Bioaugmentation of Soil with Pseudomonas monteilii Strain Eliminates Inhibition of Okra (Abelmoschus esculentus) Seed Germination by m-Cresol.

Cresols are ubiquitous in nature due to their bulk production and end uses in various industrial processes as well as due to their natural presence. They are highly toxic to both fauna and flora and are included in the list of priority pollutants. In the present study, the effect of m-cresol on germination of ten different crop seeds was tested and the seeds of okra and eggplant were found to be very sensitive, okra being the most vulnerable. Okra seeds lost its viability in the presence of m-cresol, which was proportionate to its concentration as indicated by the standard 2,3,5-tetrazoliumtrichloride (TTC) test. Marked decrease in protease and amylase activities was observed in germinating seeds exposed to the compound. The inhibitory effect of m-cresol on germination was eliminated effectively by bioaugmentation of the soil with the cresol-degrading Pseudomonas monteilii S-CSR-0014. Normal germination and seedling vigor were obtained when the seeds were sown four and eight days after the soil inoculation with the bacterial cells, whereas the seeds sown immediately did not show proper germination. The inoculated bacterium degraded m-cresol efficiently from the spiked soil and exhibited concomitant growth. It can be concluded that m-cresol-contaminated soils could be effectively bioremediated to render the soil suitable for normal seed germination and healthy seedling growth of sensitive crops.

Enhanced degradation of phenol by Pseudomonas sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes.

Phenol is one of the major toxic pollutants in the wastes generated by a number of industries and needs to be eliminated before their discharge. Although microbial degradation is a preferred method of waste treatment for phenol removal, the general inability of the degrading strains to tolerate higher substrate concentrations has been a bottleneck. Immobilization of the microorganism in suitable matrices has been shown to circumvent this problem to some extent. In this study, cells of Pseudomonas sp. CP4, a laboratory isolate that degrades phenol, cresols, and other aromatics, were immobilized by entrapment in Ca-alginate and agar gel beads, separately and their performance in a fluidized bed bioreactor was compared. In batch runs, with an aeration rate of 1 vol(-1) vol(-1) min(-1), at 30°C and pH 7.0 ± 0.2, agar-encapsulated cells degraded up to 3000 mg l(-1) of phenol as compared to 1500 mg l(-1) by Ca-alginate-entrapped cells whereas free cells could tolerate only 1000 mg l(-1). In a continuous process with Ca-alginate entrapped cells a degradation rate of 200 mg phenol l(-1) h(-1) was obtained while agar-entrapped cells were far superior and could withstand and degrade up to 4000 mg phenol l(-1) in the feed with a maximum degradation rate of 400 mg phenol l(-1) h(-1). The results indicate a clear possibility of development of an efficient treatment technology for phenol containing waste waters with the agar-entrapped bacterial strain, Pseudomonas sp. CP4.

Substantially enhanced degradation of hexachlorocyclohexane isomers by a microbial consortium on acclimation.

Widespread contamination of the environment, globally, has been caused by extensive and indiscriminate use of hexachlorocyclohexane (HCH) as an insecticide since the 1940s, threatening the biota including humans, and there is an urgent need to eliminate it, preferably through bioremediation technologies. A gamma-HCH-degrading microbial consortium was isolated by enrichment of a soil sample from a sugar cane field having a long history of technical grade HCH application. On acclimation the degrading ability improved substantially. The consortium, which took 10 days to degrade 25 microg mL(-1) of gamma-HCH, initially could mineralize even 300 microg mL(-1) of the substrate within 108 h on acclimation. With 300 microg mL(-1) substrate, the rate of degradation, as calculated for the early exponential phase, was 216 microg mL(-1) day(-1), the highest reported so far. An amount of 400 microg mL(-1) of gamma-HCH, however, was mineralized partially with only 78% Cl(-) release. No apparent accumulation of intermediary metabolites was observed up to 300 microg mL(-1) substrate, indicating a fast rate of mineralization. Aeration, mesophilic temperatures (20-35 degrees C), and near neutral pH (6.0-8.0) were favorable conditions for degradation. The presence of glucose at 1000 microg mL(-1) retarded the degradation, whereas cellulose and sawdust at 1600 microg mL(-1) and glucose at 100 microg mL(-1) did not show any marked effect. The consortium also mineralized alpha-, beta-, and delta-HCH efficiently. The consortium consisted of nine bacterial strains and a fungal strain, and individually they were able to degrade 10 microg mL(-1) of gamma-HCH. This mixed culture holds high potential for deployment in bioremediation of HCH-contaminated soils, waste dumpsites, and water bodies.

