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Applied Microbiology and Biotechnology (v.52, #6)

Utilisation of biomass for the supply of energy carriers by P. A. M. Claassen; J. B. van Lier; A. M. Lopez Contreras; E. W. J. van Niel; L. Sijtsma; A. J. M. Stams; S. S. de Vries; R. A. Weusthuis (pp. 741-755).

Because biomass is a widely available, renewable resource, its utilisation for the production of energy has great potential for reducing CO₂ emissions and thereby preventing global warming. In this mini-review the `state of the art' of several fermentation processes is discussed, starting with the most advanced process of ethanol production. This is followed by methane production, an established process for waste water purification which is gaining more attention because of the inherent energy production. Subsequently ABE fermentation is discussed and finally the biological production of hydrogen. The last section proposes a new way to assess and compare the different processes by relating their merit to `work content' values and `lost work' instead of the combustion values of their products. It is argued that, especially when dealing with energy from biomass, the application of this methodology will provide a uniform valuation for different processes and products. The described fermentation processes enable the supply of pure energy carriers, either gaseous or liquid, from biomass, yet the introduction of these processes is hampered by two major problems. The first is related to technological shortcomings in the mobilisation of fermentable components from the biomass. The second, having a much greater impact, is linked with socio-economics: until full externality costs are attributed to fossil fuels, accounting for their role in pollution and global warming, the competitiveness of the processes described here will hardly stand a chance.

Fungal transposons: from mobile elements towards molecular tools by F. Kempken (pp. 756-760).

Over the last few years an increasing number of transposons has been identified and characterized in filamentous fungi. This includes members of all known eukaryotic classes of transposable elements. Most interestingly, transposons have also been identified in fungi that are used in biotechnology which could provide new tools for strain improvement or gene tagging. Transposons have already been used for diagnostic and population analysis of fungal strains. The first attempts towards transposon-aided gene tagging have been promising and future efforts will almost certainly add a new and powerful method for gene identification in filamentous fungi.

DNA hydrolysis by inorganic catalysts by R. Ott; R. Krämer (pp. 761-767).

Non-enzymatic reagents that efficiently promote the hydrolytic cleavage of DNA currently receive much attention since they have many potential applications in molecular biology. This review focuses on recent progress in the hydrolysis of the phosphodiester backbone of DNA by metal ions and metal complexes. Pioneering work on the sequence-selective DNA scission by an artificial restriction enzyme, which is prepared by covalent attachment of a cerium(IV) complex to an antisense-deoxyoligonucleotide, is discussed.

Solid-state fermentation for xylanase production by Thermoascus aurantiacus using response surface methodology by M. C. de O. Souza; I. C. Roberto; A. M. F. Milagres (pp. 768-772).

We investigated xylanase production by Thermoascus aurantiacus using semisolid fermentation. Multivariant statistical approaches were employed to evaluate the effects of several variables (initial moisture in the medium, cultivation time, inoculum level, and bagasse mass) on xylanase production. The initial moisture content and bagasse mass were the most important factors affecting xylanase activity. The xylanase activity produced by the fungus under the optimized conditions (81% moisture content and 17 g bagasse) was found to be 2700 U per gram of initial dry matter, whereas its value predicted by a polynomial model was 2400 U per gram of initial dry matter.

Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions by W. Sabra; A. -P. Zeng; S. Sabry; S. Omar; W. -D. Deckwer (pp. 773-780).

