Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria

Bioaugmentation has previously been unreliable for the in situ clean-up of contaminated soils because of problems with poor survival and the rapid decline in activity of the bacterial inoculum. In an attempt to solve these problems, a 500-l batch fermenter was investigated for its ability to deliver inoculum repeatedly to contaminated soils via irrigation lines. In a field experiment, mesocosms were filled with 350 kg soil containing 100 mg kg⁻¹ atrazine, and inoculated one, four or eight times with an atrazine-degrading bacterial consortium that was produced in the fermenter. After 12 weeks, no significant degradation of atrazine had occurred in soil that was inoculated only once; whereas, mesocosms inoculated four and eight times mineralized 38% and 72% of the atrazine respectively. Similar results were obtained in a laboratory experiment using soil contaminated with 100 mg kg⁻¹ [¹⁴C]atrazine. After 35 days, soil that was inoculated once with 10⁸ cfu ml⁻¹ of the consortium or with the atrazine-degrading bacterium, Pseudomonas sp. strain ADP, mineralized 17% and 35% of the atrazine respectively. In comparison, microcosms inoculated every 3 days with the consortium or with Pseudomonas sp. (ADP) mineralized 64% or 90% of the atrazine over this same period. Results of these experiments suggest that repeated inoculation from an automated fermenter may provide a strategy for bioaugmentation of contaminated soil with xenobiotic-degrading bacteria. Received: 20 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Degradation of phenol by polymer entrapped microorganisms

Summary A Pseudomonas sp. which was isolated from phenol-containing soil was immobilized in alginate and polyacrylamide-hydrazide (PAAH) and cultivated in a special airlift fermenter.The immobilized Pseudomonas sp. was able to degrade phenol at initial concentrations up to 2 g/l in less than 2 days, although the free cells did not grow at this concentration.The immobilization materials act as a protective cover against phenol, PAAH being more effective than alginate. The degradation activity as well as the outgrowth of bacteria can be manipulated by the concentration of the immobilization material, the temperature and the nitrogen content in the medium.The cells grew predominantly in microcolonies in the outer area of the beads when nitrogen was available as 1.0g NH₄NO₃/l and 0.5g (NH₄)₂SO₄/l.Prof. Dr. A. Fiechter dedicated to his 60th birthday     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Use of exogenous specialised bacteria in the biological detoxification of a dump site‐polychlorobiphenyl‐contaminated soil in slurry phase conditions

The possibility of biologically detoxifying a contaminated soil from an Italian dump site containing about 1500 mg/kg (in dry soil) of polychlorinated biphenyls was studied in the laboratory in this work. The soil, which contained indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids at concentration of about 300 CFU per g of air‐dried soil, was amended with inorganic nutrients, saturated with water and treated in aerobic 3‐L batch slurry reactors (soil suspension at 20% w/v). Either Pseudomonas sp. CPE1 strain, capable of cometabolising low‐chlorinated biphenyls into chlorobenzoic acids, or a bacterial co‐culture capable of aerobically dechlorinating polychlorobiphenyls constituted by this bacterium and the two chlorobenzoic acid degrading bacteria Pseudomonas sp. CPE2 strain and Alcaligenes sp. CPE3 strain, were used as inocula (final concentration of about 10⁸ CFU/mL for each bacterium), in the absence and in the presence of biphenyl (4 g/kg of air dried soil). Significant soil polychlorobiphenyl depletions were observed in all the reactors after 119 days of treatment. The soil inoculation with the sole CPE1 was found to slightly enhance the polychlorobiphenyl depletions (about 20%) and the soil detoxification; the effect was higher in the presence of biphenyl. The use of the polychlorobiphenyl mineralising bacterial co‐culture as inoculum resulted in a strong enhancement of the depletions of both the soil polychlorobiphenyls (from 50 to 65%) and of the original soil ecotoxicity. The bacterial biomass inoculated was found to implant into the soil; the higher specialised biomass availability thus reached in the inoculated soil was probably responsible of a more extensive biodegradation of polychlorobiphenyls and therefore of the higher detoxification yields observed in the inoculated reactors. The soil ecotoxicity, measured through two different soil contact assays, i.e., the Lepidium sativum germination test and the Collembola mortality test, was often found to decrease proportionally with the soil polychlorobiphenyl concentration. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 240–249, 1999.     

