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Applied Microbiology and Biotechnology (v.51, #5)
Biotechnology of succinic acid production and markets for derived industrial products by J. G. Zeikus; M. K. Jain; P. Elankovan (pp. 545-552).
Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to supply over 2.7 × 108 kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO2 is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000 tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate fermentation economics are discussed.
Recent trends in the biochemistry of surfactin by F. Peypoux; J. M. Bonmatin; J. Wallach (pp. 553-563).
The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m−1 to 27 mN m−1 at a concentration as low as 20 μM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a β-hydroxy fatty acid with 13–15 C atoms. In solution, the molecule exhibits a characteristic “horse saddle” conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4′-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries.
Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli by B. Xu; M. Jahic; G. Blomsten; S.-O. Enfors (pp. 564-571).
Industrial 20-m3-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes.
Colonisation of phase II compost by biotypes of Trichoderma harzianum and their effect on mushroom yield and quality by H. S. S. Sharma; M. Kilpatrick; F. Ward; G. Lyons; L. Burns (pp. 572-578).
Colonisation assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b and Th3 inoculated into two distinct compost types at spawning became established by first flush assessment; the extension rate of two Th2 isolates was over 1000 times that of Th1 and Th3. Results subsequently confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. Analysis of compost type also indicated that the speed and magnitude of T. harzianum colonisation was influenced by key compost characteristics, most notably, moisture, ash content and degree of fermentation. This study has shown that compost parameters which have a positive influence on Agaricus growth and productivity also resulted in increased compost colonisation by T. harzianum. Commercially acceptable yields obtained from uninoculated compost confirmed that production of a high quality, productive substrate does not confer inherent immunity to colonisation by T. harzianum.
Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture by H. J. Cruz; A. S. Ferreira; C. M. Freitas; J. L. Moreira; M. J. T. Carrondo (pp. 579-585).
In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K Glc Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K Gln Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism.
A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution by J. Q. Liu; M. Odani; T. Dairi; N. Itoh; S. Shimizu; H. Yamada (pp. 586-591).
A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l−1) l-threo-MTPS was obtained from 200 mM (45.5 g l−1) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess.
Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase by D. Barriault; M. Sylvestre (pp. 592-597).
Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2′-, 3,3′-, 4,4′-di- and 2,2′,5,5′-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases.
Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51 by M. D. Vollmer; U. Schell; V. Seibert; S. Lakner; M. Schlömann (pp. 598-605).
The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k cat/K m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate.
Purification and mode of action of two different arabinoxylan arabinofuranohydrolases from Bifidobacterium adolescentis DSM 20083 by K. M. J. Van Laere; C. H. L. Voragen; T. Kroef; L. A. M. Van den Broek; G. Beldman; A. G. J. Voragen (pp. 606-613).
Two novel arabinofuranohydrolases (AXH-d3 and AXH-m23) were purified from Bifidobacterium adolescentis DSM 20083. Both enzymes were induced upon growth of Bi. adolescentis on xylose and arabinoxylan-derived oligosaccharides. They were only active with arabinoxylans and therefore denoted as arabinoxylan arabinofuranohydrolases. Their optimal activity was at pH 6 and 30–40 °C. They were very specific in their mode of action and were clearly different from AXH-m from Aspergillus awamori. AXH-m23 released only arabinosyl groups, which were linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan oligomers. AXH-d3 hydrolysed C-3-linked arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arab- inoxylan-derived oligosaccharides. No activity was observed with C-2-linked arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against C-2- and C-3-linked arabinofuranosyl residues of singly substituted xylopyranosyl residues. The combination of AXH-d3 and AXH-m showed low debranching activity with highly substituted glucurono-arabinoxylans. However, arabinoxylan from wheat flour was debranched almost completely.
Development of a bioconversion process for production of cis-1S,2R-indandiol from indene by recombinant Escherichia coli constructs by J. Reddy; C. Lee; M. Neeper; R. Greasham; J. Zhang (pp. 614-620).
