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Applied Microbiology and Biotechnology (v.64, #2)
Principles of biorefineries by B. Kamm; M. Kamm (pp. 137-145).
Sustainable economic growth requires safe, sustainable resources for industrial production. For the future re-arrangement of a substantial economy to biological raw materials, completely new approaches in research and development, production and economy are necessary. Biorefineries combine the necessary technologies between biological raw materials and industrial intermediates and final products. The principal goal in the development of biorefineries is defined by the following: (biomass) feedstock-mix + process-mix → product-mix. Here, particularly the combination between biotechnological and chemical conversion of substances will play an important role. Currently the “whole-crop biorefinery”, “green biorefinery” and “lignocellulose-feedstock biorefinery” systems are favored in research and development.
Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid by L. Sijtsma; M. E. de Swaaf (pp. 146-153).
Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called ω-3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.
Divergence of mobile genetic elements involved in the distribution of xenobiotic-catabolic capacity by H. Nojiri; M. Shintani; T. Omori (pp. 154-174).
Bacteria adapt rapidly to environmental stimuli, such as exposure to xenobiotics. Mobile genetic elements (MGEs) play a major role in such bacterial adaptation, via the dispersal of catabolic capacity; and, in fact, genes that encode the degradation enzymes for xenobiotics are often located on MGEs. The list of reported catabolic MGEs keeps growing as researchers continue to isolate and characterize xenobiotic degraders and the corresponding catabolic genes. Major catabolic MGEs include (conjugative) plasmids, transposons, and conjugative transposons. Catabolic transposons can be divided into class I elements (composite transposons) and class II elements (Tn3 family transposons). This review includes a comprehensive list of naturally occurring discrete catabolic MGEs, together with a brief description for each. While MGEs are often rather large, genome-wide or large-scale sequence analyses have provided useful information on the whole genetic structure of MGEs, with clues to their function (transfer, maintenance, catabolism, etc.) and behavior in a complex natural environment. This review also gives an insight into MGE functions, based on the complete sequencing of several catabolic plasmids and two Pseudomonas genomes.
Biotechnological advantages of laboratory-scale solid-state fermentation with fungi by U. Hölker; M. Höfer; J. Lenz (pp. 175-186).
Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O2, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.
Artificial carrier for oxygen supply in biological systems by E. S. Dey; O. Norrlöw; Y. Liu (pp. 187-191).
Several poly (dimethylsiloxanes) (PDMS) copolymers of dimethylsiloxane (DMS) with ethylene or propylene oxide were tested as artificial carriers for the delivery of oxygen to biological systems. Copolymers with a DMS content of 33% or lower enhanced glucose oxidation by 200% in contrast to the 25% increase produced by the same concentration of perfluorodecalin. When 0.05% of the copolymer with 18% DMS was included in the growth media of Bacillus thuriginensis, the biomass (growth rate) increased 1.5-fold. With 0.1% of this copolymer, actinorhodin production by Streptomyces coelicolor A3 (2) occurred in half the normal time and with an increased yield. In conclusion, these PDMS copolymers are a good alternative to perfluorodecalin as oxygen carriers in biotechnological processes.
Homogeneous batch cultures of Aspergillus oryzae by elimination of wall growth in the Variomixing bioreactor by B. Larsen; B. Rask Poulsen; N. T. Eriksen; J. J. Lønsmann Iversen (pp. 192-198).
A novel principle for mixing and aeration in stirred bioreactors, named Variomixing, was developed. Four baffles are rotated intermittently at a rotational speed slower or similar to the speed of a centrally placed axial flow impeller. Rotational speeds of the baffles and impeller of 5–10 and 500–600 rpm, respectively, results in the highly turbulent flow regime characteristic of conventional bioreactors with high mixing and mass transfer capacities. Stagnant zones around crevices and crannies in which wall growth may commence are avoided since the baffles are never completely at rest. Increasing the rotational speed of the baffles (5 s every 5 min), so that it follows the speed of the impeller (500–600 rpm), cancels the effect of the baffles and a deep vortex and high peripheral liquid flow rates at the reactor wall develop. The vortex ensures that also the head-space of the reactor wall is flushed and any deposits removed. The filamentous fungus Aspergillus oryzae has been grown in batch cultures in the Variomixing bioreactor. Compared to conventional laboratory-scale bioreactors, in which more than 30% of all biomass was found attached to walls, less than 2% of the total A. oryzae biomass was found on the walls in the Variomixing bioreactor.
