Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.64, #5)

Occurrence, biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle by M. Ishii; S. Chuakrut; H. Arai; Y. Igarashi (pp. 605-610).

The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO₂ fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon. In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO₂ fixation. It has been shown in A. brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA. The subunit structure of the acyl-CoA carboxylase was shown to be α₄β₄γ₄. Gene cloning revealed that the genes encoding the three subunits are adjacent to each other. accC encodes the β-subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the γ-subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the α-subunit (62 kDa subunit, carboxyltransferase subunit). Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately. Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented.

Polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha cells: a computer simulation by L. Jurasek; R. H. Marchessault (pp. 611-617).

Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA. It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on published experimental data. The results are applicable to R. eutropha cells or other microorganisms and transgenic plants, where polyhydroxybutyrate production is made possible by heterologous expression systems. The simulation starts at the outset of the PHA accumulation phase when the cells are small and contain no PHA granules. In the presence of abundant glucose, the cell responds to phosphorus limitation by producing 3-hydroxybutyryl-CoA which undergoes polymerization on the few PHA synthase molecules present in the cytoplasm. The amphiphilic PHA synthase–PHA complex attracts additional PHA synthase molecules and granules begin to grow from these initiation sites. Phosphorus limitation and the appearance of PHA in the cytoplasm also stimulate production of phasin molecules that attach themselves to the growing granules. As the granules grow bigger, they begin to touch each other and move to optimize their packing. The phasin coat prevents the granules from coalescing. The size of the cell increases and its prolate ellipsoid shape becomes closer to spherical. The accumulation process stops either when the supply of glucose is exhausted or when the granules become tightly packed within the cell, so that access to their surface is limited. All important variables, such as cell dimensions, granule size, counts of granule-associated molecules, PHA yield, degree of polymerization of the PHA molecules, etc., are recorded in real time during the simulation. Examples of virtual experiments with the cell and their results are shown.

Compactin—a review by R. Chakravarti; V. Sahai (pp. 618-624).

Compactin, a hypocholesterolemic molecule, is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase, which is a regulatory enzyme for cholesterol biosynthesis. The structural similarity and high affinity of the acid form of compactin and HMG, the natural substrate of enzyme, results in specific and effective inhibition of this enzyme. Inhibition results in reduced levels of mevalonic acid in the body, leading to pleiotropic effects. Various fungi have been used for the commercial production of compactin. Using different strategies for improving production levels, yields have been increased to around 900 times of the amount originally produced. Recently, the gene sequence responsible for compactin production has been cloned and sequenced. This review deals with the chemistry, mode of action, pharmacology, biosynthesis, and production of compactin. A comparative study of various reports dealing with the production of compactin is also included.

Secretory and extracellular production of recombinant proteins using Escherichia coli by J. H. Choi; S. Y. Lee (pp. 625-635).

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.

Effects of CO₂ on the formation of flavour volatiles during fermentation with immobilised brewer’s yeast by H.-Y. Shen; S. De Schrijver; N. Moonjai; K. J. Verstrepen; F. Delvaux; F. R. Delvaux (pp. 636-643).

Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems. However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages. One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO₂, which has inhibitory effects on yeast growth and metabolism. This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO₂ from the liquid medium, which results in a low level of dissolved CO₂ during fermentation. Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system. By sparging a CO₂ flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10–30% during the active fermentation stage, while the fermentation rate was unaffected. The uptake of branched-chain amino acids was reduced by 8–22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively. The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO₂ during fermentation with immobilised yeast.

Arabinoxylan and mono- and dimeric ferulic acid release from brewer’s grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens by C. B. Faulds; G. Mandalari; R. LoCurto; G. Bisignano; K. W. Waldron (pp. 644-650).

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewer’s grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5′ benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewer’s grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.

Liquid culture mass production of biocontrol nematodes, Heterorhabditis bacteriophora (Nematoda: Rhabditida): improved timing of dauer juvenile inoculation by S.-A. Johnigk; F. Ecke; M. Poehling; R.-U. Ehlers (pp. 651-658).