The atzABC Genes Encoding Atrazine Catabolism Are Located on a Self-Transmissible Plasmid in Pseudomonas sp. Strain ADP

Pseudomonas sp. strain ADP initiates atrazine catabolism via three enzymatic steps, encoded by atzA, -B, and -C, which yield cyanuric acid, a nitrogen source for many bacteria. In-well lysis, Southern hybridization, and plasmid transfer studies indicated that the atzA, -B, and -C genes are localized on a 96-kb self-transmissible plasmid, pADP-1, in Pseudomonas sp. strain ADP. High-performance liquid chromatography analyses showed that cyanuric acid degradation was not encoded by pADP-1. pADP-1 was transferred to Escherichia coli strains at a frequency of 4.7 × 10⁻². This suggests a potential molecular mechanism for the dispersion of the atzABC genes to other soil bacteria.     

Isolation of bacterial consortia for degradation of p-nitrophenol from agricultural soil

bacterial consortium has been isolated containing Pseudomonas spp. strains S1 and S2, which was able to degrade p‐nitrophenol (PNP). The strains were isolated from agricultural soil contaminated with organophosphorus pesticides. Pseudomonas spp. strain S2 could convert p‐nitrophenol to 4‐nitrocatechol (4NC) after pre‐exposure to phenol, when PNP was used as the only carbon source in the medium. Pseudomonas spp. strain S2, when mixed with strain S1 in the ratio 1:5 respectively, decolorised PNP completely.     

Effect of Bacteriophage on Colonization of Sugarbeet Roots by Fluorescent Pseudomonas spp

The colonization potential of two fluorescent Pseudomonas strains (M11/4, B2/6) that exhibit antifungal activity in vitro was studied on the roots of sugarbeet plants in a clay loam soil. The cell density of the introduced bacteria declined on the root system over a 16-day test period in nonsterile soil. Strain B2/6 declined at a significantly faster rate compared with M11/4. This loss in viability and difference in colonization ability between M11/4 and B2/6 was not observed in sterile soil. Nutrient deprivation induced by indigenous microorganisms was excluded as a key factor involved in the decline of the introduced bacteria on the basis that strains M11/4 and B2/6 retained viability when subjected to nutrient starvation conditions over a 16-day period. Experiments designed to test whether antagonism by indigenous microorganisms was responsible for the decline in the introduced fluorescent Pseudomonas sp. population revealed the presence of large numbers of bacteriophage in the soil capable of lysing strain B2/6. Reconstitution experiments carried out with sugarbeet seedlings inoculated independently with strains M11/4 and B2/6 and grown in sterile soil to which a soil phage filtrate had been added showed a significant decrease in the viability of strain B2/6 relative to M11/4. Phage antagonistic toward strain B2/6 were detected in 43% of soils taken from the major sugarbeet growing regions of Ireland.     

Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.     

Selecting inocula for the biodegradation of organic compounds at low concentrations

The inability of many organisms to degrade pollutants at low concentrations is a problem when selecting inocula for bioremediation of sites with these low concentrations. Thus, a study was conducted to determine the effect of low concentrations of p-nitrophenol (PNP) on growth of four PNP-degrading bacteria and their abilities to metabolize low concentrations of the compound in culture and samples from an oligotrophic lake. PNP did not increase the growth rates of Flavobacterium sp. M4, Pseudomonas sp. K, Flavobacterium sp. M1, and Pseudomonas sp. SP3 at concentrations of less than 2, 4, 10, and 100 ng/ml, respectively, when it was the sole added carbon source in culture, but it stimulated multiplication at higher concentrations. In liquid culture with the nitro compound as sole added carbon source, the four bacteria extensively mineralized PNP at 50 and 100 ng/ml, and three of the four degraded much of the substrate at 25 ng/ml. Pseudomonas sp. SP3 mineralized more than 20% but the two Flavobacterium strains converted less than 10% of the substrate to C02 at 10 ng/ml, and none of the three mineralized more than 5% at 1 and 5 ng PNP/ml. Under conditions where more than 99% of the radioactivity from ¹⁴C-PNP added at 1 ng/ml remained in solution, two of the isolates formed organic products. Pseudomonas sp. K had no activity at 1, 5, and 10 ng/ml. In contrast, when each of the bacteria was separately inoculated into samples of water from an oligotrophic lake and from a well in which PNP was not biodegraded, the bacteria were able to mineralize as little as 1 ng PNP/ml. The addition to a salts solution of 10 ng of glucose per ml resulted in mineralization of PNP at concentrations too low to be mineralized when the nitro compound was the sole source of added carbon. Bacteria may thus be able to mineralize substrates in natural waters at concentrations below those suggested by tests conducted in culture media, possibly because of the availability of other carbon sources for the bacteria.Offprint requests to: M. Alexander.     

