Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

Summary The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1^–1.h^–1 for several weeks. Correspondence to: H.-J. Rehm     

Approaching chlorpyrifos bioelimination at bench scale bioreactor

Chlorpyrifos (CP) is one of the most commonly applied insecticides for control of pests and insects. The inappropriate use of this kind of chemicals has caused heavy contamination of many terrestrial and aquatic ecosystems thus representing a great environmental and health risk. The main purpose of this work is to investigate novel microbial agents (Pseudomonas stutzeri and the previously obtained consortium LB2) with the ability to degrade CP from polluted effluents. This goal was achieved by operating at different lab scales (flask and bioreactor) and operation modes (batch and fed-batch). Very low degradation and biomass levels were detected in cultures performed with the consortium LB2. In contrast, near complete CP degradation was reached by P. stutzeri at the optimal conditions in less than 1 month, showing a depletion rate of 0.054 h^?1. The scale-up at bench scale stirred tank bioreactor allowed improving the specific degradation rate in ten folds and total CP degradation was obtained after 2 days. Moreover, biomass and biodegradation profiles were modelled to reach a better characterization of the bioremediation process.     

Enhanced-rate biodegradation of organophosphate neurotoxins by immobilized nongrowing bacteria

Pesticide wastes generated from livestock dipping operations containing the organophosphate (OP) insecticide coumaphos (CP) are well suited for disposal by biodegradation since they are highly concentrated (approximately 1 g/L), generally contained, and lack additional toxic components. In this study, a significantly enhanced efficiency of degrading CP in cattle dip waste (CDW) is reported using a dense, nongrowing cell population that functions without the addition of nutrients required for growing cell cultures. A recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), which is capable of active hydrolysis of OP neurotoxins including CP, was cultivated in a rich medium containing all essential nutrients. Cells were harvested and utilized in lab scale experiments in the form of either freely suspended cells or cells immobilized within a macroporous gel matrix, poly(vinyl alcohol) (PVA) cryogel. Significantly higher degradation rates were achieved with either suspended or immobilized OPH(+) cells compared to rates with the microbial consortium naturally present in CDW. Of the two nongrowing cell systems, the detoxification rate with immobilized cells was approximately twice that of freely suspended cells, and kinetic studies demonstrated that a higher maximum reaction rate was achieved with the immobilized cell system. A comparative study using both the CDW and pure CP substrates with free cells indicated that the CDW contained one or more factors that reduced the bioavailability of CP. The immobilized cells retained their activity over a 4-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability.     

Hydrocarbon degradation capacity and population dynamics of a microbial consortium obtained using a sequencing batch reactor in the presence of molasses

A native microbial consortium capable of degrading hydrocarbons was employed as an inoculum source in a sequencing batch reactor (SBR) using molasses as a carbon source. The microbial biomass in the SBR was able to grow in the presence of molasses, degrading 88% of the reducing sugar. Moreover, the consortium produced in the SBR was capable of maintaining 75% of the capacity for biodegradation of oil with respect to the original capacity of the native microbial consortium. Monitoring of the microbial population structure was accomplished using PCR-DGGE. The results indicated that the microbial populations grown in molasses were stable during crude oil degradation, as judged by comparison to the population structure of the native microbial consortium. The results obtained demonstrated that molasses could be used as a carbon source to promote the growth of biomass with oildegrading capacity.     

Effect of Added Heavy Metal Ions on Biotransformation and Biodegradation of 2-Chlorophenol and 3-Chlorobenzoate in Anaerobic Bacterial Consortia