Alginate production by Azotobacter vinelandii was studied in batch and continuous cultures under microaerobic conditions. In batch culture at a pO₂ of 2–3% (air saturation) alginate production was enhanced by decreasing the PO³⁻ ₄ level in the medium. Alginate yield from biomass (Y _P/X) reached the highest value of 0.66 g/g at the lowest phosphate level (100 mg/l), compared to 0.40 g/g and 0.25 g/g at higher phosphate levels (200 mg/l and 400 mg/l, respectively). In contrast, biomass formation behaved differently and the growth yield (Y _X/S) decreased with decreasing PO₄ ³⁻ concentrations. Moreover, the respiratory quotient (RQ) of the culture was dependent on the initial phosphate concentration, especially in the phosphate-limited phase of growth. As the initial phosphate level decreased from 400 mg/l to 100 mg/l, the average RQ value of the culture declined from 1.46 to 0.89. The low RQ value is very close to the theoretical optimum RQ, calculated to be 0.8 on the basis of the stoichiometry of the metabolic pathways for alginate formation from sucrose. This optimum RQ was also confirmed in continuous culture at different dilution rates. Independent of the dilution rate, a pO₂ value of 2–5% (air saturation) was found to be optimal for alginate production, the corresponding RQ values being 0.80–0.84. In addition, the molecular mass and composition of alginate were also found to be affected by both phosphate and oxygen concentrations. In conclusion, the RQ appears to be a useful parameter for optimum control of alginate production with this microorganism.

Outdoor culture of a cyanobacterium with a vertical flat-plate photobioreactor: effects on productivity of the reactor orientation, distance setting between the plates, and culture temperature by K. Zhang; N. Kurano; S. Miyachi (pp. 781-786).

The ability of a photobioreactor to fix CO₂ was evaluated with the thermophilic cyanobacterium, Synechocystis aquatilis SI-2. The reactor consisted of three to five flat plates of transparent acrylic plastic standing upright and in parallel and giving a 0.015-m light path. The reactor was 0.8 m high and 1 m long with 9 l working volume. The effects of the orientation of the vertical bioreactor, distance between the plates, and culture temperature on the productivity of biomass were investigated during the summer of 1998 in Kamaishi (39°N, 142°E), Japan. When the illuminated surface reactor was placed in an east–west-facing orientation, the biomass productivity was roughly 1.4-fold higher than that obtained in a north–south-facing orientation, because the former received more solar energy than the latter. The productivity based on the overall land area was the same for plates set either 0.25 m or 0.5 m apart. However, the volumetric productivity of the reactor in which the plates were set 0.25 m apart was lower than that when the plates were set 0.5 m apart, since the former plates received relatively lower solar irradiation because of severe mutual shading. When the culture temperature was maintained in its optimal range (37–43 °C), the productivity was 50% greater than that obtained in a culture at ambient temperature (20–44 °C). The biomass productivity and CO₂ fixation rate were investigated under various experimental conditions. The maximum rate of 53 g CO₂ m⁻² day⁻¹ was achieved in the temperature-regulated culture with the reactor set in an east–west-facing orientation, the distance between plates being 0.25 m.

Axenic aerobic biofilms inhibit corrosion of copper and aluminum by A. Jayaraman; D. Ornek; D. A. Duarte; C. -C. Lee; F. B. Mansfeld; T. K. Wood (pp. 787-790).

The corrosion behavior of unalloyed copper and aluminum alloy 2024 in modified Baar's medium has been studied with continuous reactors using electrochemical impedance spectroscopy. An axenic aerobic biofilm of either Pseudomonas fragi K or Bacillus brevis 18 was able to lessen corrosion as evidenced by a consistent 20-fold increase in the low-frequency impedance value of copper as well as by a consistent four- to seven-fold increase in the polarization resistance of aluminum 2024 after six days exposure compared to sterile controls. This is the first report of axenic aerobic biofilms inhibiting generalized corrosion of copper and aluminum. Addition of the representative sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris (to simulate consortia corrosion behavior) to either the P. fragi K or B. brevis 18 protective biofilm on copper increased the corrosion to that of the sterile control unless antibiotic (ampicillin) was added to inhibit the growth of SRB in the biofilm.

Activity and stability of chemically modified Candida antarctica lipase B adsorbed on solid supports by B. C. Koops; E. Papadimou; H. M. Verheij; A. J. Slotboom; M. R. Egmond (pp. 791-796).

