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Applied Microbiology and Biotechnology (v.65, #6)
Valuable products from biotechnology of microalgae by Otto Pulz; Wolfgang Gross (pp. 635-648).
The biotechnology of microalgae has gained considerable importance in recent decades. Applications range from simple biomass production for food and feed to valuable products for ecological applications. For most of these applications, the market is still developing and the biotechnological use of microalgae will extend into new areas. Considering the enormous biodiversity of microalgae and recent developments in genetic engineering, this group of organisms represents one of the most promising sources for new products and applications. With the development of sophisticated culture and screening techniques, microalgal biotechnology can already meet the high demands of both the food and pharmaceutical industries.
RNA interference: potential therapeutic targets by S. Jana; C. Chakraborty; S. Nandi; J. K. Deb (pp. 649-657).
One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.
Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent by Appadurai Senthuran; Vasanthe Senthuran; Rajni Hatti-Kaul; Bo Mattiasson (pp. 658-663).
The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.
Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using plastic composite support bioreactors by Susan E. Urbance; Anthony L. Pometto III; Alan A. DiSpirito; Yuksel Denli (pp. 664-670).
Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems. For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation. Agitation was controlled at 125 rpm and 150 rpm. Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h−1 and analyzed for succinic acid production and glucose consumption (g l−1). For PCS bioreactors, the highest final succinic acid concentrations (10.1 g −1, 10.4 g l−1) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h−1. PCS disks were evaluated in a repeat-batch biofilm reactor. Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor. The maximum concentration of succinic acid produced was 40 g l−1. Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A. succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h−1 (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l−1 (86.7%).
Design of mineral medium for growth of Actinomadura sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources by Zuzana Technikova-Dobrova; Fabrizio Damiano; Salvatore M. Tredici; Giovanni Vigliotta; Roberta di Summa; Luigi Palese; Anna Abbrescia; Nestor Labonia; Gabriele V. Gnoni; Pietro Alifano (pp. 671-677).
Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.
Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides by Heather R. Luckarift; Howard Dalton; Narain D. Sharma; Derek R. Boyd; Robert A. Holt (pp. 678-685).
The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.
A novel N-carbamoyl-l-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-l-cysteine amidohydrolase expressed in Escherichia coli by Tetsuo Ohmachi; Megumi Narita; Maki Kawata; Akiko Bizen; Yoshiharu Tamura; Yoshihiro Asada (pp. 686-693).
N-carbamoyl-l-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50°C, respectively. The enzyme required Mn2+ ion for activity expression and was inhibited by EDTA, Hg2+ and sulfhydryl reagents. The enzyme was strictly specific for the l-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-l-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel l-carbamoylase, different from the known l-carbamoylases.
Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon Thermococcus chitonophagus by Evi Andronopoulou; Constantinos E. Vorgias (pp. 694-702).
Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.
Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria by Kohtaro Kirimura; Koji Harada; Hidekazu Iwasawa; Takeomi Tanaka; Yuichiro Iwasaki; Toshiki Furuya; Yoshitaka Ishii; Kuniki Kino (pp. 703-713).
Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52°C. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e.g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2′-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30–52°C, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50°C and DBT desulfurization (2-HBP production) at 40°C in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.
Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus by Wai Prathumpai; Simon J. Flitter; Mhairi McIntyre; Jens Nielsen (pp. 714-719).
Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Yxp total), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7±0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3±0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (rp total, the sum of extracellular and intracellular lipase productivity) was found to be 1.60±0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h−1, compared with a total specific lipase productivity of 1.10±0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
Screening for novel lipolytic enzymes from uncultured soil microorganisms by Seon-Woo Lee; Keehoon Won; He Kyoung Lim; Jin-Cheol Kim; Gyung Ja Choi; Kwang Yun Cho (pp. 720-726).
The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C4) but not p-nitrophenyl palmitate (C16).
Adhesion of a Pseudomonas putida strain isolated from a paper machine to cellulose fibres by A. Rochex; D. Lecouturier; I. Pezron; J.-M. Lebeault (pp. 727-733).
The adhesion to cellulose fibres of a strain of Pseudomonas putida isolated from a paper machine was studied under different environmental conditions. The physicochemical properties of both P. putida cells and cellulose fibres were also determined to better understand the adhesion phenomenon. Adhesion was rapid (1 min) and increased with time, cell concentration and temperature (from 25 to 40°C), indicating that bacterial adhesion to cellulose fibres is essentially governed by a physicochemical process. The P. putida cell surface was negatively charged, as shown by electrophoretic mobility measurements, and was hydrophilic due to a strong electron-donor character, as shown by the microbial adhesion to solvents method. Cellulose fibres were shown to be hydrophilic by contact angle measurements using the capillary rise method. These results suggest the importance of Lewis acid-base interactions in the adhesion process. In various ionic solutions (NaCl, KCl, CaCl2 and MgCl2), adhesion increased with increasing ionic strength up to 10–100 mM, indicating that, at low ionic strength, electrostatic interactions were involved in the adhesion process. An increase in the C/N ratio of the growth medium (from 5 to 90) decreased adhesion but this could not be related to changes in physicochemical properties, suggesting that other factors may be involved. In practice, temperature, ionic strength and nitrogen concentration must be taken into consideration to reduce bacterial contamination in the paper industry.
Effect of acetic acid present in bagasse hydrolysate on the activities of xylose reductase and xylitol dehydrogenase in Candida guilliermondii by Luanne Helena Augusto Lima; Maria das Graças de Almeida Felipe; Michele Vitolo; Fernando Araripe Gonçalves Torres (pp. 734-738).
The first two steps in xylose metabolism are catalyzed by NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21) and NAD(P)-dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9), which lead to xylose→xylitol→xylulose conversion. Xylitol has high commercial value, due to its sweetening and anticariogenic properties, as well as several clinical applications. The acid hydrolysis of sugarcane bagasse allows the separation of a xylose-rich hemicellulosic fraction that can be used as a substrate for Candida guilliermondii to produce xylitol. However, the hydrolysate contains acetic acid, an inhibitor of microbial metabolism. In this study, the effect of acetic acid on the activities of XR and XDH and on xylitol formation by C. guilliermondii were studied. For this purpose, fermentations were carried out in bagasse hydrolysate and in synthetic medium. The activities of XR and XDH were higher in the medium containing acetic acid than in control medium. Moreover, none of the fermentative parameters were significantly altered during cell culture. It was concluded that acetic acid does not interfere with xylitol formation since the increase in XR activity is proportional to XDH activity, leading to a greater production of xylitol and its subsequent conversion to xylulose.
Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media by Jason T. Popesku; Ajay Singh; Jian-Shen Zhao; Jalal Hawari; Owen P. Ward (pp. 739-746).
Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4′-azoxytetranitrotoluene and 2′,4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled 14C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.
Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms by Rongjun Zuo; Thomas K. Wood (pp. 747-753).
A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.