Anaerobic dechlorination of pentachlorophenol in fixed-film and upflow anaerobic sludge blanket reactors using different inocula

Longterm performance and stability of two upflow anaerobic sludge blanket (UASB) reactors inoculated with granular sludge and treating a synthetic waste water containing pentachlorophenol (PCP) and phenol were studied. A similar system consisting of two fixed-film reactors inoculated with anaerobic digested sewage sludge were further studied. One reactor in each series received glucose in addition to the phenols. Dechlorination of PCP proceeded via two different dominating pathways in the respective reactor systems, suggesting that two distinct microbial populations were present, probably originating from the different inocula. Dechlorinating activity was maintained for more than 18 months in the UASB reactors and was generally higher than in the fixed-film reactors. In the fixed-film reactors, dechlorination of PCP suddenly decreased after 15.5 months of operation compared to earlier performance. Since no operational parameters had been changed, this indicated that the enriched culture was unstable on a longterm basis. Addition of yeast extract to the medium restored activity. General process stability in both reactor systems was clearly enhanced by the addition of glucose and was superior in the UASB/granular sludge system. The better performance and the higher stability in the UASB/granular sludge reactor highlights the importance of thorough screening of inocular prior to start-up of processes treating waste waters containing xenobiotic compounds.Abbreviations PCP pentachlorophenol - TeCP tetrachlorophenol - TCP trichlorophenol - DCP dichlorophenol - UASB upflow anaerobic sludge blanket - HRT hydraulic retention time     

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.     

Nitrification performance and nitrifier community composition of a chemostat and a membrane-assisted bioreactor for the nitrification of sludge reject water

Nitrification performance of a chemostat and a membrane-assisted bioreactor (MBR) was assessed at pilot scale for the treatment of sludge reject waters with NH₄⁺-N concentrations up to 600 mg/L and low organic content (COD<200 mg/L). To prevent nitrifier washout the 1-m³ chemostat was operated at 20°C with minimum hydraulic retention time of F=2 days. At the 0.71 m³ MBR, F was successively reduced to 6.2 h. Complete sludge retention was achieved by means of a 2-m² 100,000-Dalton PES ultrafiltration membrane. Operation in crossflow mode with flow velocities from v_F=2.4-3.7 m/s and transmembrane pressures (p=0.5-1.2 bar yielded a long-term permeate flux of 110 L/(m²2h). In the MBR, nitrification rates up to 2,500 g N/(m³2d) were measured with biomass concentrations between 4 and 15 g TSS/L. Despite low TSS values, about 0.2 g/L of the chemostat was able to nitrify 180 g N/(m³2d). The microbial community composition differed considerably between the two reactors as determined by fluorescent in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes. For both reactors, the relative abundance of ammonia and nitrite oxidisers measured by FISH was consistent with results from dynamic simulation of the nitrification process.     

荧光定量PCR监测五氯酚对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响

通过特异引物扩增环境中氨氧化细菌16S rDNAV2保守区域,将该片段克隆到T-easy载体上,PCR产物经测序和定量PCR扩增体系鉴定,证实PCR扩增产物为氨氧化细菌16S rDNA保守序列,以含该序列的重组质粒作为定量PCR监测氨氧化细菌数量的DNA标准品。用荧光定量PCR技术比较了五氯酚(PCP)对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响。结果表明,不加PCP的反应器中,好氧颗粒污泥和活性污泥中氨氧化细菌的数量分别为4.28×107±5.44×106cells/(g干污泥)和2.51×109±8.61×108cells/(g干污泥)。随着PCP浓度的增加(0~50mg/L),PCP对氨氧化细菌数量的影响不大(P>0.05),而且,污泥中氨氧化细菌的数量与氨氮的去除率无直接的正相关关系(P>0.05),PCP主要是抑制氨氧化细菌的代谢活性导致污泥氨氮去除效率降低。     

A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase

A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358. The two-stage bioreactor system was successfully operated for 20 days. PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell. Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop. Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air. Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min. The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d. These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams.     

Desulfitobacterium hafniense Is Present in a High Proportion within the Biofilms of a High-Performance Pentachlorophenol-Degrading, Methanogenic Fixed-Film Reactor

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 μM day⁻¹, a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.     

