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Applied Microbiology and Biotechnology (v.55, #4)
Modification of lignin for the production of new compounded materials by A. Hüttermann; C. Mai; A. Kharazipour (pp. 387-394).
The cell walls of woody plants are compounded materials made by in situ polymerization of a polyphenolic matrix (lignin) into a web of fibers (cellulose), a process that is catalysed by polyphenoloxidases (laccases) or peroxidases. The first attempt to transform the basic strategy of this natural process for use in human craftsmanship was the ancient lacquer method. The sap of the lacquer tree (Rhus verniciflua) contains large amounts of a phenol (urushiol), a polysaccharide and the enzyme laccase. This oil-in-water emulsion solidifies in the presence of oxygen. The Chinese began using this phenomenon for the production of highly creative artwork more than 6,000 years ago. It was the first example of an isolated enzyme being used as a catalyst to create an artificial plastic compound. In order to apply this process to the production of products on an industrial scale, an inexpensive phenol must be used, which is transferred by an enzyme to active radicals that react with different components to form a compounded material. At present, the following approaches have been studied: (1) In situ polymerization of lignin for the production of particle boards. Adhesive cure is based on the oxidative polymerization of lignin using phenoloxidases (laccase) as radical donors. This lignin-based bio-adhesive can be applied under conventional pressing conditions. The resulting particle boards meet German performance standards. By this process, 80% of the petrochemical binders in the wood-composite industry can be replaced by materials from renewable resources. (2) Enzymatic copolymerization of lignin and alkenes. In the presence of organic hydroperoxides, laccase catalyses the reaction between lignin and olefins. Detailed studies on the reaction between lignin and acrylate monomers showed that chemo-enzymatic copolymerization offers the possibility to produce defined lignin-acrylate copolymers. The system allows control of the molecular weights of the products in a way that has not been possible with chemical catalysts. This is a novel attempt to enzymatically induce grafting of polymeric side chains onto the lignin backbone, and it enables the utilization of lignin as part of new engineering materials. (3) Enzymatic activation of the middle-lamella lignin of wood fibers for the production of wood composites. The incubation of wood fibers with a phenol oxidizing enzyme results in oxidative activation of the lignin crust on the fiber surface. When such fibers are pressed together, boards are obtained which meet the German standards for medium-density fiber boards (MDF). The fibers are bound together in a way that comes close to that by which wood fibers are bound together in naturally grown wood. This process will, for the first time, yield wood composites that are produced solely from naturally grown products without any addition of resins.
Microbial metabolites as eco-friendly agrochemicals for the next millennium by Sanjai Saxena; Akhilesh K. Pandey (pp. 395-403).
As a result of the increasing environmental and health-related problems caused by the synthetic agrochemicals currently used, suitable and non-hazardous innovative alternatives are being sought. Antagonism and allelopathy, both in nature and in agro-ecosystems, have attracted these researchers' attention, with the main goal of using these phenomena in the biological control of weeds. This article presents a review on the use and efficacy of microbial secondary metabolites which have potential as natural herbicides, either directly or as templates for bio-rational eco-friendly agrochemicals (allelochemicals). Their merits as alternatives to synthetic chemicals and biological control agents have been highlighted for an holistic approach in integrated pest/weed management.
Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal by Chuangui Wang; Jianyong Wu; Xingguo Mei (pp. 404-410).
An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l–1 was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1±7.5 mg l–1, which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l–1 day–1, which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.
Application of oxygen vectors to Claviceps purpurea cultivation by M Menge; J Mukherjee; T Scheper (pp. 411-416).
The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.
Kinetic analysis of growth and xanthan gum production with Xanthomonas campestris on sucrose, using sequentially consumed nitrogen sources by Fabien Letisse; Paule Chevallereau; Jean-Luc Simon; Nic D. Lindley (pp. 417-422).
A batch fermentation strategy using Xanthomonas campestris ATCC 13951 for xanthan gum production has been established in which all essential medium components are supplied at the onset. This has been achieved using sucrose as sole sugar feedstock. Sequential consumption of nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) was observed to facilitate the further optimisation of the medium. Biomass accumulation was limited by phosphate availability. Xanthan yields of more than 60% (grams of xanthan per gram of sugar) have been obtained with constant acetyl content. However, pyruvyl substitution decreased as the growth rate declined, due to the metabolic constraints specific to phosphate depletion. High rates of carbon conversion into xanthan were observed throughout the culture and the ATP/ADP ratio was not affected by the decline in the specific growth rate.
Production of β-carotene by a mutant of Rhodotorula glutinis by P. Bhosale; R. Gadre (pp. 423-427).
Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of β-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of β-carotene was 14% (w/w) of the total carotenoid content (17 µg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more β-carotene (2,048 µg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of β-carotene (33±3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66±2 mg/l) with β-carotene as the major component (55±2%, w/w).
Evaluation of a vertical flat-plate photobioreactor for outdoor biomass production and carbon dioxide bio-fixation: effects of reactor dimensions, irradiation and cell concentration on the biomass productivity and irradiation utilization efficiency by K. Zhang; S. Miyachi; N. Kurano (pp. 428-433).
Outdoor culture of the thermophilic cyanobacterium Synechocystis aquatilis SI-2 with a vertical flat-plate photobioreactor (VFPP) was studied during the period of January to August of 1999 in the northern region of Japan (Kamaishi, Iwate, 39°N, 142°E). The aim of this study was to investigate the CO2 fixation ability of the VFPP device under various irradiation conditions. An average biomass productivity of over 30 g m–2 day–1, which corresponds to a CO2 fixation rate of 50 g m–2 day–1, was achieved during this period with a 192-l scale culture. The effects on biomass productivity of the light path, height of the reactor, cell concentration and irradiation were also investigated. Variation of the optimal cell concentration to achieve the highest productivity for outdoor operation is discussed. A cell concentration of 1–2 g l–1 was found to be most suitable for the irradiation range of 1–12 MJ m–2 day–1 under the experimental conditions used.
Evaluation of plastic-composite supports in repeated fed-batch biofilm lactic acid fermentation by Lactobacillus casei by Ana Velázquez; Anthony L. Pometto III; Kai-Lai G. Ho; Ali Demirci (pp. 434-441).
A customized stirred-tank biofilm reactor was designed for plastic-composite supports (PCS). In repeated-batch studies, the PCS-biofilm reactors outperformed the suspended-cell reactors by demonstrating higher lactic acid productivities (2.45 g l–1 h–1 vs 1.75 g l–1 h–1) and greater glucose consumption rates (3.27 g l–1 h–1 vs 2.09 g l–1 h–1). In the repeated fed-batch studies, reactors were spiked periodically with concentrated glucose (75%) to maintain a concentration of ~80 g of glucose l–1 in the bioreactor. In suspended-cell fermentations with 10 g of yeast extract (YE) l–1 and zero, one, two, and three glucose spikes, the lactic acid productivities were 2.64, 1.58, 0.80, and 0.62 g l–1 h–1, respectively. In comparison, biofilm reactors with 7 g of YE l–1 and zero, one, two, and three glucose spikes achieved lactic acid productivities of 4.20, 2.78, 0.66, and 0.94 g l–1 h–1, respectively. The use of nystatin (30 U ml–1) subdued the contaminating yeast population with no effect on the lactic acid productivity of the biofilm reactors, but it did affect productivity in the suspended-cell bioreactor. Overall, in repeated fed-batch fermentations, the biofilm reactors consistently outperformed the suspended-cell bioreactors, required less YE, and produced up to 146 g of lactic acid l–1 with 7 g of YE l–1, whereas the suspended-cell reactor produced 132 g l–1 with 10 g of YE l–1.
Production of glucose–fructose oxidoreductase and ethanol by Zymomonas mobilis ATCC 29191 in medium containing corn steep liquor as a source of vitamins by M. Silveira; E. Wisbeck; I. Hoch; R. Jonas (pp. 442-445).
Different concentrations of corn steep liquor (CSL) were tested in the cultivation of Zymomonas mobilis. Cell growth, ethanol production, and the formation of glucose–fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL), the enzymes responsible for the bio-production of gluconic acid and sorbitol, were examined. The cell yields using 25 g CSL l–1 and 40 g CSL l–1 (Y X/S≈0.031 g g–1) were close to that obtained with 5 g yeast extract (YE) l–1. With 5 g CSL l–1 and 15 g CSL l–1, the nutritional limitation led to smaller Y X/S. Using 100 g CSL l–1 produced an inhibitory effect on cell growth. Similar ethanol yields (92–95%) were calculated for each concentration of CSL and also for YE medium. The highest specific GFOR/GL activities (13.2–13.5 U g–1 dry cell) were reached with 25 g CSL l–1 and 40 g CSL l–1, values comparable to that achieved with 5 g YE l–1. The results confirm that CSL is an effective and cheap supplement for Z. mobilis medium, increasing the economic potential of a large-scale bio-production of sorbitol and gluconic acid by untreated Z. mobilis cells. The economic feasibility of the process is discussed.
Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment by Hiroshi Tarui; Masahiro Murata; Ikuko Tani; Shigeo Imanishi; Shigemichi Nishikawa; Toshio Hara (pp. 446-453).