Recombinant Escherichia coli cells expressing the toluene dioxygenase (TDO) genes from Pseudomonas putida convert indene to cis-1S,2R-indandiol, a potentially important intermediate for the chemical synthesis of the HIV-1 protease inhibitor, Crixivan. A bioconversion process was developed through optimization of medium composition and reaction conditions at the shake-flask and 23-l fermentor scales. A cis-1,2-indandiol productivity of approx. 1000 mg/l was achieved with construct TDO123, which represents a 50-fold increase over the initial titer. Varying the bioconversion conditions did not change the enantiomeric excess (e.e.) for the 1S,2R enantiomer from about 30%, suggesting that toluene dioxygenase intrinsically converts indene to 1S,2R- and 1R,2S-indandiols at a ratio of 2:1. Further inclusion of the Pseudomonas dehydrogenase gene in construct D160-1 led to the production of chirally pure cis-1S,2R-indandiol (e.e. > 99%) as a result of the selective degradation of the 1R,2S enantiomer, with the overall yield (650 mg/l) proportionally reduced. A single stage process was developed for D160-1 and scaled up to the 23-l fermentor, achieving a cis-1S,2R-indandiol titer of 1200 mg/l.
Construction of a flocculent Saccharomyces cerevisiae fermenting lactose by L. Domingues; J. A. Teixeira; N. Lima (pp. 621-626).
A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for β-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs.
High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium by G. Miksch; R. Neitzel; K. Friehs; E. Flaschel (pp. 627-632).
The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.
The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum by L. Karaffa; E. Sándor; J. Kozma; C. P. Kubicek; A. Szentirmai (pp. 633-638).
Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed.
Polyphosphate formation by Acinetobacter johnsonii 210A: effect of cellular energy status and phosphate-specific transport system by E. W. J. van Niel; J. H. de Best; E. P. W. Kets; C. F. C. Bonting; G. J. J. Kortstee (pp. 639-646).
In acetate-limited chemostat cultures of Acinetobacter johnsonii 210A at a dilution rate of 0.1 h−1 the polyphosphate content of the cells increased from 13% to 24% of the biomass dry weight by glucose (100 mM), which was only oxidized to gluconic acid. At this dilution rate, only about 17% of the energy from glucose oxidation was calculated to be used for polyphosphate synthesis, the remaining 83% being used for biomass formation. Suspensions of non-growing, phosphate-deficient cells had a six- to tenfold increased uptake rate of phosphate and accumulated polyphosphate aerobically up to 53% of the biomass dry weight when supplied with only orthophosphate and Mg2+. The initial polyphosphate synthesis rate was 98 ± 17 nmol phosphate min−1 mg protein−1. Intracellular poly-β-hydroxybutyrate and lipids served as energy sources for the active uptake of phosphate and its subsequent sequestration to polyphosphate. The H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide caused low ATP levels and a severe inhibition of polyphosphate formation, suggesting the involvement of polyphosphate kinase in polyphosphate synthesis. It is concluded that, in A. johnsonii 210A, (i) polyphosphate is accumulated as the energy supply is in excess of that required for biosynthesis, (ii) not only intracellular poly-β-hydroxybutyrate but also neutral lipids can serve as an energy source for polyphosphate-kinase-mediated polyphosphate formation, (iii) phosphate-deficient cells may accumulate as much polyphosphate as activated sludges and recombinants of Escherichia coli designed for polyphosphate accumulation.
Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum by B. S. Lee; E. Holler (pp. 647-652).
Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO3. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO3, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C.
An image analysis method for determination of spatial colonization patterns of bacteria in plant rhizosphere by D. P. Roberts; D. Y. Kobayashi; P. D. Dery; N. M. Short Jr (pp. 653-658).