Biodegradation of wood in crude oil-polluted soil

A field investigation (April–November) in Nigeria showed that biodegradation of obeche (Triplochiton scleroxylon) wood blocks was initially retarded in crude oil-contaminated soil but later became enhanced as indicated by loss of compression resistance. Further indication of this pattern was the detection of soft-rot cavities and basidiomycete fungi after 2–3 months exposure when compared to control blocks in uncontaminated soil. Laboratory tests with Pleurotus sp., Trametes sp., Gloeophyllum sp. (basidiomycetes) and Chaetomium sp. (soft-rot fungus) confirmed that degradation of crude oil-coated obeche blocks was markedly retarded without the presence of hydrocarbon-degrading bacteria. The filtrate of hydrocarbon-degrading Pseudomonas sp. grown in mineral salt/crude oil medium for 3–4 weeks supported growth of the test fungi better than in carboxymethyl cellulose medium but less than in potato dextrose broth. Similarly, wood blocks immersed in the filtrate became significantly more susceptible to fungal degradation. Pseudomonas sp. from stationary phase growth in crude oil medium depleted residual sugar in basidiomycete-degraded sawdust with a concomitant marked increase in its population. It may be concluded that readily metabolizable products of crude oil degradation by soil organisms and the removal of residual sugar which may have prevented catabolite repression of cellulases, culminated in increased attack on the wood by soil-borne wood-decomposing organisms.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Uptake modes of octadecane by Pseudomonas sp. DG17 and synthesis of biosurfactant

Aims: In order to gain more insight into the uptake modes of octadecane by bacteria. Methods and Results: A strain that could utilize octadecane well was isolated from crude oil contaminated soil, and named as Pseudomonas sp. DG17 by 16S rDNA analysis. Culture growth result showed that Pseudomonas sp. DG17 grew well in the addition of 200 and 400 mg l^?1 of octadecane, which showed that physical contact between substrate and bacteria was important in the substrate biodegradation. Meanwhile, Pseudomonas sp. DG17 produced rhamnolipids biosurfactant that contains 10 congeners, thus causing the surface tension of the culture medium decline and facilitating the contact between hydrocarbon and bacteria. Scanning‐electron‐microscopy results showed that a disruption of the surface membranes in certain zones was observed in some of the cells grown in 400 mg l^?1 octadecane at 176 h compared with the cells in exponential phase at 72 h due to the production of biosurfactant‐rhamnolipid. Conclusions: These results indicated the possibility that the direct contact with insoluble octadecane droplets occurred before the contact with pseudosolubilization smaller oil droplets. Significance: This report throws more light on the uptake mechanisms of octadecane by bacteria, and proposes the possibility that role of biosurfactant is to increase the contact between hydrocarbon and bacteria by changing the cell membrane structure which needs studied in depth. Impact of Study: Results of this study are useful in the bioremediation of petroleum polluted soil.     

Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and freePseudomonas sp. UG14Lr cells in creosote-contaminated soil slurries

The effects of nutrient amendment and alginate encapsulation on survival of and phenanthrene mineralization by the bioluminescentPseudomonas sp. UG14Lr in creosote-contaminated soil slurries were examined. UG14Lr was inoculated into creosote-contaminated soil slurries either as a free cell suspension or encapsulated in alginate beads prepared with montmorillonite clay and skim milk. Additional treatments were free-cell-inoculated slurries amended with sterile alginate beads, free-cell-inoculated and uninoculated slurries amended with skim milk only, and uninoculated, unamended slurries. Mineralization was determined by measuring¹⁴CO₂ released from radiolabelled phenanthrene. Survival was measured by selective plating and bioluminescence. Inclusion of skim milk was found to enhance both survival of and phenanthrene mineralization by free and encapsulated UG14Lr cells.     

不同来源溶藻菌的分离、鉴定及溶藻效果比较

为了探究从何种类型的自然生境中更易分离得到溶藻微生物,采用高氏1号培养基分别从水库底泥、湖泊底泥、农田土壤、林地土壤等四种来源共36份样品中分离了7 600株菌,并最终从中筛选得到了5株溶铜绿微囊藻(Microcystis aeruginosa)的溶藻菌,其中4株为假单胞菌(Pseudomonas sp.),1株为黄杆菌(Flavobacterium sp.),5株菌溶藻效率的变化范围为62%~95%。结果表明,当采用高氏1号培养基作为分离培养基时,湖泊底泥和水库底泥中的成功筛选概率最高,农田土壤次之,而林地土壤中则难以筛选得到,假单胞菌是较容易筛选得到的溶藻菌。     

Potential use of endophytic root bacteria and host plants to degrade hydrocarbons