Toxicity and biodegradation of formaldehyde in anaerobic methanogenic culture

Formaldehyde is present in several industrial wastewaters including petrochemical wastes. In this study, the toxicity and degradability of formaldehyde in anaerobic systems were investigated. Formaldehyde showed severe toxicity to an acetate enrichment methanogenic culture. As low as 10 mg/L (0.33 mM) of formaldehyde in the reactor completely inhibited acetate utilization. Formaldehyde, however, was degraded while acetate utilization was inhibited. Degradation of formaldehyde (Initial concentration /=60 mg/L), formaldehyde degradation was inhibited and partial degradation was possible. The initial formaldehyde to biomass ratio, S(0)/X(0), was useful to predict the degradation potential of high formaldehyde concentrations in batch systems. When S(0)/X(0) /= 0.29, formaldehyde at higher than 60 mg/L was only partially degraded. The inhibition of formaldehyde degradation in batch systems could be avoided by repeated additions of low concentrations of formaldehyde (up to 30 mg/L). Chemostats (14-day retention time) showed degradation of 74 mg/L-d (1110 mg/L) of influent formaldehyde with a removal capacity of 164 mg/g VSS-day. A spike of 30 mg/L (final concentration in the chemostat) formaldehyde to the chemostat caused only a small increase in effluent acetate concentration for 3 days. But a spike of 60 mg/L (final concentration in the chemostat) formaldehyde to the chemostat resulted in a dramatic increase in acetate concentration in the effluent. The results also showed that the acetate enrichment culture was not acclimated to formaldehyde even after 226 days. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 727-736, 1997.     

The effect of Ni(II) on properties of bulking activated sludge and microbial analysis of sludge using 16S rDNA gene

This study investigated the effect of nickel on properties and microbial community of bulking activated sludge when 60-240 mg/L Ni(II) was dosed continuously in a sequencing batch reactor (SBR) over 350 days. Results showed that 120 mg/L nickel did not significantly inhibited removal of organic pollutant by activated sludge. However, the system was completely upset when 240 mg/L Ni(II) was dosed. Improvement of settling and dewatering ability was also observed with the addition of Ni(II). In addition, investigations by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rDNA of bacteria strain demonstrated that Ni(II) significantly affected microbial community of bulking activated sludge, judging from the elimination of original species and emergence of possible new nickel-resistant bacteria. The effect of nickel on shift of microbial community was an important cause resulted in the improvement of sludge properties in this bulking activated sludge system.     

Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

Degradation of pentachlorophenol by <Emphasis Type="Italic">Kocuria</Emphasis> sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600 mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.     

澳洲青苹组织培养再生体系的研究

通过对澳洲青苹茎尖培养及植株再生体系中无菌培养物的建立、初代培养、继代培养、生根培养和移栽五个技术环节的研究,建立了澳洲青苹组织培养快速繁殖技术体系。结果表明：(1)BA0．3～0．6mg／L NAA0．1～0．3mg／L的MS培养基能促进澳洲青苹茎尖生长或大量侧芽的分化,不定芽生长健壮;(2)无根幼苗在含有IBA0．6～1．0mg／L NAA0．1mg／L的1／2MS培养基上暗培养一周后,再进行自然光培养,30d生根率可达到78%以上;(3)生根苗经50mg／L NAA浸渍1～2h后,移入蛭石：腐殖土：田园土=2：1：1的移栽基质中,保温保湿,成活率达80％以上;(4)进一步建立了自然光下生根、练苗一次完成的同步化体系,缩短了繁育时间,简化了步骤,降低了成本。此技术体系为澳洲青苹优良种苗快速繁殖提供了一条新途径。     

Studies on the effect of inoculation of activated sludge with bacteria actively degrading hydrocarbons on the biodegradation of petroleum products

Eighteen strains of bacteria were isolated from activated sludge purifying petroleum-refining wastewaters. These strains were plated on solidified mineral medium supplemented with oil fraction in concentration 1000 mg/l. Four of the strains that grew best in the presence of oil were selected for further studies. The strains were identified based on Bonde's scheme and microscopic observations. Three of them belonged to the genus Arthrobacter and one to the genus Micrococcus. Stationary cultures of single strains and their mixtures were set up in mineral medium containing oil (sterile and non-sterile) as sole carbon source in concentration 1000 mg/l. The oils were found to be removed the most efficiently by a mixture of the strains. After 14 days of culture the amount of oil was utilized by from 63 to 95%. In the next stage of the studies the bacteria were used to inoculate activated sludge. Stationary cultures of the activated sludge were set up in mineral medium with oil. The utilisation of petroleum products by non-inoculated activated sludge (control), activated sludge inoculated with a single strain or a mixture of all four strains was examined. In both inoculated activated sludge cultures approximately 80% of the oils were removed, compared to 60% in the control activated sludge. Therefore, inoculated activated sludge showed 20% higher effectiveness of removal of petroleum derivatives.     