Heterorhabditis bacteriophora is used in biological control of soil-borne insect pests in horticulture and turf. Mass production is carried out in monoxenic liquid cultures pre-incubated with the symbiont of the nematodes, the bacterium Photorhabdus luminescens, before nematode dauer juveniles (DJ) are inoculated. As a response to bacterial food signals, the DJ recover from the developmentally arrested dauer stage, grow to adults and produce DJ offspring. Variable DJ recovery after inoculation into cultures of P. luminescens often causes process failure due to low numbers of adult nematodes in the medium. In order to enhance DJ recovery, improve nematode population management and increase yields, the optimal timing for DJ inoculation was sought. The process parameter pH and respiration quotient (RQ) were recorded in order to test whether changes can be used to identify the best moment for DJ inoculation. When DJ were inoculated during the lag and early logarithmic growth phases of P. luminescens cultures, DJ recovery was low and almost no nematode reproduction was obtained. High populations of P. luminescens phase variants were recorded. Recovery and yields increased when DJ were inoculated during the latter log phase during which the RQ dropped to values <0.8 and the pH reached a maximum. The highest DJ recovery and yields were observed in cultures that were inoculated during the late stationary growth phase. This period started with the increase of the pH after its distinct minimum at pH <8.0. Thus optimal timing for DJ inoculation can be defined through monitoring of the pH in the P. luminescens culture.

A new non-aerated illuminated packed-column reactor for the development of sulfide-oxidizing biofilms by I. Ferrera; O. Sánchez; J. Mas (pp. 659-664).

This paper describes an illuminated reactor that allows the spontaneous development of biofilms aimed at the treatment of sulfide-containing streams. The reactor operates as a sulfidostat and is composed of an illuminated packed-column, in which microorganisms are exposed to constant low substrate concentrations, thereby avoiding inhibition due to high sulfide concentrations. The control system allows highly polluted streams to be oxidized by the microbial biofilm while ensuring the quality of the effluent produced. Both monospecies and multispecies biofilms have been developed. Biofilms undergo changes in light irradiance and sulfide load while providing a consistent reduction of the sulfide levels, down to micromolar concentrations. Both types of biofilm developed differ from stirred reactors in that their specific activities are lower, constituting systems with a slow dynamic behavior and, therefore, they are less sensitive to sudden disturbances.

DNA isolation from soil samples for cloning in different hosts by I. M. Kauffmann; J. Schmitt; R. D. Schmid (pp. 665-670).

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.

CYP175A1 from Thermus thermophilus HB27, the first β-carotene hydroxylase of the P450 superfamily by F. Blasco; I. Kauffmann; R. D. Schmid (pp. 671-674).

The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli. The gene product of CYP175A1 added hydroxyl groups to both β rings of β-carotene to form zeaxanthin (β,β-carotene-3,3′-diol) in E. coli, which produces β-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. In addition, spectroscopic methods revealed that E. coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone. The predicted amino acid sequence of the enzyme from T. thermophilus does not show substantial similarity with other known β-carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3). It is concluded that CYP175 A1 represents a new type of β-carotene hydroxylase of the P450 superfamily.

Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain by M. C. Limón; M. R. Chacón; R. Mejías; J. Delgado-Jarana; A. M. Rincón; A. C. Codón; T. Benítez (pp. 675-685).

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

Behaviour of dehydrated baker’s yeast during reduction reactions in a biphasic medium by L. Cappaert; C. Larroche (pp. 686-690).

The behaviour of dry baker’s yeast (Saccharomyces cerevisiae type II, Sigma) used as biocatalyst without preliminary growth for the synthesis of 2-heptanol from 2-heptanone in a biphasic system is presented. Cells undergo intracellular trehalose consumption with a stoichiometric ethanol production during the first 15 h of the process. This metabolism is then replaced by acetate accumulation. These reactions are disconnected from the biocatalytic reaction and do not provide reduced cofactors. 2-Heptanone is metabolised by two pathways. The first leads to 2-heptanol (molar yield close to 55%, enantioselectivity higher than 99%, with a slight decrease at the end of the process) and the second corresponds to material incorporation into the biomass. This latter phenomenon is assumed to provide the biocatalyst with the reduced cofactors needed for the reduction process. Overall, the process yielded ca. 1.4 g/l 2-heptanol in 50 h reaction, which is close to that observed with fresh cells previously grown for 15 h.

Influence of the substrate on the ultrastructure of Pleurotus pulmonarius fruit body primordia by C. Sánchez (pp. 691-694).

Pleurotus pulmonarius fruit body primordia (FBP) formed on potato extract agar (PEA) and wheat straw-based media (WS) were studied. When grown on PEA, FBP hyphae had a large number of vacuoles and less stainable cytoplasmic material. In contrast, when grown on WS, there was abundant cytoplasmic material and fewer vacuoles. FBP grown on WS had a more well-defined mushroom shape than those grown on PEA. The average hyphal diameters on PEA and WS were 4.15 and 3.52 µm, respectively; the average hyphal lengths were 11.75 and 11.80 µm, respectively. The control mechanisms regulating formation of the normal shape and size of FBP might be more dependent on the amount of cytoplasmic material than on the physical volume of the hyphal compartment. Since the ultrastructure of the hyphal compartment, which makes up the substance of the FBP, depends on the substrate in which the FBP has been grown, the composition of the substrate may be important for manipulating the nutritive and organoleptic characteristics of the mushroom crop.