The effect of various covalent chemical modifications on the transesterification activity and stability of adsorbed lipase B from Candida antarctica (CALB) was studied in 2-butanone and o-xylene. CALB species modified with either polyethylene glycol 2000 monomethyl ether (MPEG), polyethylene glycol 300 mono-octyl ether (OPEG) or n-octanol (OCT) were used in combination with a hydrophobic (Accurel) and a hydrophilic (Duolite) support. The thermostabilities of adsorbed CALB in both solvents, and that of free CALB in o-xylene were not influenced by the modifications. In contrast, the thermostability of free CALB in 2-butanone decreased 2.5-fold after MPEG modification and increased 1.5-fold after modification with OPEG and n-octanol, compared to that of native CALB. The activities of the native and modified CALB species were up to 9-fold higher after adsorption onto Accurel than those of the corresponding free enzymes. Adsorption of these enzyme species onto Duolite only resulted in a 2- to 3-fold increase in the activity of OPEG- and OCT-modified CALB. The modified CALB species adsorbed onto Accurel show similar or up to 2-fold lower activities than do native adsorbed CALB species, while 1.5- to 6-fold higher activities were found for modified CALB species adsorbed onto Duolite. We propose that hydrophobic modifiers induce conformational changes of CALB during adsorption on a hydrophobic support whereas all three modifiers protect CALB from structural alterations during adsorption onto a hydrophilic support.

β-Carbon stereoselectivity of N-carbamoyl-d-α-amino acid amidohydrolase for α,β-diastereomeric amino acids by J. Ogawa; A. Ryono; S. -X. Xie; R. M. Vohra; R. Indrati; H. Miyakawa; T. Ueno; Y. Ikenaka; H. Nanba; S. Takahashi; S. Shimizu (pp. 797-801).

N-Carbamoyl-d-α-amino acid amidohydrolase (d-carbamoylase) was found to distinguish stereochemistry not only at the α-carbon but also at the β-carbon of N-carbamoyl-d-α-amino acids. The enzyme selectively acted on one of the four stereoisomers of N-carbamoyl-α,β-diastereomeric amino acids. This simultaneous recognition of two chiral centers by d-carbamoylase was useful for the fine stereoselective synthesis of α,β-diastereomeric amino acids such as threonine, isoleucine, 3,4-methylenedioxyphenylserine and β-methylphenylalanine. The stereoselectivity for the β-carbon was influenced by the pH of the reaction mixture and by the bulk of the substituent at the β-carbon.

Purification and characterization of β-amylase from Curculigo pilosa by M. H. Dicko; M. J. F. Searle-van Leeuwen; G. Beldman; O. G. Ouedraogo; R. Hilhorst; A. S. Traoré (pp. 802-805).

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes.

Metal-induced expressing of mammal Metallothionein-1 gene in cyanobacteria to promote cadmium-binding preferences by X. -X. Guo; D. -J. Shi; X. -D. Xu; Y. -X. Ouyang; B. -G. Ru (pp. 806-810).

The metal(zinc)-inducible smtA gene promoter (smt O-P) from cyanobacteria was applied for the expression of mouse MT-1 cDNA in the filamentous cyanobacterium Anabaena sp. PCC 7120 to enhance its metal-binding capability and to change its main binding specificity from zinc to cadmium. Shuttle expression vector pKT-MRE transformed the cyanobacterial cells by triparent conjugal transfer. Positive clones were screened and identified by streptomycin, DNA dot blot, SDS-PAGE and Western blot analysis. Photosynthetic oxygen evolution and metal atom absorption indicated that under the cadmium stress the metal-induced expression of foreign mMT-1 doubled their cadmium resistance and developed cells showing a much higher preference to absorb cadmium other than zinc in medium. The cadmium content in cell extract rose from 11% to 36%, and the cadmium cleared from media by transgenic cells rose from 18% to 62%. There was only a slight enhancement for zinc binding in the wild or transgenic type.

Characterization of IS1676 from Rhodococcus erythropolis SQ1 by P. A. Lessard; X. M. O'Brien; N. A. Ahlgren; S. A. Ribich; A. J. Sinskey (pp. 811-819).

