Robust linuron degradation in on-farm biopurification systems exposed to sequential environmental changes

On-farm biopurification systems (BPS) treat pesticide-contaminated wastewater of farms through biodegradation. Adding pesticide-primed soil has been shown to be beneficial for the establishment of pesticide-degrading populations in BPS. However, no data exist on the response of pesticide-degrading microbiota, either endogenous or introduced with pesticide-primed soil, when BPS are exposed to expected less favorable environmental conditions like cold periods, drought periods, and periods without a pesticide supply. Therefore, the response of microbiota mineralizing the herbicide linuron in BPS microcosm setups inoculated either with a linuron-primed soil or a nonprimed soil to a sequence of such less favorable conditions was examined. A period without linuron supply or a drought period reduced the size of the linuron-mineralizing community in both setups. The most severe effect was recorded for the setup containing nonprimed soil, in which stopping the linuron supply decreased the linuron degradation capacity to nondetectable levels. In both systems, linuron mineralization rapidly reestablished after conventional operation conditions were restored. A cold period and feeding with a pesticide mixture did not affect linuron mineralization. The changes in the linuron-mineralizing capacity in microcosms containing primed soil were associated with the dynamics of a particular Variovorax phylotype that previously had been associated with linuron mineralization. This study suggests that the pesticide-mineralizing community in BPS is robust in stress situations imposed by changes in environmental conditions expected to occur on farms. Moreover, it suggests that, in cases where effects do occur, recovery is rapid after restoring conventional operation conditions.     

Dynamics of the linuron hydrolase libA gene pool size in response to linuron application and environmental perturbations in agricultural soil and on-farm biopurification systems

libA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizing Variovorax sp. strain SRS16. In order to assess the contribution of libA to linuron degradation in environmental settings, libA abundance was monitored in response to the application of linuron and to environmental perturbations in agricultural soil microcosms and microcosms simulating the matrix of on-farm biopurification systems. libA numbers were measured by real-time PCR and linked to reported data of Variovorax community composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup, libA numbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particular Variovorax phylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence of libA can be linked to the linuron mineralization capacity and that libA is primarily hosted by Variovorax phylotype A strains. However, the results also suggest that, apart from libA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.     

Method for rapid screening of pesticide mineralization in soil

A method has been developed for the analysis of (14)CO(2) evolution from the mineralization of (14)C-labelled organic compounds in soil samples. The new method is less space demanding and substantially cuts down laborious manual work compared to the traditional incubation bottle method used. Furthermore, the use of scintillation cocktail is largely reduced with the new method. In the new method, (14)CO(2) is trapped in filter paper held in the lid of a 20 ml glass vial by surface tension. The trapping solution used is Ca(OH)(2), which fixates CO(2) in the filter paper and the analysis of trapped (14)CO(2) is done using the Cyclone trade mark Storage Phosphor system. The lids are placed in a 32 well holder and exposed to a phosphor screen prior to scanning in a Cyclone trade mark scanner. The new filter method has been tested and compared to results obtained using the traditional method. The results show good agreement but due to a smaller capacity for CO(2) with the filter method compared to the traditional method, the interval between sampling has to be shorter using the filter method when the CO(2) development is high. The detection limits for the filter method is higher compared to the traditional method. With the filter method, the level of radioactivity has to exceed 300 dpm before detection is possible, while the same limit for the traditional method is around 30 dpm. On the other hand, the gas trapping faster and the efficiency is higher with the filter method.     

Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil

The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil. A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass. Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass. Within 30 days, PCP-degrading bacteria increased from 10⁵ cfu/g to 10⁸ cfu/g soil. Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution. The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l. This high level of tolerance was attributed to the beneficial effect of the soil particles. Once produced, the activated soil biomass remained active for 5 weeks at 20 °C and for up to 3 months when kept at 4 °C. The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms. Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration. The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Received: 31 March 1997 / Received revision: 22 July 1997 / Accepted: 25 August 1997     

