Screening of hexavalent chromium biosorbent from marine algae

A high-chromate-selective biosorbent with high adsorption capacity was sought by examining the chromate adsorption capacities of 48 species of red, brown, or green marine algae sampled from the east coast of Korea. Screening showed a red marine alga to have the most excellent adsorption characteristics among them, and it was identified as Pachymeniopsis sp. The period at which Pachymeniopsis sp. was sampled did not affect the adsorption capacity of the alga, but the optimum period for mass collection was April to May. The alga also showed high selectivity for chromate since its adsorption capacity for other heavy metal ions such as cadmium and manganese ions was relatively low. An investigation of the adsorption isotherm of dried powder of Pachymeniopsis sp. for chromate adsorption at 25 °C showed a Langmuir-type dependence. The maximum chromate adsorption capacity of the selected alga was about 225 mg/g. Desorption of the adsorbed chromate from Pachymeniopsis sp. was done by treating the sample with 1 N NaOH. It was confirmed that ion exchange type adsorption was observed with an anion exchanger but not with a cation exchanger. It is therefore believed that the chromate adsorption is based on anionic exchange of Pachymeniopsis sp. Received: 6 October 1999 / Accepted: 4 February 2000     

Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria

Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 μM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c ₃(M _r. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c ₃ from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c ₇ from Desulfuromonas acetoxidans. Results with cytochromes c ₃ and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate. Received: 14 April 2000 / Received revision: 6 July 2000 / Accepted: 9 July 2000     

Extracellular reduction of selenite by a novel marine photosynthetic bacterium

A novel purple nonsulfur bacterium strain NKPB030619, which has resistance to over 5 mM selenite, was isolated from a marine environment. An initial concentration of 1.1 mM selenite, added to the medium, was decreased to under 0.05 mM within 5 days. The color of the cell suspension turned red within 2 days. The red coloration gradually decreased and black precipitates appeared during 2 weeks of cultivation. Under these conditions, two main types of deposit were formed extracellularly. These deposits were thought to contain red amorphous selenium and black vitreous selenium. The selenite reduction to elemental selenium in this bacterium was induced by the introduction of light and l-malic acid under anaerobic conditions. These results suggest that selenite reduction is coupled with photosynthesis and l-malic acid can serve as the indirect electron donor for its reduction. Phylogenetic analysis based on the 16S rDNA sequence showed that NKPB0360619 belongs to the α subdivision of Proteobacteria and is classified into the Rhodobacter species. The highest similarity of 86.2% was observed with R. sphaeroides. Received: 13 August 1996 / Received last revision: 6 May 1997 / Accepted: 11 May 1997     

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms

An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions. Received: 20 October 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

Halogenating activities detected in Antarctic macroalgae

 Halogenating activities were determined in samples of 18 cultivated species of brown, red and green macroalgae from the Antarctic. Activities for the halogenating organic compounds with bromide, iodide and chloride were found. Investigated red algae (rhodophytes) showed higher brominating and iodinating activities compared to brown (phaeophytes) and green (chlorophytes) algae. The highest brominating and iodinating activities were measured in the red algae Plocamium cartilagineum (1.11±0.01 U g^-1 wet algal weight and 0.18 U g^-1 wet algal weight, respectively) and Myriogramme mangini (3.62±0.17 U g^-1 wet algal weight and 4.5 U g^-1 wet algal weight, respectively). Chlorinating activities were detected in the red alga Plocamium cartilagineum only (0.086 U g^-1 wet algal weight). Received: 12 February 1996/Accepted: 20 June 1996     

Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp. strain HZM-1

A zinc-resistant bacterium, Brevibacterium sp. strain HZM-1 which shows a high Zn²⁺-adsorbing capacity, was isolated from the soil of an abandoned zinc mine. Kinetic analyses showed that Zn²⁺ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM. The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn²⁺ biosorbents. The cells could also adsorb heavy metal ions such as Cu²⁺. HZM-1 cells could remove relatively low levels of the Zn²⁺ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions. Bound Zn²⁺ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn²⁺-adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH. Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn²⁺ from natural environments. The cells could grow in the presence of significant concentrations of ZnCl₂ (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn²⁺-contaminated aqueous systems. Received: 1 February 2000 / Received revision: 31 March 2000 / Accepted: 1 May 2000     

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Microbial and cytoplasmic membrane-based potentiometric biosensors for direct determination of organophosphorus insecticides

Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp. on the surface of a glass pH electrode. The ability of Flavobacterium sp. to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation. The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp. In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed. This stoichiometry forms the electrochemical basis of the potentiometric biosensors. Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer. Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase. The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability. Received: 11 February 2000 / Received revision: 25 May 2000 / Accepted: 26 May 2000     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation

The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described. This matrix showed promise for application in iron oxidation under strongly acidic conditions. The influence on the colonisation process of T. ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied. Results showed that immobilisation of T. ferrooxidans cells was affected by changes of temperature between 30 °C and 40 °C and in pH from 1.4 to 2.0. Received: 25 January 2000 / Received version: 20 April 2000 / Accepted: 1 May 2000     

Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp.

The marine photosynthetic bacterium Chromatium sp. successfully removed orthophosphate when grown phototrophically. The phosphate-uptake rate was almost constant at more than 5.0 mg- PO₄ ³⁻/l in synthetic medium. Addition of seawater causes flocculation of this strain. The successful use of seawater as an inexpensive source of magnesium could prove to be effective in the removal of photosynthetic bacterial cells from a medium. A semicontinuous culture system was used for the removal of low concentrations of phosphate and the phosphate-uptake activity of Chromatium sp. was maintained under 0.1 day⁻¹ dilution rate. This strain was also able to remove high concentrations of phosphate from domestic sewage. Received 24 May 1996 / Received revision: 5 August 1996 / Accepted: 6 September 1996     

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed. Received: 27 September 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N′-diacetylchitobiose were 1.06 mM and 44.4 μmol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N′-diacetylchitobiose, indicating that ChiA is a chitobiosidase. Received: 29 October 1999 / Received revision: 16 March 2000 / Accepted: 24 March 2000     

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day⁻¹ l⁻¹ gel) at the highest dilution rate (0.25 day⁻¹) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer. Received: 22 October 1999 / Received revision: 14 February 2000 / Accepted: 18 February 2000     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. Received: 27 December 1996 / Received last revision: 10 June 1997 / Accepted: 14 June 1997     

Nitrate reduction by Citrobacter diversus under aerobic environment

A new aerobic denitrifier, Citrobacter diversus, was isolated from both nitrification and denitrification sludge. To monitor the variation in the concentration of nitrogen oxides, aerobic denitrification by C. diversus was carried out in a batch reactor. When the nitrate concentration was greater than 180 mg N l⁻¹, the nitrate reduction rate became stable. The effect of the C/N ratio on the denitrification activity was also investigated. The results showed that the optimum denitrification activity was obtained when the C/N ratio was 4–5. The range of the C/N ratio was higher than that for traditional anoxic denitrification. The effect of the dissolved oxygen concentration was further studied; and it was found that the range of dissolved oxygen concentrations, both for specific growth rates and for specific denitrification rates, was 2–6 mg⁻¹. From these results, it can be concluded that both the concentration of dissolved oxygen and the C/N ratio are key factors in the aerobic denitrification by C. diversus. Received: 23 November 1999 / Received revision: 4 February 2000 / Accepted: 13 February 2000     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996