Abstract

Microbe-assisted phytoremediation depends on competent root-associated microorganisms that enhance remediation efficiency of organic compounds. Endophytic bacteria are a key element of the root microbiome and may assist plant degradation of contaminants. The aim of this study was to investigate the application of four hydrocarbon-degrading endophytic strains previously isolated from an oil sands reclamation area. Strains EA1-17 (Stenotrophomonas sp.), EA2-30 (Flavobacterium sp.), EA4-40 (Pantoea sp.), and EA6-5 (Pseudomonas sp.) were inoculated in white sweet clover growing on soils amended with diesel at 5,000, 10,000, and 20,000?mg·kg^?1. Our results indicate that plant growth inhibition caused by diesel fuel toxicity was overcome in inoculated plants, which showed significantly higher plant biomass. Analysis of soil F2 and F3 hydrocarbon fractions also revealed that these soils were remediated by inoculated plants when diesel was applied at 10,000?mg·kg^?1 and 20,000?mg·kg^?1. In addition, quantification of hydrocarbon-degrading genes suggests that all bacterial strains successfully colonized sweet clover plants. Overall, the endophytic strain EA6-5 (Pseudomonas sp.), which harbored hydrocarbon-degrading genes, was the most effective candidate in phytoremediation experiments and could be a strategy to increase plant tolerance and hydrocarbon degradation in contaminated (e.g., diesel fuel) soils.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.     

Salt-tolerant and plant growth-promoting bacteria isolated from Zn/Cd contaminated soil: identification and effect on rice under saline conditions

The bacteria of PDMCd0501, PDMCd2007, and PDMZnCd2003 were isolated from a Zn/Cd contaminated soil. They were classified as salt-tolerant bacteria in this experiment. The bacteria had indole-3-acetic acids (IAA) production, nitrogen fixation, and phosphate solubilization, under 8% (w/v) NaCl condition. Biochemical test (API 20E) and 16S rDNA sequencing identified PDMCd2007 and PDMCd0501 as Serratia sp. and PDMZnCd2003 was Pseudomonas sp. The effect of Pseudomonas sp. PDMZnCd2003 on the germination and seedlings of Oryza sativa L.cv. RD6 was determined under a salinity of 0–16 dS/m. The salinity levels of 4–16 dS/m affected to decrease germination and seedlings of rice. Comparison between uninoculated and inoculated system, however, Pseudomonas sp. PDMZnCd2003 had a negative impact on the rice growth. This unexpected effect was a case that should be concerned and studied further before application as a plant growth-promoting bacteria (PGPB).     

Simultaneous Degradation of Atrazine and Phenol by Pseudomonas sp. Strain ADP: Effects of Toxicity and Adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na₂-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ~35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.     

Simultaneous biodegradation of pyridine and quinoline by two mixed bacterial strains

Experiments were conducted to provide data on the effectiveness of bioaugmentation in the removal of pyridine and quinoline from different wastewaters. A pyridine-degrading bacterial strain (Paracoccus sp. BW001) and a quinoline-degrading strain (Pseudomonas sp. BW003) were isolated from the activated sludge of a coking wastewater treatment plant. In this study, a consortium of these two bacterial strains was used as inoculum to simultaneously degrade pyridine and quinoline in three types of wastewaters: sterile synthetic, domestic, and industrial. In addition, variation of the bacterial community structures during degradation was monitored by denaturing gradient gel electrophoresis and amplicon length heterogeneity polymerase chain reaction techniques. The results of our experiments indicate that pyridine and quinoline can be removed efficiently using this inoculum but that the degradation process results in the production of ammonium as a by-product. Also, in the two actual wastewaters investigated, we observed that several autochthonous strains of bacteria in both the domestic and industrial wastewater were tolerant of pyridine and quinoline and grew rapidly.     

Synergistic interaction between two bacterial isolates in the degradation of carbofuran

The dominant bacteriaPseudomonas sp. andArthrobacter sp. were isolated from the standing water of carbofuran-retreatedAzolla plot.Arthrobacter sp. hydrolysed carbofuran added to the mineral salts medium as a sole source of carbon and nitrogen while no degradation occurred withPseudomonas sp. Interestingly, when the medium containing carbofuran was inoculated with bothArthrobacter sp. andPseudomonas sp., a synergistic increase in its hydrolysis and subsequent release of CO₂ from the side chain was noticed. This synergistic interaction was better expressed at 25° C than at 35° C. Likewise, related carbamates, carbaryl, bendiocarb and carbosulfan were more rapidly degraded in the combined presence of both bacterial isolates.