The effect of added Cd(II), Cu(II), Cr(VI), or Hg(II) at 0.01 to 100 ppm on metabolism in anaerobic bacterial consortia which degrade 2-chlorophenol (2CP), 3-chlorobenzoate (3CB), phenol, and benzoate was examined. Three effects were observed, including extended acclimation periods (0.1 to 2.0 ppm), reduced dechlorination or biodegradation rates (0.1 to 2.0 ppm), and failure to dechlorinate or biodegrade the target compound (0.5 to 5.0 ppm). 3CB biodegradation was most sensitive to Cd(II) and Cr(VI). Biodegradation of benzoate and phenol was most sensitive to Cu(II) and Hg(II), respectively. Adding Cr(VI) at 0.01 ppm increased biodegradation rates of phenol (177%) and benzoate (169%), while Cd(II) and Cu(II) at 0.01 ppm enhanced biodegradation rates of benzoate (185%) and 2CP (168%), respectively. Interestingly, with Hg(II) at 1.0 to 2.0 ppm, 2CP and 3CB were biodegraded 133 to 154% faster than controls after an extended acclimation period, suggesting adaptation to Hg(II). Metal ions were added at inhibitory, but sublethal, concentrations to investigate effects on metabolic intermediates and end products. Phenol accumulated to concentrations higher than those in controls only in the 2CP consortium with added Cu(II) at 1.2 ppm but was subsequently degraded. There was no effect on benzoate, and little effect on acetate intermediates was observed. In most cases, methane yields were reduced by 23 to 97%. Thus, dehalogenation, aromatic degradation, and methanogenesis in these anaerobic consortia showed differential sensitivities to the heavy metal ions added. These data indicate that the presence of heavy metals can affect the outcome of anaerobic bioremediation of aromatic pollutants. In addition, a potential exists to use combinations of anaerobic bacterial species to bioremediate sites contaminated with both heavy metals and aromatic pollutants.     

Utilization of microbial community potential for removal of chlorpyrifos: a review

Chlorpyrifos (CP) is the most commonly used pesticide in agricultural fields worldwide. Exposure to CP and its metabolites creates severe neuron-disorders in human beings. Improper handling and uncontrolled application of CP by farmers have lead to the contamination of surface and ground water bodies. Biodegradation offers an efficient and cost effective method for the removal of CP and other toxic organophosphorus pesticides from the contaminated environment. The degradation of CP by various microorganisms has been investigated by several researchers over the past few years. This review presents a critical summary of the recent published results on the biodegradation of CP. A diverse range of bacterial species such as Agrobacterium sp., Alcaligenes faecalis, Enterobacter sp. Arthrobacter sp. Bacillus pumilus, Pseudomonas sp. etc., fungal species like Trichoderma viridae, Aspergillus niger, Verticillium sp., Acremonium sp. Cladosporium cladosporiodes, etc. and certain algal species viz. Chlorella vulgaris, Spirulina platensis, Synechocystis sp., etc., have been shown to degrade CP. The efficacy of these communities for CP degradation in batch and continuous modes has also been discussed but more studies are required on continuous reactors. Also, the available published information on kinetics of biodegradation of CP along with the available results on molecular biological approaches are discussed in this work.     

CP1菌株的分离、筛选及其对毒死蜱的降解

从生产毒死蜱的农药生产厂曝气池中分离、筛选到降解毒死蜱且能以毒死蜱为唯一碳源生长的微生物菌株,命名为CP1。根据该菌株的Biolog特性鉴定和16S rRNA序列相似性分析,初步鉴定该菌为苍白杆菌属(Ochrobactrum sp.)。利用正交实验和Box-Behnken响应面法对影响CP1菌株降解毒死蜱的主要因素进行优化分析,得到菌株CP1对毒死蜱的最适降解条件为:农药浓度100 mg/L,pH值7.0,温度为28.5°C。优化后,CP1对毒死蜱的降解率由最初的70.26%提高到75.18%。毒死蜱降解优化试验提高了CP1菌株对毒死蜱的生物降解性能。     

Control of carbon and ammonium ratio for simultaneous nitrification and denitrification in a sequencing batch bioreactor

This study shows how the carbon and nitrogen (C/N) ratio controls the simultaneous occurrence of nitrification and denitrification in a sequencing batch reactor (SBR). Data demonstrated that a low C/N ratio resulted in a rapid carbon deficit, causing an unbalanced simultaneous nitrification–denitrification (SND) process in SBR. When the initial COD/NH₄⁺-N ratio was adjusted to 11.1, the SND-based SBR achieved complete removal of NH₄-N and COD without leaving any NO₂⁻-N in the effluent. The nitrogen removal efficiency decreases gradually with increasing ammonium-loading rate to the SND–SBR system. Altogether, data showed that appropriate controls of carbon and nitrogen input are required to achieve an efficient SND–SBR. An established SND technology can save operation time and energy, and might replace the traditional two-stage biological nitrification and denitrification process.     