Behaviour of Mycobacterium sp. NRRL B-3805 whole cells in aqueous, organic-aqueous and organic media studied by fluorescence microscopy by C. C. C. R. de Carvalho; A. Cruz; B. Angelova; P. Fernandes; M. N. Pons; H. M. Pinheiro; J. M. S. Cabral; M. M. R. da Fonseca (pp. 695-701).

The present work aimed at quantifying the viability and morphological changes occurring during the time course of the side-chain cleavage of β-sitosterol, in aqueous, two-phase organic-aqueous and organic media by free resting cells of Mycobacterium sp. NRRL B-3805. The solvent used was bis(2-ethylhexyl) phthalate (BEHP). A 66.3% reduction in cell viability was observed after 24 h when the cells were incubated in phosphate buffer only, but the percentage of viable cells was constant thereafter. In biphasic systems with BEHP, cell viability was maintained at higher values in the first 48 h, during which complete degradation of substrate was achieved. The availability of oxygen, which should be higher in the biphasic system than in the aqueous system, and of a carbon and energy source, thus seem important for the cells to retain their viability. In biphasic systems, cells tended to shrink and decrease their surface roughness, i.e. to decrease their surface area, possibly as a way to protect themselves from mechanical stress due to the presence of organic-aqueous interfacial forces, which resulted in disaggregation of cell clusters. A method used to visualise BEHP droplets with a standard optical microscope showed that the cells adhered to the surface of the solvent droplets, but no cells were observed inside these. In pure BEHP medium, cells retained their viability level for at least 150 h, independently of a pre-incubation period, which did not seem to induce any adaptation effect. Solvent biocompatibility, higher oxygen availability and reduced interfacial stress could have contributed to this maintenance of viability.

Quinone-respiration improves dechlorination of carbon tetrachloride by anaerobic sludge by F. J. Cervantes; L. Vu-Thi-Thu; G. Lettinga; J. A. Field (pp. 702-711).

The impact of humic acids and the humic model compound, anthraquinone-2,6-disulfonate (AQDS), on the biodegradation of carbon tetrachloride (CT) by anaerobic granular sludge was studied. Addition of both humic acids and AQDS at sub-stoichiometric levels increased the first-order rate of conversion of CT up to 6-fold, leading to an increased production of inorganic chloride, which accounted for 40–50% of the CT initially added. Considerably less dechlorination occurred in sludge incubations lacking humic substances. By comparison, very limited dechlorination occurred in sterile controls with autoclaved sludge. Accumulation of chloroform (1–10%) and dichloromethane (traces) also accounted for the CT converted. The accumulation of a chlorinated ethene, perchloroethylene (up to 9% of added CT), is also reported for the first time as an end-product of CT degradation. A humus-respiring enrichment culture (composed primarily of a Geobacter sp.) derived from the granular sludge also dechlorinated CT, yielding products similar to the AQDS-supplemented granular sludge consortium. The dechlorination of CT by the Geobacter enrichment was dependent on the presence of AQDS or humic acids, which were reduced during the assays. The reduced form of AQDS, anthrahydroquinone-2,6-disulfonate, was shown to cause the chemical reduction of CT when incubated in sterile medium. The results taken as a whole indicate that the formation of reduced humic substances by quinone-respiring microorganisms can contribute to the reductive dechlorination of CT.

Biodegradation of alachlor by soil streptomycetes by L. Durães Sette; L. A. Mendonça Alves da Costa; A. J. Marsaioli; G. P. Manfio (pp. 712-717).

Streptomycetes resistant to the herbicide alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl) acetanilide] were used in degradation assays to characterize the products of alachlor biodegradation. Of six strains tested, Streptomyces sp. LS166, LS177, and LS182 were able to grow at an alachlor concentration of 144 mg l⁻¹ and degraded approximately 60–75% of the alachlor in 14 days, as evaluated by high performance liquid chromatography. The alachlor biodegradation products were identified by gas chromatography-mass spectrometry based on mass spectral data and fragmentation patterns. All compounds detected in these assays were similar for all streptomycetes strains tested, and involved dechlorination with subsequent N-dealkylation and cyclization of the remaining N-substituent with one of the ethyl groups to produce indole and quinoline derivatives. The enzymatic pathway used by Streptomyces sp. LS182 did not generate DEA (2′,6′-diethylaniline), a carcinogenic derivative of alachlor reported in other studies. Given the high degradation rates observed here, the Streptomyces strains tested may be useful in the degradation/detoxification processes of alachlor.