Improvement of sludge digestate biodegradability by thermophilic bioaugmentation

The sludge digestate stabilized by mesophilic anaerobic digestion was further degraded through thermophilic anaerobic digestion using 0–10 % (v/v) of thermophilic, proteolytic Coprothermobacter proteolyticus, and/or methanogenic granular sludge. The results demonstrated that the temperature shift to thermophilic condition promoted abiotic solubilization of proteins and reactivated the fermentative bacteria and methanogens indigenous in the sludge digestate, resulting in a final methane yield of 6.25 mmol-CH₄/g-volatile suspended solid (VSS) digestate. The addition of C. proteolyticus accelerated the hydrolysis and fermentation of proteins and polysaccharides in the digestate during the early stage of thermophilic anaerobic digestion and stimulated methane production by syntrophic cooperation with methanogenic granular sludge. In the treatment with granular sludge and inoculated with 10 % (v/v) of C. proteolyticus, a final methane yield of 7 mmol-CH₄/g-VSS digestate was obtained, and 48.4 % proteins and 27.0 % polysaccharides were degraded. The dissolved proteins were contributed by abiotic factor, C. proteolyticus, and indigenous digestate bacteria, respectively, by around 16, 28, and 56 %.     

Decontamination of a polychlorinated biphenyls-contaminated soil by phytoremediation-assisted bioaugmentation

A 70 day pot experiment was conducted for the cleaning-up of a PCBs-contaminated soil (104 mg kg^?1 soil DW) using bioaugmentation with Burkholderia xenovorans LB400 (LB400) assisted or not by the use of tall fescue (Festuca arundinacea). The total cultivable bacteria of the soil were higher with the presence of plants. Real-time PCR showed that LB400 (targeting 16S–23S rRNA ITS) survived with abundance related to total bacteria (targeting 16S rRNA) being higher with fescue (up to a factor of three). Bioaugmentation had a positive effect on fescue biomass and more specifically on roots (by a factor of three). PCB dissipation (sum of congeners 28, 52, 101, 118, 153, 180) averaged 13 % (bioaugmented-planted) up to 32 % (non bioaugmented-planted), without any significant difference between treatments. Basically our results demonstrated that indigenous bacteria were able to dissipate PCBs (26.2 % dissipation). PCB dissipation was not related to the abundance of LB400 or to the total bacterial counts. Bioaugmentation or fescue altered the structure of the bacterial community of the soil, not the combination of both. Principal component analysis showed that bioaugmentation tended to improve the control of the process (lower variability in PCB dissipation). Opposite to that bioaugmentation increased the variability of the structure of the bacterial community.     

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5·0 mg g^?1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2·50 × 10⁹ g^?1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cyω10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol‐contaminated areas.     

Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia

Biodegradation of a mixture of PAHs was assessed in forest soil microcosms performed either without or with bioaugmentation using individual fungi and bacterial and a fungal consortia. Respiratory activity, metabolic intermediates and extent of PAH degradation were determined. In all microcosms the low molecular weight PAH’s naphthalene, phenanthrene and anthracene, showed a rapid initial rate of removal. However, bioaugmentation did not significantly affect the biodegradation efficiency for these compounds. Significantly slower degradation rates were demonstrated for the high molecular weight PAH’s pyrene, benz[a]anthracene and benz[a]pyrene. Bioaugmentation did not improve the rate or extent of PAH degradation, except in the case of Aspergillus sp. Respiratory activity was determined by CO₂ evolution and correlated roughly with the rate and timing of PAH removal. This indicated that the PAHs were being used as an energy source. The native microbiota responded rapidly to the addition of the PAHs and demonstrated the ability to degrade all of the PAHs added to the soil, indicating their ability to remediate PAH-contaminated soils.     