Long-term effect of weak nitrogen limitation on polyhydroxyalkanoates production of propionate-fed activated sludge operated at long sludge retention time

A sequencing batch reactor (SBR) submitted to aerobic dynamic feeding condition was fed with weakly nitrogen-limited substrate (COD/N ratio of 60?mg/mg, propionate as the sole carbon source), and operated at 15?days sludge retention time (SRT). Then, the nitrogen-limited sludges at different cultivating periods (45th, 120th, 195th and 240th days) were harvested to conduct aerobic batch experiments of PHAs production under various nitrogen concentrations. Experimental results indicated that PHAs accumulating behavior of the propionate-fed sludge declined significantly after long-term cultivation, implying that weak nitrogen limitation and long SRT operation cannot achieve a stable PHAs producing performance of the sludge. Supplementing enough nitrogen was consequently a requisite to guarantee the stability of the propionate-fed PHAs accumulating culture. Besides, the sludge with higher PHAs content in SBR did not ensure a better PHAs producing capability in the batch PHAs production phase. Batch experiments also indicated that the propionate-fed sludge with 0.7?N?mmol/L exhibited a better PHAs producing capability than with other nitrogen concentrations.     

Influence of total solid and inoculum contents on performance of anaerobic reactors treating food waste

The aim of this paper was to analyze the biomethanization process of food waste (FW) from a university campus restaurant in six reactors with three different total solid percentages (20%, 25% and 30% TS) and two different inoculum percentages (20-30% of mesophilic sludge). The experimental procedure was programmed to select the initial performance parameters (total solid and inoculum contents) in a lab-reactor with V: 1100mL and, later, to validate the optimal parameters in a lab-scale batch reactor with V: 5000mL. The best performance for food waste biodegradation and methane generation was the reactor with 20% of total solid and 30% of inoculum: give rise to an acclimation stage with acidogenic/acetogenic activity between 20 and 60 days and methane yield of 0.49L CH4/g VS. Also, lab-scale batch reactor (V: 5000mL) exhibit the classical waste decomposition pattern and the process was completed with high values of methane yield (0.22L CH4/g VS). Finally, a protocol was proposed to enhance the start-up phase for dry thermophilic anaerobic digestion of food waste.     

Growth and enrichment of pentachlorophenol-degrading microorganisms in the nutristat, a substrate concentration-controlled continuous culture.

The nutristat, a substrate concentration-controlled continuous culture, was used to grow pentachlorophenol (PCP)-degrading microorganisms. The PCP concentration control system consisted of on-line measurement of the PCP concentration in the culture vessel with a tangential filter and a flowthrough spectrophotometer. With PCP concentrations between 45 and 77 microM, a stable situation was established in the nutristat, with an average dilution rate of 0.035 +/- 0.003 h-1. Compared with those of fed-batch cultures and chemostat cultures, the growth rates of microorganisms in the PCP nutristat were significantly higher, leading to considerable time savings in the enrichment procedure. In addition, PCP accumulation to severe inhibitory levels in the culture is prevented because the set point determines the (maximum) PCP concentration in the culture. The use of the nutristat as a tool for the growth of bacteria that degrade toxic compounds is discussed.     

Glyphosate degradation by immobilized bacteria: laboratory studies showing feasibility for glyphosate removal from waste water.

To evaluate immobilized bacteria technology for the removal of low levels of glyphosate (N-phosphonomethylglycine) from aqueous industrial effluents, microorganisms with glyphosate-degrading activity obtained from a fill and draw enrichment reactor inoculated with activated sludge were first exposed to glyphosate production wastes containing 500-2000 mg glyphosate/L. The microorganisms were then immobilized by adsorption onto a diatomaceous earth biocarrier contained in upflow Plexiglas columns. The columns were aerated, maintained at pH 7.0-8.0, incubated at 25 degrees C, supplemented with NH4NO3 (50 mg/L), and exposed to glyphosate process wastes pumped upflow through the biocarrier. Glyphosate degradation to aminomethylphosphonic acid was initially > 96% for 21 days of operation at flows yielding hydraulic residence times (HRTs) as short as 42 min. Higher flow rate studies showed > 98% removal of 50 mg glyphosate/L from the waste stream could be achieved at a HRT of 23 min. Glyphosate removal of > 99% at a 37-min HRT was achieved under similar conditions with a column inoculated with a pure culture of Pseudomonas sp. strain LBr, a bacterium known to have high glyphosate-degrading activity. After acid shocking (pH 2.8 for 18 h) of a column of immobilized bacteria, glyphosate-degrading activity was regained within 4 days without reinoculation. Although microbial growth and glyphosate degradation were not maintained under low organic nutrient conditions in the laboratory, the low levels of degradable carbon (45-94 mg/L) in the industrial effluent were sufficient to support prolonged glyphosate-degrading activity. The results demonstrated that immobilized bacteria technology is effective in removing low levels of glyphosate in high-volume liquid waste streams.     