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5′-(C/T)TA(A/G)-3′ in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacteriumkansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available.

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene by J. Overhage; H. Priefert; J. Rabenhorst; A. Steinbüchel (pp. 820-828).

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD⁺-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (ΩKm) or gentamycin (ΩGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3′-O-phosphotransferase gene and the gentamycin- 3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by ΩKm or ΩGm, and the functional vdh gene was replaced by vdhΩKm or vdhΩGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhΩKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources by W. H. van Zyl; A. Eliasson; T. Hobley; B. Hahn-Hägerdal (pp. 829-833).

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l⁻¹ d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l⁻¹ d-glucose or raffinose was used as co-substrate together with 50 g l⁻¹ d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation.

Reduction of aryl acids by white-rot fungi for the biocatalytic production of aryl aldehydes and alcohols by A. Hage; H. E. Schoemaker; J. A. Field (pp. 834-838).

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols.

The effects of exopolymers on cell morphology and culturability of Leuconostoc mesenteroides during starvation by D. -S. Kim; H. S. Fogler (pp. 839-844).

Biofilm formation by bacterial cells can be used to modify the subsurface permeability for the purpose of microbial enhanced oil recovery, bio-barrier formation, and in situ bioremediation. Once injected into the subsurface, the bacteria undergo starvation due to a decrease in nutrient supply and diffusion limitations in biofilms. To help understand the starvation response of bacteria in biofilms, the relationship between exopolymer formation and cell culturability was examined in a batch culture. The average cell diameter was observed to decrease from 0.8 μm to 0.35 μm 3 days after starvation began. Cell chain fragmentation was also observed during starvation. Cells that underwent starvation in the presence of insoluble exopolymers showed a slower rate of decrease in cell diameter and in cell chain length than cells without insoluble exopolymers. The rate of decrease in the average cell diameter and cell chain length were determined using a first order decay model. Cells starved in the presence of exopolymers showed greater culturability than cells starved without exopolymers. After 200 days starvation, 2.5 × 10⁻³% cells were culturable, but no increase in cell number was observed. During starvation, the exopolymer concentration remained constant, an indication that the exopolymer was not consumed by the starving bacteria as an alternative carbon or energy source.

Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region by L.-S. P. Tran; L. Szabó; T. Ponyi; L. Orosz; T. Sík; A. Holczinger (pp. 845-852).

The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5′ end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomycescerevisiae gene coding for a serine/threonine protein kinase (RCK1).

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water by E. Katsivela; D. Bonse; A. Krüger; C. Strömpl; A. Livingston; R. -M. Wittich (pp. 853-862).

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04).

Analysis of the relative abundance of different types of bacteria capable of toluene degradation in a compost biofilter by P. Juteau; D. Rho; R. Larocque; A. LeDuy (pp. 863-868).

A microbial community of a compost biofilter treating toluene vapors was investigated using serum-bottle assays and mineral-agar plates. Toluene was not consumed in the absence of oxygen. However, filter-bed extracts exposed to toluene vapor as the only carbon source produced distinct colony types (phenotypic groups) that were counted separately. Strains from each group were isolated and checked for toluene-degradation activity in serum bottles. Only 15% of colonies were true toluene degraders. This population was divided into 11 genotypic groups based on DNA fingerprints. Identification of a member of each group using 16S rRNA gene-sequence comparison showed that they belonged to seven genera: Acinetobacter, Azoarcus, Mycobacterium, Nevskia, Pseudomonas, Pseudonocardia and Rhodococcus. Together, members of the genera Pseudonocardia and Rhodococcus were 34 times more numerous than all the others. We hypothesized that these two organisms are K-strategists (adapted to a resource-restricted and crowded environment) and that the compost biofilter is a K-environment. This would explain why they are not outnumbered by faster growers like Pseudomonas or Acinetobacter species, which would be r-strategists (adapted to a resource-abundant and uncrowded environment).

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Applied Microbiology and Biotechnology (v.52, #6)