Utilization of the buffering capacity of corn steep liquor in bacterial cellulose production by Acetobacter xylinum by N. Noro; Y. Sugano; M. Shoda (pp. 199-205).
Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC). Using a pH sensor for the accurate control of pH, which is one of the most critical factors for efficient BC production, is difficult especially in a baffled shake-flask and an airlift reactor. The buffering capacity of corn steep liquor (CSL) was estimated by measuring β (buffering capacity) values in advance and was used to maintain the pH within the optimal range during the production of BC. When CSL was added to either a shake-flask, a stirred-tank reactor or an airlift reactor, BC production was almost the same as that in cultivations where pH was controlled manually or by a pH sensor.
Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis by F. Aulenta; S. Rossetti; M. Majone; V. Tandoi (pp. 206-212).
The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 μmol PCE [g volatile suspended solids (VSS)]−1 day−1. Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 ± 0.08 mmol Cl− l−1 day−1. From the specific activity of D. ethenogenes [i.e. 0.055 ± 0.008 mmol Cl− (mg VSS)−1 day−1], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0±10.4 mg VSS·l−1 (41.5±11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.
Characterisation of cellobiose dehydrogenases from the white-rot fungi Trametes pubescens and Trametes villosa by R. Ludwig; A. Salamon; J. Varga; M. Zámocky; C. K. Peterbauer; K. D. Kulbe; D. Haltrich (pp. 213-222).
Cellobiose dehydrogenase (CDH) is an extracellular haemoflavoenzyme that is produced by a number of wood-degrading and phytopathogenic fungi and it has a proposed role in the early events of lignocellulose degradation and wood colonisation. In the presence of a suitable electron acceptor, e.g. 2,6-dichloro-indophenol, cytochrome c, or metal ions, CDH oxidises cellobiose to cellobionolactone. When screening 11 different Trametes spp. for the formation of CDH activity, all the strains investigated were found to secrete significant amounts of CDH when cultivated on a cellulose-containing medium. Amongst others, Trametes pubescens and Trametes villosa were identified as excellent, not-yet-described, producer strains of this enzyme activity that has various potential applications in biotechnology. CDH from both strains was purified to apparent homogeneity and subsequently characterised. Both monomeric enzymes have a molecular mass of approximately 90 kDa (gel filtration) and a pI value of 4.2–4.4. The best substrates are cellobiose and cellooligosaccharides; additionally, lactose, thiocellobiose, and xylobiose are efficiently oxidised. Glucose and maltose are poor substrates. The preferred substrate is cellobiose with a K m value of 0.21 mM and a k cat value of 22 s−1 for CDH from T. pubescens; the corresponding values for the T. villosa enzyme are 0.21 mM and 24 s−1, respectively. Both enzymes showed very high activity with one-electron acceptors such as ferricenium, ferricyanide, or the azino-bis-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical.
An enzymatic assay for metabolites of perfluoro-tagged 5-hydroxytryptophan by T. Snyder-Leiby; S. Dingman; R. Thomas; C. Guo (pp. 223-227).
l-5-hydroxytryptophan (5-HTP) with two types of multiple 19F-atom tags bonded at various positions onto the indole ring (positions 4, 6, or 7) was exposed to aromatic l-amino acid decarboxylase (AADC) in lysates of Escherichia coli JM109 which had been transformed with the plasmid pKKAADCII. Resulting samples were analyzed with HPLC. In the first study, which investigated a straight-chain seven-atom tag, a novel peak, putatively perfluoro-tagged serotonin, was detected. A second study demonstrated that 5-HTP was converted to 5-HT in transformed E.coli lysate but not in untransformed lysate. A third study, investigating a tag with nine fluorine atoms all in the same nuclear environment, identified the isomer serving as the best substrate for AADC. This novel molecule had the tag bonded at the 6 position on the indole ring. Isomers that fit into the active site of AADC are likely to follow the biosynthetic path for serotonin in vivo and are potentially useful in 19F magnetic resonance spectroscopy studies. The enzymatic assay described here provides an efficient and cost-effective tool for screening new compounds.