A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter–Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the post-translational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 µg of glycosylated gp120/ml of reaction mixture.
Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae by S. Takahashi; M. Ueda; A. Tanaka (pp. 454-462).
The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-α-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20–57) were significant for in vivo folding and secretion of active mROL.
Expression level of heterologous proteins in Pichia pastoris is influenced by flask design by F. Villatte; A. Hussein; T. Bachmann; R. Schmid (pp. 463-465).
The yeast Pichia pastoris is a convenient production system that enables expression of heterologous proteins in high amounts. As a fermentation method, shaking flasks are very popular because of their simplicity of handling and their low cost. We compared the expression level of the enzyme acetylcholinesterase in a transformed strain of P. pastoris grown in different flasks, presenting various designs but all with the same volume. A several-thousand-fold difference appeared in the expression levels; and the results could not be explained by differences between the flasks in the oxygenation of the medium. The data show that flask design is an important factor to consider for optimising fermentation processes.
Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum by Champika D. Wijayarathna; Masaaki Wachi; Kazuo Nagai (pp. 466-470).
Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2) ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.
Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses by S. Shibasaki; M. Ueda; T. Iizuka; M. Hirayama; Y. Ikeda; N. Kamasawa; M. Osumi; A. Tanaka (pp. 471-475).
The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as α-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 104–105 molecules of α-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.
Preparation of an organic solvent-tolerant strain from baker's yeast by T. Kawamoto; T. Kanda; A. Tanaka (pp. 476-479).
By using immobilized baker's yeast repeatedly in isooctane with occasional reactivation by cultivation, we succeeded in the preparation of an organic solvent-tolerant strain, named KK21, which could grow in the presence of isooctane. This is the first report on an organic solvent-tolerant strain from baker's yeast. Strain KK21 showed high tolerance to organic solvents and maintained a high and stable activity on continuous reduction of n-butyl 3-oxobutanoate in an isooctane-medium two-phase system. Although the morphology of strain KK21 was the same as that of baker's yeast, the saturated fatty acid occupancy (SFA occupancy), which is defined as the percentage of saturated fatty acids in the total fatty acids of phospholipids, of strain KK21 was significantly higher than that of parental baker's yeast when strain KK21 was grown in the presence of isooctane, suggesting that a decrease in fluidity of the cell membrane might play an important role in the tolerance to organic solvents.
The fungicidal and phytotoxic properties of benomyl and PPM in supplemented agar media supporting transgenic arabidopsis plants for a Space Shuttle flight experiment by Anna-Lisa Paul; Charles Semer; Thomas Kucharek; Robert J. Ferl (pp. 480-485).
Fungal contamination is a significant problem in the use of sucrose-enriched agar-based media for plant culture, especially in closed habitats such as the Space Shuttle. While a variety of fungicides are commercially available, not all are equal in their effectiveness in inhibiting fungal contamination. In addition, fungicide effectiveness must be weighed against its phytotoxicity and in this case, its influence on transgene expression. In a series of experiments designed to optimize media composition for a recent shuttle mission, the fungicide benomyl and the biocide "Plant Preservative Mixture" (PPM) were evaluated for effectiveness in controlling three common fungal contaminants, as well as their impact on the growth and development of arabidopsis seedlings. Benomyl proved to be an effective inhibitor of all three contaminants in concentrations as low as 2 ppm (parts per million) within the agar medium, and no evidence of phytotoxicity was observed until concentrations exceeded 20 ppm. The biocide mix PPM was effective as a fungicide only at concentrations that had deleterious effects on arabidopsis seedlings. As a result of these findings, a concentration of 3 ppm benomyl was used in the media for experiment PGIM-01 which flew on shuttle Columbia mission STS-93 in July 1999.
Constitutive and inducible hydroxylase activities involved in the degradation of naphthalene by Cunninghamella elegans by B. Faber; A. Schonewille; R. van Gorcom; J. Duine (pp. 486-491).
The non-ligninolytic fungus Cunninghamella elegans was investigated for its ability to produce naphthalene hydroxylase (NAH) and naphthol hydroxylase (NOH) activities under various conditions. When the organism was cultivated on a rich growth medium, the mycelia exhibited significant constitutive NAH activity in the late exponential growth phase, but not in the early-exponential-growth-phase. On incubating the early-exponential-growth-phase mycelia with naphthalene, NAH activity was increased five-fold; however, this increase did not occur in the presence of the protein synthesis inhibitor cycloheximide. Since incubation of the late-phase mycelia with naphthalene did not lead to a higher degradation rate of naphthalene, mycelia in this physiological state have apparently lost the ability to induce synthesis of the enzyme exhibiting NAH activity. This is not due to an overall inability to perform de novo protein synthesis, since NOH activity, non-constitutive at all growth phases, could be induced by incubating late-phase mycelia with naphthalene. Whether inducible and constitutive NAH activity originate from one and the same enzyme remains to be elucidated. It is suggested that naphthalene oxidizing enzyme(s) may also oxidize pyrene, but not anthracene or benzo[a]pyrene, although the latter are degradable by C. elegans.
