adachi; Kazunobu Matsushita (pp. 741-751).

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.

Keywords: Acetic acid bacteria; Acetobacter pasteurianus ; Quinoprotein alcohol dehydrogenases; Acetic acid fermentation

Microbial community of acetate utilizing denitrifiers in aerobic granules by Sunil S. Adav; Duu-Jong Lee; J. Y. Lai (pp. 753-762).

The aerobic sludge granules cultivated at high organic loading rates could effectively convert 100–700 mg l⁻¹ nitrite to nitrogen gas with 400 or 1,200 mg l⁻¹ dosed acetate. The denitrifying microbial community structure of the so-cultivated granules was investigated by 16S rRNA gene sequences and localized using fluorescence in situ hybridization (FISH). The 16S rRNA gene phylotypes in the clone library and FISH probes used exhibited high diversity among the bacteria and denitrifying communities, with the members of Betaproteobacteria predominant that were closely related to families Comamonadaceae, Nitrosomonadaceae, Alcaligenaceae, and Rhodocyclaceae. The confocal laser scanning microscope and staining test revealed that active microbial community principally distributed at 200–250 μm beneath the outer surface, embedded in extracellular polymeric substances.

Keywords: Denitrifiers; Aerobic granules; Microbial community; Acetate

Removing heavy metals from synthetic effluents using “kamikaze” Saccharomyces cerevisiae cells by Lavinia Ruta; Codruta Paraschivescu; Mihaela Matache; Sorin Avramescu; Ileana Cornelia Farcasanu (pp. 763-771).

One key step of the bioremediation processes designed to clean up heavy metal contaminated environments is growing resistant cells that accumulate the heavy metals to ensure better removal through a combination of biosorption and continuous metabolic uptake after physical adsorption. Saccharomyces cerevisiae cells can easily act as cation biosorbents, but isolation of mutants that are both hyperaccumulating and tolerant to heavy metals proved extremely difficult. Instead, mutants that are hypersensitive to heavy metals due to increased and continuous uptake from the environment were considered, aiming to use such mutants to reduce the heavy metal content of contaminated waters. In this study, the heavy metal hypersensitive yeast strain pmr1∆ was investigated for the ability to remove Mn²⁺, Cu²⁺, Co²⁺, or Cd²⁺ from synthetic effluents. Due to increased metal accumulation, the mutant strain was more efficient than the wild-type in removing Mn²⁺, Cu²⁺, or Co²⁺ from synthetic effluents containing 1–2 mM cations, with a selectivity $$ { ext{Mn}}^{{{ ext{2}} + }} > { ext{Co}}^{{{ ext{2}} + }} ~ > { ext{Cu}}^{{{ ext{2}} + }} $$ and also in removing Mn²⁺ and Cd²⁺ from synthetic effluents containing 20–50 μM cations, with a selectivity Mn²⁺ > Cd²⁺.

Keywords: Saccharomyces cerevisiae ; PMR1 ; Heavy metal; Bioremediation

Enhanced biological denitrification of high concentration of nitrite with supplementary carbon source by Sunil S. Adav; Duu-Jong Lee; J. Y. Lai (pp. 773-778).

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l⁻¹ nitrite at a rate of 0.27 mg NO₂–N g⁻¹ VSS h⁻¹. Notably, 1,500 mg l⁻¹ acetate and 700 mg l⁻¹ methanol or ethanol enhanced denitrification rates for 200 mg l⁻¹ nitrite at 2.07, 1.20, and 1.60 mg NO₂–N g⁻¹ VSS h⁻¹, respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37–41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol > acetate > ethanol on molar basis or acetate > ethanol > methanol on an added weight basis.

Keywords: Denitrification; Nitrite; Nitrite reductase; Carbon source

Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting by Léa Cabrol; Luc Malhautier; Franck Poly; Anne-Sophie Lepeuple; Jean-Louis Fanlo (pp. 779-790).

In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNA yield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.

Keywords: DNA extraction; Cell dispersion; DGGE; Biofiltration; Diversity; Microbial community structure

Bacterial diversity in water injection systems of Brazilian offshore oil platforms by Elisa Korenblum; Érika Valoni; Mônica Penna; Lucy Seldin (pp. 791-800).

Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.

Keywords: Bacterial diversity; Offshore oil platforms; Water injection systems; 16S rRNA gene

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample by Christian G. Ramos; André M. Grilo; Sílvia A. Sousa; Marta L. Barbosa; Helena Nadais; Jorge H. Leitão (pp. 801-806).

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.

Keywords: Molecular characterization of microbial populations; Clone RFLP analysis; Primer design; Molecular methodology to characterize microbial populations; 16S rRNA gene sequence analysis

Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii by Sun Young Park; Bokhui Lee; Kwang-Su Park; Youhoon Chong; Moon-Young Yoon; Sung-Jong Jeon; Dong-Eun Kim (pp. 807-812).

An inorganic pyrophosphatase (PPases) was cloned from the hyperthermophilic archaeon Pyrococcus horikoshii and was expressed in and purified from Escherichia coli. The recombinant inorganic pyrophosphatase (PhPPase) exhibited robust catalytic activity of the hydrolysis of pyrophosphate into two orthophosphates at high temperatures (70°C to 95°C). Thermostable pyrophosphatase activity was applied into polymerase chain reaction (PCR) due to its ability to push chemical equilibrium toward the synthesis of DNA by removing pyrophosphate from the reaction. A colorimetric method using molybdate and reducing agents was used to measure PCR progress by detecting and quantifying inorganic phosphate in the PhPPase-coupled PCR mixture. Compared to PCR mixtures without PhPPase, the thermostable PhPPase enhanced the amount of PCR product in the same number of cycles. Thus, thermostable PPase may overcome the limitations of thermodynamically unfavorable DNA polymerization in PCR by yielding more products.

Keywords: Hyperthermophilic archaeon; Inorganic pyrophosphatase; Polymerase chain reaction; Thermostability

Monitoring Escherichia coli growth in M63 media by ultrasonic noninvasive methods and correlation with spectrophotometric and HPLC techniques by Carlos Sierra; Luis Elvira; José L. García; Pablo Resa; Beatriz Galán (pp. 813-821).

A low-intensity ultrasonic technique (that is noninvasive, nondestructive, and online) has been developed to monitor the growth of Escherichia coli in glucose minimal media under aerobic and anaerobic conditions. Ultrasonic time of flight (TOF) variations were correlated with microorganism growth and the disappearance of nutrients and their subsequent conversion into different metabolites. Spectrophotometric growth data and high-performance liquid chromatography (HPLC) analysis of released and consumed metabolites were compared with the ultrasonic data demonstrating that the ultrasound device presented can provide supplementary real-time noninvasive information about the metabolic processes taking place in the culture. A semiempirical model under aerobic conditions was developed to explain the TOF variations as a function of the glucose concentration and was modified to take into account the growth inhibition due to the initial glucose concentration. The inhibition effect was obtained by fitting HPLC measurements to the logistic function of Boltzmann. Under aerobic and anaerobic culture conditions, the metabolic processes were correlated with TOF experimental variations through a theoretical approach based on ultrasonic propagation in ternary mixtures.

Keywords: Ultrasound; Time of flight; Escherichia coli ; Growth; Aerobic; Anaerobic

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Applied Microbiology and Biotechnology (v.85, #3)

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Applied Microbiology and Biotechnology (v.85, #3)