A method that allows the rapid visualization of bacterial spatial colonization patterns on roots for the determination of general colonization trends was developed. This method, which analyzes images of roots, and bioluminescence-enhanced images of bacterial colonization patterns on these roots, was used to study the colonization patterns of seed-applied Enterobacter cloacae strain E6 on 3-day-old cucumber plants. Conventional dilution-plating methods indicated that E6 colonized cucumber tap roots in high populations and that these populations significantly decreased as the distance from the seed increased. In addition to confirming these observations, image analysis indicated that colonization by E6 significantly decreased on lateral roots as the distance increased horizontally away from the tap root, and that this bacterium did not evenly cover the most densely colonized regions of the cucumber root system. Results from these experiments indicate that the majority of E6 populations on cucumber roots after seed application are limited to the upper regions of the tap root and that E6 does not effectively colonize other regions of the root system.
Characterization of Lactobacillus collinoides response to heat, acid and ethanol treatments by J. M. Laplace; N. Sauvageot; A. Hartke; Y. Auffray (pp. 659-663).
Tolerance to stress and cross-protection in Lactobacillus collinoides were examined after exposure to ethanol, acid or heat shock. Ethanol and heat-adapted cells demonstrate induced homologous␣tolerance and cross-resistance to acid stress. No cross-protection of acid-adapted cells against ethanol and heat stresses was observed. Heat was the only pretreatment leading to cross-protection against the two other stresses. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins; the synthesis of some of these polypeptides being induced by more than one condition. The greatest overlap was observed between ethanol and heat treatments. Ten proteins were found to be common to these stresses.
An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria by T. Kondo; T. Ikeda (pp. 664-668).
In order to measure the substrate-oxidizing activity of intact cells of Acetobacter pasteurianus no. 2, a given amount of the bacterial cells was immobilized on a carbon-paste electrode, and the current at the electrode was measured in a buffer solution. When Fe(CN)3− 6 was added to the buffer solution, an anodic current was observed at 0.5 V (against Ag/AgCl). Further, when ethanol was added to the solution, the current started to increase to reach a steady-state within 3 min. The electrode had a good response to acetaldehyde and lactic acid as well as ethanol. Culture conditions affected the current response to various substances; the response of the electrode modified with the cells grown in static culture was much higher than that of the electrode with the cells grown in shaking culture, and the electrode with ethanol-grown cells had a high response to ethanol and acetaldehyde compared with that of the electrode with glucose-grown cells. The increase in the amount of the current after the addition of ethanol (ΔI EtOH) was linearly proportional to the total number of immobilized cells per electrode in the range 1.0 × 104–1.0 × 108 cells. The ΔI EtOH values were measured with the electrode prepared with a fixed volume of the cell suspensions taken from the culture at 6-h intervals; the dependence of the ΔI EtOH value on time agreed well with the cell growth measured by colony counting and turbidity in the lag and logarithmic phase. After the logarithmic phase, the value of ΔI EtOH sharply decreased, resembling to the growth measured by colony counting, rather than by turbidity.
Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose by K. Hofvendahl; E. W. J. van Niel; B. Hahn-Hägerdal (pp. 669-672).
Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase.
Characterisation of a starch-hydrolysing enzyme of Aspergillus niger by C. Suresh; A. K. Dubey; S. Srikanta; S. Umesh Kumar; N. G. Karanth (pp. 673-675).
A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries.
Transformation of N ′,N ′-dimethyl-N -(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor by C. Jolivalt; A. Raynal; E. Caminade; B. Kokel; F. Le Goffic; C. Mougin (pp. 676-681).
Transformation of N′,N′-dimethyl-N-(hydroxyphenyl)ureas was assayed in the presence of purified laccase produced by the fungus Trametes versicolor. The para- and ortho-hydroxyphenyl derivatives were enzymatically transformed, whereas the meta derivative was not. The performance of laccase-mediated transformation depended on the pH, with an optimum for the para-derivative degradation rate at pH 5. The pH also influenced the nature of the reaction products. The chemical was exclusively oxidised into p-benzoquinone at pH 3 and into mainly N′,N′-dimethyl-N-[(2,5-cyclohexadiene-1-one)-4-ylidene]urea at pH 6. The ortho- derivative was transformed essentially into insoluble purple compounds, probably appearing as polymers resulting from coupling of the parent compound.