Degradation of 3-chlorobenzoate and phenol singly and in mixture by a mixed culture of two ortho-pathway-following Pseudomonas strains.

The compatibility and efficiency of two ortho-cleavage pathway-following pseudomonads viz. the 3-chlorobenzoate (3-CBA)-degrader, Pseudomonas aeruginosa 3mT (3mT) and the phenol-degrader, P. stutzeri SPC-2 (SPC-2) in a mixed culture for the degradation of these substrates singly and simultaneously in mixtures was studied. Another phenol-degrading strain, Pseudomonas sp. SoPC-5 (SoPC-5) that utilizes a meta-cleavage mode also was tried in co-culture with 3mT. The former combination was found to be a better degrader of both the substrates when present alone. But, with inoculum levels of 0.15 mg cell dry wt each of 3mT/SPC-2 or 3mT/SoPC-5 growth with 2 mM each of 3-CBA and phenol was slow with a lag of 24 h and degradation being incomplete. However, with higher inocula in the ratios 1:1, 1:2, and 2:1, i.e., 0.3 + 0.3, 0.3 + 0.6, and 0.6 + 0.3 mg cell dry wt of 3mT and SPC-2, respectively complete degradation of both the substrates occurred. Degradation of 3-CBA was complete with the release of stoichiometric amounts of chloride (Cl(-)) when concentrations of phenol/3-CBA were varied as 2:2, 2:4, and 4:2 mM, i.e., even when the concentration of the more toxic co-substrate 3-CBA was higher than phenol effective simultaneous degradation occurred at the inoculums ratio of 1:1 (0.3 mg dry cell wt. of each strain). These studies clearly indicated the better suitability of ortho-cleavage-utilizing strains as partners in a mixed culture than those follow different modes.

Determination of benzo(a)pyrene by GC/MS/MS in retail olive oil samples available in Qatar.

A survey was carried out for the presence of benzo(a)pyrene (B(a)P) in olive oils following reports that some batches of Spanish olive-pomace oil and its products contained high levels of B(a)P. Three types of olive oils (1) virgin olive oil, (2) refined olive oil and (3) olive-pomace oil, originating from France, Greece, Italy, the Lebanon, Spain, Tunisia and Turkey, and available on Qatar market, were analysed for B(a)P. Determination was carried out by extraction from a cyclohexane solution with N,N-dimethylformamide: water (9:1), back extraction with cyclohexane, followed by clean-up on a silica gel column and quantification by GC/MS/MS. The recovery and limit of quantitation of B(a)P in olive oils by this method were estimated as 88% and 0.5 microg kg(-1), respectively. B(a)P was detected in amounts below the permitted level of 2 microg kg(-1) in all 31 virgin olive oil samples and in 13 refined olive oil samples. However, all seven samples of Spanish olive-pomace oil produced and packed in Spain and three samples of olive-pomace oil produced in Spain, but packed in Saudi Arabia, were found to be contaminated with B(a)P, the level ranging from 3.1 to 70.8 microg kg(-1). In two samples of olive-pomace oil originating from Greece, B(a)P was not detected.

Protection of tomato seed germination from the inhibitory effect of 2,4,5-trichlorophenoxyacetic acid by inoculation of soil with Burkholderia cepacia AC1100.

The effect of 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) on the germination and seedling vigor of different crop seeds was tested. Seeds of rice, maize, sorghum, finger millet, and horse gram were comparatively more tolerant to the chemical with no marked effect up to a concentration of 200 mg 2,4,5-T L(-)(1) as tested by the filter paper method. Tomato and brinjal (egg plant) were highly susceptible. Even at 5 and 10 mg 2,4,5-T L(-)(1), marked reduction in the germination and seedling vigor of tomato and egg plant, respectively, was observed. At 20 and 30 mg L(-)(1), the germination of tomato and egg plant seeds, respectively, were completely inhibited on filter paper, whereas the inhibitory concentrations in soil was 40 mg 2,4,5-T kg(-)(1) soil. Several abnormalities were observed in the chemically affected seedlings. Protease activity of the seeds germinating in the presence of the chemical was drastically reduced. Bioremediation of the chemically contaminated soil with Burkholderia cepacia AC1100, by inoculation of the soil 7 days before sowing the seeds, completely protected the seeds, resulting in normal germination and an improved seedling vigor.