Biodegradation and ecotoxicity of soil contaminated by pentachlorophenol applying bioaugmentation and addition of sorbents

Biodegradation of pentachlorophenol (PCP) in soil by autochthonous microorganisms and in soil bioaugmented by the bacterial strain Comamonas testosteroni CCM 7530 was studied. Subsequent addition of organomineral complex (OMC) or lignite as possible sorbents for PCP immobilization has been investigated as well. The OMC was prepared from humic acids (HAs) isolated from lignite by binding them onto zeolite. Biodegradation of PCP and number of colony forming units (CFUs) were determined in the three types of soil, Chernozem, Fluvisol, and Regosol, freshly spiked with PCP and amended separately with tested sorbents. The enhancing effect of sorbent addition and bioaugmentation on PCP biodegradation depended mainly on the soil type and the initial PCP concentration. Microbial activity resulted in biotransformation of PCP into certain toxic substances, probably lower chlorinated phenols that are more soluble than PCP, and therefore more toxic to present biota. Therefore, it was necessary to monitor soil ecotoxicity during biodegradation. Addition of the OMC resulted in a more significant decrease of soil toxicity in comparison with addition of lignite. Lignite and OMC appear to be good traps for PCP with potential application in remediation technology.     

Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration

A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6-TCP with stoichiometric release of Cl⁻. In cultures of Alc. eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation. Experiments conducted with soil microcosms gave evidence that : the degradative process had a biotic nature and was accompanied by microbial growth ; the soil used presented an intrinsic degradative capacity versus 2,4,6-TCP ; the specialized organism used as inoculum was effective in degrading 2,4,6-TCP in a short time. These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil.     

Phytoremediation of pesticide wastes in soil

Soils at agrochemical dealer sites often are contaminated with pesticide residues from decades of accidental and incidental spillage. We have determined that prairie grasses native to the Midwestern U.S. are suitable for phytoremediation because they are tolerant of most herbicides and of climatic extremes, such as heat, cold, drought, and flooding. A mixed stand of big bluestem, switch grass, and yellow indiangrass develops a rhizosphere with microflora that can readily detoxify pesticide residues. Specific atrazine-degrading bacteria or the free enzyme atrazine chlorohydrolase also can enhance the rate of biotransformation of atrazine in soil. Metolachlor degradation can be accelerated significantly by the prairie grass/rhizosphere effect. Several grasses used in filter strips have also been evaluated for their pesticide-degradation capabilities. The prairie grasses also have been demonstrated to reduce the rates of leaching of pesticides through intact soil columns, since less water leaches out of vegetated soil columns compared to non-vegetated soil columns. The evaluation of the degree of success of remediation has relied heavily on chemical residue analysis, but recent studies on biological endpoints have shown promise for providing more ecologically relevant indications of the potential exposure of organisms to pesticides in the soil. Earthworm 8-day bioaccumulation assays and root growth assays have shown the value of assessing the bioavailability of the residues. Mass balance experiments have utilized radiolabeled atrazine and metolachlor to ascertain the complete metabolism and binding profile of those two pesticides in phytoremediation studies.     

Microbial consortium bioaugmentation of a polycyclic aromatic hydrocarbons contaminated soil

In this study we evaluated the capacity of a defined microbial consortium (five bacteria: Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, Microbacteriaceae bacterium, Naphthalene-utilizing bacterium; and a fungus identified as Fusarium oxysporum) isolated from a PAHs contaminated landfarm site to degrade and mineralize different concentrations (0, 250, 500 and 1000 mg kg(-1)) of anthracene, phenanthrene and pyrene in soil. PAHs degradation and mineralization was evaluated by gas chromatography and respirometry, respectively. The microbial consortium degraded on average, 99%, 99% and 96% of the different concentrations of anthracene, phenanthrene and pyrene in the soil, in 70 days, respectively. This consortium mineralized 78%, on average, of the different concentrations of the 3 PAHs in soil after 70 days. Contrarily, the autochthonous soil microbial population showed no substantial mineralization of the PAHs. Bacterial and fungal isolates from the consortium, when inoculated separately to the soil, were less effective in anthracene mineralization compared to the consortium. This signifies synergistic promotion of PAHs mineralization by mixtures of the monoculture isolates (the microbial consortium).     