生物膜法和SBR法相结合处理难降解制药废水的研究

采用生物膜法和SBR法相结合的废水处理工艺处理含抗生素类等难降解的制药废水 ,对生物膜的耐冲击负荷能力、生物膜对进水可生化性的影响、生物膜对好氧SBR活性污泥性能的影响、pH对系统去除效果的影响等工艺条件进行研究 ,并通过与传统SBR处理工艺的对比试验 ,进一步揭示了生物膜法和SBR法相结合的处理工艺强的耐冲击负荷能力。     

Degradation pathway, toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR

The present study deals with cultivation of 2,4,6-trichlorophenol (TCP) degrading aerobic granules in two SBR systems based on glucose and acetate as co-substrate. Biodegradation of TCP containing wastewater starting from 10 to 360 mg L⁻¹ with more than 90% efficiency was achieved. Sludge volume index decreases as the operation proceeds to stabilize at 35 and 30 mL g⁻¹ while MLVSS increases from 4 to 6.5 and 6.2 g L⁻¹ for R1 (with glucose as co-substrate) and R2 (with sodium acetate as co-substrate), respectively. FTIR, GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocatechol pathway and the cleavage may be at ortho-position. Haldane model for inhibitory substrate was applied to the system and it was observed that glucose fed granules have a high specific degradation rate and efficiency than acetate fed granules. Genotoxicity studies shows that effluent coming from SBRs was non-toxic.     

Degradation of xenobiotic compounds in situ: Capabilities and limits

Abstract: Exploiting microorganisms for remediation of waste sites is a promising alternative to groundwater pumping and above ground treatment. The objective of in situ bioremediation is to stimulate the growth of indigenous or introduced microorganisms in regions of subsurface contamination, and thus to provide direct contact between microorganisms and the dissolved and sorbed contaminants for biotransformation. Subsurface microorganisms detected at a former manufactured gas plant site contaminated with coal tars mineralized significant amounts of naphthalene (8–43%) and phenanthrene (3–31%) in sediment-water microcosms incubated for 4 weeks under aerobic conditions. Evidence was obtained for naphthalene mineralization (8–13%) in the absence of oxygen in field samples. These data suggest that biodegradation of these compounds is occurring at the site, and the prospects are good for enhancing this biodegradation. Additional batch studies demonstrated that sorption of naphthalene onto aquifer materials reduced the extent and rate of biodegradation, indicating that desorption rate was controlling the biodegradation performance.     

Microbiological Activities of Coenzyme Forms of Vitamin B12

A novel quinoline-degrading strain, named K4, was isolated from activated sludge of a coking wastewater treatment plant and identified as Brevundimonas sp. on the basis of its 16s rDNA gene sequence analysis. Its optimum temperature and pH for quinoline degradation were 30 °C and pH 9.0, respectively, and during the biodegradation process, at 100 mg/L initial quinoline concentration, an inoculation amount of 8% (OD600 of 0.23) was the optimal strain concentration. In addition, the kinetics of free K4 strains for quinoline degradation showed that it followed a zero-order equation. Furthermore, compared with free K4 strains, immobilized K4 strains’ potential for quinoline degradation was investigated by adding both of them into SBR reactors for actual coking wastewater treatment on operation over 15 days. The results showed that bioaugmentation by both free and immobilized K4 strains enhanced quinoline removal efficiency, and especially, the latter could reach its stable removal after a shorter accommodation period, with 94.8% of mean quinoline removal efficiency.     

Biodegradation of methyl tert-butyl ether as a sole carbon source by aerobic granules cultivated in a sequencing batch reactor

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) were successfully developed in a well-mixed sequencing batch reactor (SBR). Treatment efficiency of MTBE in the reactor during the stable operations exceeded 99.8%, and effluent MTBE was consistently below 800 mug/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentrations from 25 to 400 mg/L, peaked at 18.2 mg-MTBE/g-VSS h, and declined with further increases in MTBE concentration as substrate inhibition effects became significant. There was a good fit between these biodegradation data and the Haldane equation (R (2) = 0.976). Microbial community DNA profiling was carried out using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction amplified 16S rDNA. The aerobic granule was found to contain a wide diversity of microorganisms. More than 70% similarity among the samples in the time period examined indicated a highly stable microbial community as the reactor reached the stable operation.     