Isolation and acclimation of a microbial consortium for improved aerobic degradation of alpha-hexachlorocyclohexane.

A microbial consortium that can utilize alpha-hexachlorocyclohexane (alpha-HCH) as a sole source of carbon and energy was isolated from soil and sewage through a novel technique involving an initial enrichment in a glass column reactor followed by a shake flask enrichment. This consortium took 14 days to completely mineralize 5 and 10 microg mL(-)(1) alpha-HCH in mineral salts medium in shake flasks. The degradative ability of this consortium improved very markedly on acclimation by successive and repeated passages through media containing increasing concentrations of alpha-HCH. The acclimated consortium could degrade 100 microg mL(-)(1) of alpha-HCH within 72 h at a degradation rate of 58 microg mL(-)(1) day(-)(1) with concomitant release of stoichiometric amounts of chloride. Accumulation of any intermediary metabolites was not detected in the culture broth as tested by TLC and GC, implying complete mineralization of the substrate. The acclimated consortium contained eight bacterial strains and a fungus. The individual strains and the different permutations and combinations of them, however, were able to utilize only 10 microg mL(-)(1) of alpha-HCH. Mesophilic temperatures (20-30 degrees C) and near-neutral pH (6.0-8.0) were most favorable for alpha-HCH degradation. Among the auxiliary carbon sources tested, ethanol, benzoate, and glucose (at higher concentrations) retarded the degradation of alpha-HCH, whereas the addition of cellulose, sawdust, and low concentrations of glucose (<200 microg mL(-)(1)) and acetone enhanced the rate of degradation.

Pathways for 3-chloro- and 4-chlorobenzoate degradation in Pseudomonas aeruginosa 3mT.

A bacterial isolate, Pseudomonas aeruginosa 3mT exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l(-1)) and 4-chlorobenzoate (4-CBA 12 g l(-1)) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against 4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoate was readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.

Simultaneous degradation of 3-chlorobenzoate and phenolic compounds by a defined mixed culture ofPseudomonas spp.

A mixed culture of a chlorobenzoate-(3-CBA)-degradingPseudomonas aeruginosa, strain 3mT, and a phenol/cresols-degradingPseudomonas sp., strain CP4, simultaneously and efficiently degraded mixtures of 3-CBA and phenol/cresols. However, strains 3mT and CP4 usedortho- andmeta-ring cleavage pathways, respectively. Degradation of 3-CBA was complete when the 3-CBA was equal in amount to or less than that of phenol. CP4/3mT inoculum ratios (w/w) of 1:1 or 1:2 gave the most effective degradation of both the substrates in the mixture. The mixed culture degraded equimolar mixtures of 3-CBA/phenol up to 10MM. Equimolar mixtures of 3-CBA ando-, m- orp-cresol were also degraded by the mixed culture.

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4.

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.

Standardization of assay procedure and some properties of ribonuclease from Aspergillus candidus.

Screening of several fungal cultures resulted in the selection of an isolate of Aspergillus candidus which produced a considerable around of RNa-degrading enzyme in both surface and submerged methods of cultivation. The conditions for the assay of the RNAase were standardized at pH 4.5, 55 degrees C and using 0.25% yeast RNA as substrate. The enzyme was stable at pH 5.2. EDTA was found to activate the enzyme slightly. at temperatures 50-60 degrees C there was considerable loss in enzyme activity which was traced to the presence of a contaminating protease which presumably degraded the RNAase optimally at this temperature. The protease could be preferentially inactivated at or above 75 degrees C. The crude enzyme, in addition to RNAase was found to possess DNAase, nonspecific phosphodiesterase and 3'- and 5'-phosphomonoesterase activities.