Membrane-aerated biofilm reactor for the removal of 1,2-dichloroethane by Pseudomonas sp. strain DCA1 by J. C. Hage; R. T. van Houten; J. Tramper; S. Hartmans (pp. 718-725).

A membrane-aerated biofilm reactor (MBR) with a biofilm of Pseudomonas sp. strain DCA1 was studied for the removal of 1,2-dichloroethane (DCA) from water. A hydrophobic membrane was used to create a barrier between the liquid and the gas phase. Inoculation of the MBR with cells of strain DCA1 grown in a continuous culture resulted in the formation of a stable and active DCA-degrading biofilm on the membrane. The maximum removal rate of the MBR was reached at a DCA concentration of approximately 80 µM. Simulation of the DCA fluxes into the biofilm showed that the MBR performance at lower concentrations was limited by the DCA diffusion rate rather than by kinetic constraints of strain DCA1. Aerobic biodegradation of DCA present in anoxic water could be achieved by supplying oxygen solely from the gas phase to the biofilm grown on the liquid side of the membrane. As a result, direct aeration of the water, which leads to undesired coagulation of iron oxides, could be avoided.

High-diversity biofilm for the oxidation of sulfide-containing effluents by I. Ferrera; R. Massana; E. O. Casamayor; V. Balagué; O. Sánchez; C. Pedrós-Alió; J. Mas (pp. 726-734).

In the present work, we describe for the first time the utilization of a complex microbial biofilm for the treatment of sulfide-containing effluents. A non-aerated packed-column reactor was inoculated with anoxic lake sediment and exposed to light. A biofilm developed in the column and showed a stable oxidation performance for several weeks. Microbial species composition was analyzed by microscopy, pigment analysis and a bacterial 16S rRNA gene clone library. Colorless sulfur bacteria, green algae and purple sulfur bacteria were observed microscopically. Pigment composition confirmed the presence of algae and purple sulfur bacteria. The clone library was dominated by alpha-Proteobacteria (mostly Rhodobacter group), followed by gamma-Proteobacteria (Chromatiaceae-like and Thiothrix-like aerobic sulfur oxidizers) and the Cytophaga-Flavobacterium-Bacteroides group. Plastid signatures from algae were also present and a few clones belonged to both the beta- (Rhodoferax sp., Thiobacillus sp.) and delta-Proteobacteria (Desulfocapsa sp.) and to the low G+C Gram-positive bacteria (Firmicutes group). The coexistence of aerobic, anaerobic, phototrophic and chemotrophic microorganisms in the biofilm, the species richness found within these metabolic groups (42 operational taxonomic units) and the microdiversity observed within some species could be very important for the long-term functioning and versatility of the reactor.

Chemical and toxic evaluation of a biological treatment for olive-oil mill wastewater using commercial microbial formulations by M. Isidori; M. Lavorgna; A. Nardelli; A. Parrella (pp. 735-739).

Olive-oil-mill wastewater (OMW) has significant polluting properties due to its high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and phenols. In the present study, different commercial bacterial formulations were used in the biological treatment of OMW. COD and toxicity testing using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna) were employed to evaluate abatement of the organic load and reduction of the toxic potential. In addition, the four most active formulations were tested mixed pair-wise on the basis of their unique characteristics in order to evaluate the improvement of treatment. The effect of treatment was assessed by measuring COD removal, reduction of total phenols, and decreased toxicity. The results obtained with the mixed formulations showed that the maximum removal of the organic load was about 85%, whereas phenols were reduced by about 67%. The toxicity for rotifers decreased by 43% and for crustaceans by about 83%.

Comparative analysis of genetic diversity and expression of amoA in wastewater treatment processes by Y. Ebie; N. Noda; H. Miura; M. Matsumura; S. Tsuneda; A. Hirata; Y. Inamori (pp. 740-744).

The genetic diversity and expression of amoA of autotrophic ammonia oxidizers in wastewater treatment processes were investigated by RT-PCR and denaturing gradient gel electrophoresis (DGGE) in order to identify active components of ammonia-oxidizer populations in a such processes. Ammonia oxidizers, evidenced by the presence of amoA mRNA, were regarded as metabolically active. The DGGE profiles derived from amoA mRNA and from its gene, which were amplified by RT-PCR or PCR using samples collected from a bench-scale reactor treating high concentration of inorganic ammonia, were similar. In contrast, RNA and DNA-derived DGGE profiles from three domestic wastewater treatment facilities were different from each other. These data indicate that the dominant ammonia oxidizers in the bench-scale reactor exhibited ammonia-oxidizing activity, whereas some ammonia oxidizers in the domestic wastewater treatment facilities apparently did not express high levels of amoA mRNA.

Sections

Personal tools

Applied Microbiology and Biotechnology (v.64, #5)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.64, #5)