Impact of chlortetracycline on sequencing batch reactor performance for swine manure treatment

Treatment of aged (500 day, 4 °C stored) chlortetracycline (CTC; 0, 20, 40, 80 mg/L CTC)-amended swine manure using two cycle, 22 day stage anaerobic sequencing batch reactors (SBR) was assessed. Eighty milligrams per liter CTC treatment inhibited SBR treatment efficiencies, although total gas production was enhanced compared to the no-CTC treatment. The 20 and 40 mg/L CTC treatments resulted in either slight or no differences to SBR treatment efficiencies and microbial diversities compared to the no-CTC treatment, and were generally similar to no-CTC treatments upon completion of the first 22 day SBR cycle. All CTC treatments enhanced SBR gas generation, however CH₄ yields were lowest for the 80 mg/L CTC treatment (0.111 L CH₄/g tCOD) upon completion of the second SBR react cycle. After a 22 day acclimation period, the 80 mg/L CTC treatment inhibited methanogenesis due to acetate accumulation, and decreased microbial diversity and CH₄ yield compared to the no-CTC treatment.     

Improving utilization of crop residues. Evaluation of two chemicals used to improve digestibility of crop residues

Two chemical treatments involving chelating metal caustic swelling (CMCS) and sodium hydroxide (NaOH) were evaluated for their ability to affect in vitro dry matter and in vitro cellulose disappearance (IVDMD and IVCD, respectively), dry matter disappearance in sacco and the chemical composition of two low-quality crop residues, cornstalks (CS) and soya bean residue (SBR). At $\text{chemical}\text{substrate}$ ratios ranging from 0.25:1 to 5:1, linear increases in IVCD of both roughages were noted. Improvements in IVCD of CS and SBR were noted at 1:1, 3:1 and 5:1$\text{chemical}\text{substrate}$ ratios, regardless of whether the treated roughages were used immediately after treatment (fresh basis) or preserved by drying or freezing. Decreased concentrations of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL) and crude protein (CP) resulted from chemical treatment. No effects of the water component of the solvent solutions were observed. Dry matter disappearance in sacco of CMCS- and NaOH-treated roughages was increased at all $\text{chemical}\text{substrate}$ ratios tested. Treatment of either roughage with CMCS resulted in greater digestibility in sacco than did treatment with NaOH. Treatment of CS with CMCS resulted in higher rates of digestion than did treatment with NaOH. No differences in rates of digestion of SBR were noted between chemicals. Both chemicals were more effective in improving digestibility of CS than of SBR.     

Biodegradation of 4-chlorophenol by <Emphasis Type="Italic">Arthrobacter chlorophenolicus A6</Emphasis>: effect of culture conditions and degradation kinetics

Among known microbial species, Arthrobacter chlorophenolicus A6 has shown very good potential to treat phenolic wastewaters. In this study, the levels of various culture conditions, namely initial pH, agitation (rpm), temperature (°C), and inoculum age (h) were optimized to enhance 4-chlorophenol (4-CP) biodegradation and the culture specific growth rate. For optimization, central composite design of experiments followed by response surface methodology (RSM) was applied. Results showed that among the four independent variables, i.e., pH, agitation (rpm), temperature (°C), and inoculum age (h) investigated in this study, interaction effect between agitation and inoculum age as well as that between agitation and temperature were significant on both 4-CP biodegradation efficiency and culture specific growth rate. Also, at the RSM optimized settings of 7.5 pH, 207 rpm, 29.6°C and 39.5 h inoculum age, 100% biodegradation of 4-CP at a high initial concentration of 300 mg l⁻¹ was achieved within a short span of 18.5 h of culture. The enhancement in the 4-CP biodegradation efficiency was found to be 23% higher than that obtained at the unoptimized settings of the culture conditions. Results of batch growth kinetics of A. chlorophenolicus A6 for various 4-CP initial concentrations revealed that the culture followed substrate inhibition kinetics. Biokinetic constants involved in the process were estimated by fitting the experimental data to several models available from the literature.     