Characterization of the ask–asd operon in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331 by Y. Cuadrado; M. Fernández; E. Recio; J. F. Aparicio; J. F. Martín (pp. 228-236).
The first two genes of the threonine pathway, ask and asd, were cloned and sequenced from the aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331. The two genes are organized in a bicistronic operon. ask, encoding the apartokinase (ASK), is located upstream from asd. The presence of a ribosome-binding site within the ask sequence suggests that this open reading frame encodes two overlapping proteins. The formation of both subunits of the aspartokinase from a single gene was studied using antibodies raised against the C-terminal end of the aspartokinase subunits. Disruption of asd results in a significant decrease of aminoethoxyvinylglycine production, thus supporting the involvement of the ask–asd operon in the biosynthesis of this metabolite. This is the first report in which a gene cluster for the first two steps of aminoethoxyvinylglycine biosynthesis is characterized.
Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity by C. D. Skory (pp. 237-242).
Rhizopus oryzae is capable of producing high levels of lactic acid by the fermentation of glucose. Yields typically vary over 60–80%, with the remaining glucose diverted primarily into ethanol fermentation. The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate. Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment, were transformed into R. oryzae. This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in a high-copy number. Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection. Isolates transformed with ldhA containing plasmid were compared with both the wild-type parent strain and the auxotrophic recipient strain containing vector only. All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls. Integration of plasmid pLdhA48XI increased the stability of the strain, but did not seem to offer any benefit for increasing lactic acid production. Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid. Plasmid pLdhA71X, containing a large ldhA fragment (6.1 kb), routinely yielded higher levels of lactic acid than the smaller region (3.3 kb) used to construct plasmid pLdhA48XI. The greatest levels of ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII. However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared with the control.
Identification and functional characterization of a type I signal peptidase gene of Bacillus megaterium DSM319 by H. Nahrstedt; K.- D. Wittchen; M. A. Rachman; F. Meinhardt (pp. 243-249).
The sipM gene of Bacillus megaterium encoding a type I signal peptidase (SPase) was isolated and structurally characterized. RNA analysis revealed a transcript size in accordance with a bicistronic operon comprising sipM and an adjacent open reading frame. Inactivation of sipM by targeted gene disruption could not be achieved, indicating its essential role for cell viability since there might be no other type I SPase of major importance present in B. megaterium. Plasmid-assisted amplification of the gene resulted in an increase in activity of the heterologous glucanase used as an extracellular reporter, suggesting a potential bottleneck for protein secretion within this species.
Isolation of phages infecting Actinoplanes SN223 and characterization of two of these viruses by M. Jarling; K. Bartkowiak; H. Robenek; H. Pape; F. Meinhardt (pp. 250-254).
Phages infecting the industrially important Actinoplanes strain SN223 were isolated from soil samples collected at the shores of inland waters in Germany. The genome sizes range from 53 kb to 58 kb. Preliminary analyses revealed G+C contents comparable with the G/C bias of the host. Electron microscopy of three selected viruses displayed no obvious morphological differences, the phage heads being icosahedral and their tails non-contractible. Two of the phages (φAsp2, φAsp3.1) characterized in more detail are capable of provoking putative pseudolysogenic growth of the host bacterium. The carrier state for φAsp2, in which cells are tightly packed with viruses, was demonstrated by electron microscopy. The latter phage is apparently widely distributed, as it was isolated from regions which are distantly located, i.e. more than 600 km apart from each other.
SulA-independent filamentation of Escherichia coli during growth after release from high hydrostatic pressure treatment by T. Kawarai; M. Wachi; H. Ogino; S. Furukawa; K. Suzuki; H. Ogihara; M. Yamasaki (pp. 255-262).