Biodegradation of radiolabelled synthetic lignin (14C-DHP) and mechanical pulp in a compost environment by Marja Tuomela; Annele Hatakka; Sanni Raiskila; Minna Vikman; Merja Itävaara (pp. 492-499).
Mineralization of radioactive synthetic lignin (14C-DHP) was studied in a compost environment at 35, 50 and 58°C. Compost samples were successively extracted with water, dioxane and alkali, and the molecular weight distribution of some extracts was determined by gel permeation chromatography (GPC). Biodegradation of lignin-containing spruce groundwood (SGW) and pine sawdust was concurrently determined in controlled composting tests by measuring evolved CO2. The temperatures were the same as in the 14C-DHP mineralization experiment and bleached kraft paper, with a lignin content of 0.2%, was used as a reference. The mineralization of 14C-DHP was relatively high (23–24%) at 35°C and 50°C, although the mixed population of compost obviously lacks the most effective lignin degraders. At 58°C the mineralization of 14C-DHP, as well as the biodegradation of SGW and sawdust, was very low, indicating that the lignin-degrading organisms of compost were inactivated at this temperature. SGW was poorly biodegradable (<40%) in controlled composting tests compared with kraft paper (77–86%) at all temperatures, which means that lignin inhibits the degradation of carbohydrates. During the incubation, water-soluble degradation products, mainly monomers and dimers, and the original 14C-DHP were either mineralized or bound to humic substances. A substantial fraction of 14C-DHP was incorporated into humin or other insolubles.
Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration by Patrick Dabert; Bruno Sialve; Jean-Philippe Delgenès; René Moletta; Jean-Jacques Godon (pp. 500-509).
The microbial community of a conventional anaerobic–aerobic sequencing batch reactor was investigated by cloning and sequencing bacterial 16S rDNA. The 92 16S rDNA sequences analysed ranged across 50 different operational taxonomic units (OTU). The majority of these sequences were not closely related to known species. They belonged to 12 different groups, but essentially to the Cytophagales and the Proteobacteria beta, which represented 38% and 17% of the retrieved sequences respectively. No OTU numerically outnumbered the others. However, similarities were observed with previous reports on molecular characterisation of phosphorus-accumulating ecosystems, suggesting an enrichment in microorganisms belonging to the Rhodocyclus group. Thereafter, the ability of this anaerobic–aerobic microbial community to accumulate phosphorus with nitrate as its energy source was investigated. The reactor was shifted from anaerobic–aerobic running conditions to anaerobic–anoxic conditions by injection of nitrate; and its microbial community was monitored by PCR-single strand conformation polymorphism (SSCP). The reactor maintained a good phosphorus accumulation and similar SSCP microbial community patterns for a period of 17 days, suggesting that the same microbial community was able to respire both oxygen and nitrate. However, this situation was unstable, since a breakdown in phosphorus accumulation occurred thereafter.
Decolorization of molasses spent wash by the white-rot fungus Flavodon flavus, isolated from a marine habitat by Chandralata Raghukumar; Gauri Rivonkar (pp. 510-514).
Flavodon flavus (Klotzsch) Ryvarden, a basidiomycete (NIOCC strain 312) isolated from decomposing leaves of a sea grass, decolorized pigments in molasses spent wash (MSW) by 80% after 8 days of incubation, when used at concentrations of 10% and 50%. Decolorizing activity was also present in media prepared with half-strength seawater (equivalent to 15 ppt salinity). Decolorizing activity was seen in low-nitrogen medium, nutrient-rich medium and in sugarcane bagasse medium. The percentage decolorization of MSW was highest when glucose or sucrose was used as the carbon source in the low-nitrogen medium. The production of lignin-modifying enzymes, manganese-dependent peroxidase (MNP) and laccase decreased in a medium containing MSW. MNP production and MSW decolorization were inversely correlated, suggesting no role for MNP in MSW decolorization. The decolorization of MSW was not effective when F. flavus was immobilized in calcium alginate beads. Decolorization was achieved best in oxygenated cultures. Besides color, total phenolics and chemical oxygen demand were reduced by 50% in MSW treated with F. flavus, suggesting its potential in the bioremediation of effluents.