Fermented whey – an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis by T. L. Bledsoe; A. W. Cantafio; J. M. Macy (pp. 682-685).
It is critical that an inexpensive electron- donor/carbon-source be found for selenium bioremedia-tion using the selenate-respiring bacterium, Thauera selenatis. Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate. Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h−1). The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T. selenatis, treating selenium-contaminated drainage water was also demonstrated. Selenium oxyanions and nitrate were reduced by 98%.
Zn biosorption by Rhizopus arrhizus and other fungi by J. L. Zhou (pp. 686-693).
Biosorption of zinc ions by inactivated fungal mycelia was studied. Of the six fungal species, Rhizopus arrhizus, Mucor racemosus, Mycotypha africana, Aspergillus nidulans, Aspergillus niger and Schizosaccharomyces pombe, R. arrhizus exhibited the highest capacity (Q max = 213 μmol g−1 dry weight). Further experiments with different cellular fractions of R. arrhizus showed that Zn was predominantly bound to cell-wall chitin and chitosan (Q max = 312 μmol g−1 dry weight). Adsorption data were best modelled by the Langmuir isotherm, although they can be modelled by the Freundlich equation as well at relatively low aqueous concentrations. Biosorption generally decreased with increase in biosorbent particle size and its concentration. Low pH reduced Zn sorption, because of the strong competition from hydrogen ions for binding sites on fungi. The presence of ligands reduced metal uptake, chiefly by forming metal complexes of a less biosorbable nature.
Tetrachloroethene dechlorination kinetics by Dehalospirillum multivorans immobilized in upflow anaerobic sludge blanket reactors by C. Hörber; N. Christensen; E. Arvin; B. Kiær Ahring (pp. 694-699).
Tetrachloroethene (C2Cl4) dechlorination kinetics in upflow anaerobic sludge blanket (UASB) reactors was determined after introducing de novo activities into the granular sludge. These activities were introduced by immobilizing Dehalospirillum multivorans in a test reactor containing unsterile granular sludge, and in a reference reactor, R1, containing sterile granular sludge. A second reference reactor, R2, contained only unsterile granular sludge and served as a control. The kinetic experiments were performed by pulsing the reactors with C2Cl4 in a recirculating batch mode. Formate and acetate were added as electron donor and carbon source. Both reactors inoculated with D. multivorans dechlorinated C2Cl4 to an equimolar amount of C2H2Cl2 with only traces of C2HCl3 in the effluent. In the control reactor, C2HCl3 accumulated before C2H2Cl2 was produced. A computer simulation program (AQUASIM) was used to estimate the kinetic parameters. The half-saturation constants (K s) for C2Cl4 and C2HCl3 were almost equal in the reactors containing D.␣multivorans (17 μM and 18 μM for C2Cl4; 26 μM and 28 μM for C2HCl3), indicating no influence of sludge bacteria on the affinity of D. multivorans for C2Cl4 and C2HCl3. The maximum dechlorination rates (k m X B) were about twice as high in the reactor containing D.␣multivorans immobilized in sterile sludge (11 mmol C2Cl4 l sludge−1 day−1 and 27 mmol C2HCl3 l sludge−1 day−1) than in the test reactor (4.4 mmol C2Cl4 l sludge−1 day−1 and 15 mmol C2HCl3 l sludge−1 day−1). Compared to other C2Cl4-degrading systems, the dechlorination rates of the inoculated reactors and their affinities for C2Cl4 and C2HCl3 were high. Therefore, introduction of de novo activity is promising for the use of anaerobic reactors to bioremediate C2Cl4-polluted water.