To improve the efficiency of sterilization by high hydrostatic pressure treatment (HPT), it is desirable to know the biochemical process of bacteria most sensitive to the treatment. We investigated growth properties after release from HPT of exponentially growing Escherichia coli K-12 cells. We observed growth retardation after treatment (30 min at 37°C) above 75 MPa. Long filamentous cells of about eight times normal cell length were observed at 90 min growth after treatment at 75 MPa. In the subsequent period the filamentous cells divided into normal-sized cells. recA and sulA mutant strains also formed filamentous cells, indicating that filamentation was SulA-independent. Nucleoids segregated normally in the filamentous cells. Only one FtsZ ring (or none) was detected at possible division sites in the elongated cells. Western blotting analysis demonstrated that the amount of FtsZ protein was not affected by the treatment. GTP-dependent in vitro polymerization of either FtsZ protein in E. coli crude extract or purified FtsZ protein, however, was sensitive to HPT. These facts suggest that HPT at 75 MPa denatures a fraction of FtsZ molecules, and that these denatured molecules interfere with the polymerization of functional FtsZ, resulting in the significantly reduced number of FtsZ rings.
Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca by D. Romero; A. Pérez-García; M. E. Rivera; F. M. Cazorla; A. de Vicente (pp. 263-269).
Powdery mildew is one of the most important limiting factors for cucurbits production in Spain, its management being strongly dependent on chemicals. The aim of this work was to evaluate the possibility of exploiting antagonistic bacteria in the biological control of the cucurbit powdery mildew fungus Podosphaera fusca (syn. Sphaerotheca fusca). Among a collection of bacterial strains isolated from distinct cucurbit powdery mildew diseased plants and rhizospheric soils, four isolates were selected, by means of a screening method based on antibiotic production, and identified as Bacillus spp. These isolates proved to be efficacious in the control of cucurbit powdery mildew in in vitro detached leaves and seedling biocontrol assays, where reductions of disease severity of up to 80% were obtained. Furthermore, bacterial populations on melon leaves remained at similar levels (105 cfu cm−2) over the 16-day period studied and, as observed by scanning electron microscopy analysis, they were able to establish microcolonies associated with an extracellular matrix, which reveals that these isolates efficiently colonize melon phylloplane. These results indicate that the bacterial isolates selected are promising candidates for biological control agents of cucurbit powdery mildew in southern Spain.
Toward rational control of Escherichia coli O157:H7 by a phage cocktail by Y. Tanji; T. Shimada; M. Yoichi; K. Miyanaga; K. Hori; H. Unno (pp. 270-274).
Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.
Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water by R. Zuo; D. Örnek; B. C. Syrett; R. M. Green; C.-H. Hsu; F. B. Mansfeld; T. K. Wood (pp. 275-283).
Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50–90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.
Bioaugmentation of the phyllosphere for the removal of toluene from indoor air by L. De Kempeneer; B. Sercu; W. Vanbrabant; H. Van Langenhove; W. Verstraete (pp. 284-288).
The removal of airborne toluene by means of the phyllosphere of Azalea indica augmented with a toluene-degrading enrichment culture of Pseudomonas putida TVA8 was studied. The 95% disappearance time [DT95%; the time in which an initial toluene concentration of 90 ppmv (339 mg.m3) was removed in a batch experiment] was 75 h for Azalea plants. Under the same experimental conditions, DT95% of inoculated Azalea plants decreased remarkably to about 27 h. Subsequent additions of toluene further increased the removal efficiency of the bioaugmented system (DT95% decreased by a factor of four). A decrease in DT95% was also recorded after repeated incubations of non-inoculated plants, but the toluene-removal rate was remarkably low, compared with the inoculated plants. Hence, inoculation of the leaf surface appeared essential for obtaining rapid removal rates. It was not possible to obtain comparable and sustained removal of airborne toluene by inoculating artificial plant surfaces. This is, to our knowledge, the first report on bioaugmentation of the leaf surface of plants to remove gaseous pollutants from air. The results presented are promising and could be of great practical importance in the field of indoor air pollution control.