Enhancement of Pentachlorophenol Biodegradation Using Organic and Inorganic Supports

The role of soil, straw, and sawdust as supports in enhancing pentachlorophenol (PCP) mineralization by an indigenous soil consortium was examined by assessing the bioavailability of the substrate and other nutrients. PCP sorption tests were conducted in the presence of sterile supports to evaluate PCP bioavailability. Indigenous biomass, practically free of soil particles, was prepared to test the influence of sterile soil and soil components on the mineralization of increasing PCP concentrations. Organic supports such as straw and sawdust were very good sorbents for PCP, resulting in a slow, continuous desorption of substrate, high mineralization rates, and reduced toxicity to the active biomass. Soil and clay retained less PCP and desorbed it in decreasing amounts. Soil was the best amendment to enhance the mineralization of 100 mg/L PCP. Soil, soil extract, and the lowest-molecular-weight fraction of the soil extract facilitated the complete mineralization of 300 mg/L of PCP with a lag time of about 9 days, compared to 21 days for the unamended culture. Addition of soil enhanced PCP mineralization by an indigenous consortium, probably because soil particles served as an adsorbent for the contaminant to decrease its toxicity, as a support for biomass colonization, and as a source of supplementary nutrients for the biomass. This concept is of importance, particularly for the production of active and resistant biomass for the biotreatment of contaminated soils.     

Anaerobic granule development for removal of pentachlorophenol in an upflow anaerobic sludge blanket (UASB) reactor

The granulation process using synthetic wastewater containing pentachlorophenol (PCP) in four 1.1 l laboratory scale upflow anaerobic sludge blanket (UASB) reactors was studied, and the anaerobic biotransformation of PCP during the granulation process investigated. After 110 days granular sludge was developed and up to 160 and 180 mg/l of PCP was added into the reactors R1 and R2, respectively, when they were inoculated with acclimated anaerobic sludge from an anaerobic digester of a citric acid plant. The inoculum was predominately composed of bacilli and filamentous bacteria. Granulation did not occur in reactors R3 and R4 which were inoculated with acclimated anaerobic sludge from aerobic sludge of the municipal sewage treatment plant which consisted mainly of cocci. Despite similar bacilli in the granule, the filamentous bacteria from reactor R1 were thicker than those of reactor R2. The granular sludge had a maximum diameter of 2.5 and 2.2 mm, and SMA of 1.44 and 1.32 gCOD/gTVS per day for reactors R1 and R2, respectively. Over 98% chemical oxygen demand (COD) removal rate and 99% of PCP removal rate were achieved when reactors R1 and R2 were operated at PCP and COD loading rates of 150 and 7.5 g/l per day, respectively. H₂-producing acetogens were the dominant anaerobes in the granular sludge.     

五氯酚（PCP）污染土壤厌氧生物修复技术的初步研究

研究土壤泥浆反应器在投加厌氧颗粒污泥条件下修复PCP污染土壤的性能.结果表明,对PCP浓度30mg     

Removal of nitrate from industrial wastewaters in a pilot plant by nitrate-tolerant klebsiella oxytoca CECT 4460 and arthrobacter globiformis CECT 4500

Two strains, a gram-negative bacterium Klebsiella oxytoca CECT 4460 and a gram-positive, mycelium-forming bacterium Arthrobacter globiformis CECT 4500, tolerant to up to 1 M nitrate, were isolated from the grounds of a munitions factory. Under strict aerobic conditions and with appropriate C-sources, growth of these bacteria took place when the nitrate concentration in the medium was below 150 mM. Optimal growth conditions regarding the culture medium composition for the biological removal of nitrate were established in batch cultures. Then, the system was scaled up to a 40-L pilot plant and operated under continuous conditions in a factory with direct waste streams from dinitroethylene glycol production after appropriate dilution with nontreated groundwaters. The level of nitrate in the effluent was below 0.5% of the initial N-load. Nitrite and ammonium were undetectable and the level of the C-source in the effluent was below 50 mg per L. On the basis of these results, we conclude that the system worked on site satisfactorily. Copyright 1998 John Wiley & Sons, Inc.