Isolation of toxic metal-tolerant bacteria from soil and examination of their bioaugmentation potentiality by pot studies in cadmium- and lead-contaminated soil

Heavy metals, also regarded as toxic metals, are the important environmental pollutants that affect all forms of life. Accumulation of toxic metals in plants results in various biochemical, physiological and structural disturbances, leading to inhibited growth and sometimes plant death. Toxic metal contamination disturbs the soil ecology as well as the agricultural productivity. Several indigenous microbes can withstand the effect of toxic metal and play a vital role in the revival of tarnished soil. In the present study, soil samples were collected from contaminated crop field of Cachar district of Assam, India. Segregation, enumeration and identification of bacteria from soil samples were performed. Among all the tested isolates, very few were able to withstand a high concentration of Cd and Pb in nutrient agar plates. Toxic metal-tolerant bacteria were identified as Pseudomonas aeruginosa and Bacillus cereus. The isolates having a higher tolerance for Cd and Pb were taken into consideration for pot studies. P. aeruginosa strain SN4 and strain SN5 showed significant results at Cd- and Pb-contaminated soil, evidenced by the healthy growth of Oryza sativa seedlings. However, B. cereus strain SN6 showed high tolerance towards Cd and Pb, but pot experimental studies showed adverse effects on seedling germination and shoot growth of O. sativa. P. aeruginosa strains were significantly able to reduce the negative impact of Cd and Pb in the soil, thus finding an alternative in removal, recovery and remediation of toxic metal-contaminated crop field.     

Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848     

Effects of rhizosphere remediation and bioaugmentation on carbofuran removal from soil

Rhizosphere soil contains important sources of nutrients for microorganisms resulting in high number of microorganisms capable of degrading various types of chemicals in the soil. Thus, this study investigated a carbofuran dissipation in rhizosphere soils of 6 weeds namely, umbrella sedge (Cyperus iria L.), fuzzy flatsedge (C. pilosus V.), small flower umbrella plant (C. difformis L.), tall-fringe-rush hoorah grass (Fimbristylis miliacea V.), cover fern (Marsilea crenata P.), and water primrose (Jussiaea linifolia V.). Rhizosphere soil of fuzzy flatsedge showed the shortest half-life (t_1/2) of carbofuran (15 days) among other soils. So, it was selected to be used in the bioaugmentation experiment using carbofuran degrader namely Burkholderia cepacia, PCL3, as inoculum in order to examine whether they would improve carbofuran degradation in soil. The results showed that the addition of PCL3 into rhizosphere soil did not improve carbofuran degradation suggesting that microorganisms in rhizosphere soil might be capable enough to remove carbofuran from soil. The number of carbofuran degraders in the rhizosphere soils was greater than in bulk soil 10–100 times which might be responsible to a rapid degradation of carbofuran in rhizosphere soils without the addition of PCL3. The ability of PCL3 to degrade carbofuran was evident in bulk soil (t_1/2 of 12 days) and autoclaved soils (t_1/2 13–14 days) when compared to soils without an inoculation (t_1/2 of 58 days) indicated that the addition of a degrader was useful in improving carbofuran degradation in soil.     

Biostimulation and bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil

Bioremediation of weathered diesel fuel in Arctic soil at low temperature was studied both on-site in small-scale biopiles and in laboratory microcosms. The field study site was on Ellesmere Island (82°30'N, 62°20'W). Biostimulation was by fertilization with phosphorous and nitrogen. Bioaugmentation was with an enrichment culture originating from the field site. In biopiles, total petroleum hydrocarbons (TPH) were reduced from 2.9 to 0.5 mg/g of dry soil over a period of 65 days. In microcosms at 7 °C, TPH were reduced from 2.4 to 0.5 mg/g of dry soil over a period of 90 days. Inoculation had no effect on hydrocarbon removal in biopiles or in microcosms. Maximum TPH removal rates in the biopiles were approximately 90 μg of TPH g^–1 of soil day^–1, occurring during the first 14 days when ambient temperature ranged from 0 to 10 °C. The fate of three phylotypes present in the inoculum was monitored using most-probable-number PCR, targeting 16S rRNA genes. Populations of all three phylotypes increased more than 100-fold during incubation of both uninoculated and inoculated biopiles. The inoculum increased the initial populations of the phylotypes but did not significantly affect their final populations. Thus, biostimulation on site enriched populations that were also selected in laboratory enrichment cultures. Electronic Publication     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.