Removal and biodegradation of nonylphenol by immobilized Chlorella vulgaris

The removal and biodegradation of nonylphenol (NP) by alginate-immobilized cells of Chlorella vulgaris were compared with their respective free cultures. The effects of four cell densities of 10(4) per algal bead were investigated, as were the four algal bead concentrations, with regard to the removal and biodegradation of NP. Although immobilization significantly decreased the growth rate and NP's biodegradation efficiency of C. vulgaris, NP removal over a short period was enhanced. The NP removal mechanism by immobilized cells was similar to that by free cells, including adsorption onto alginate matrix and algal cells, absorption within cells and cellular biodegradation. The optimal cell density and bead concentration for the removal and biodegradation of NP was 50-100×10(4) cells algal bead(-1) and 2-4 beads ml(-1) of wastewater, respectively. These results demonstrated that immobilized C. vulgaris cells under optimal biomass and photoautotrophic conditions are effective in removing NP from contaminated water.     

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h⁻¹, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research. Received: 14 May 1998 / Received last revision: 30 July 1998 / Accepted: 31 July 1998     

Enhanced Biodegradation of Diesel Oil in Seawater Supplemented with Nutrients

The imbalance of C, N, and P caused by the spilled oil could be regulated by the addition of nitrogen and phosphorous. Moreover, different kinds of N and P sources were used in order to stimulate oil biodegradation under laboratory and field conditions, but the results were conflicting. To evaluate the effectiveness of nutrient supplementation, N sources (NO₃‐N and NH₄‐N) and P sources (PO₄‐P) were applied to the simulated diesel‐polluted seawater in the N/P ratio of 10:1 and 20:1, respectively. The results showed that the addition of nutrients increased the oil biodegradation rate and the counts of petroleum degrading bacteria (PDB) and heterotrophic bacteria (HB). A strongly positive correlation existed (the interrelated coefficient was nearly 0.9) between the percentage ratio of PDB/HB and the oil biodegradation rates, and therefore the percentage ratio of PDB/HB could be used as a good indicator to predict oil biodegradation. Among the four samples treated with nutrients, the biodegradation efficiency of the group amended with NO₃‐N and PO₄‐P in the ratio of 10:1 (10NO₃‐P group) was as much as 75.8 %, while in the 10NH₄‐P, 20NO₃‐P and 20NH₄‐P groups this value was 61.3 %, 52.4 % and 40.5, respectively. It would take natural degradation without nutrient supplementation about 78 days to achieve the result obtained within 14 days with 10NO₃‐P amendment . Chemical and microbiological analyses confirmed that the addition of nutrients in the same N/P ratio remarkably enhanced the biodegradation rate and the counts of microorganisms in the NO₃‐N treated groups, indicating that the microorganisms tend to utilize NO₃‐N rather than NH₄‐N as their growth N source. When the same kind of N source was added to the system, the promoted efficiency in the 10:1 (N/P ratio) groups was notable compared to the 20:1 groups, i.e., adding nutrients in the ratio of 10:1 is superior in the stimulation of oil biodegradation to the ratio of 20:1.     

X-ray structures of 4-chlorocatechol 1,2-dioxygenase adducts with substituted catechols: New perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity

The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols.The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates. The analysis of the present structures allowed to identify the residues selectively involved in recognition of the diverse substrates.Furthermore the structural comparison with the corresponding complexes of catechol 1,2-dioxygenase from the same Rhodococcus strain (Rho-1,2-CTD) highlights significant differences in the binding of the tested catechols to the active site of the enzyme, particularly in the orientation of the aromatic ring substituents. As an example the 3-substituted catechols are bound with the substituent oriented towards the external part of the 4-CCD active site cavity, whereas in the Rho-1,2-CTD complexes the 3-substituents were placed in the internal position. The present crystallographic study shed light on the mechanism that allows substrate recognition inside this class of high specific enzymes involved in the biodegradation of recalcitrant pollutants.