Cometabolic biodegradation of 4-chlorophenol by sequencing batch reactors at different temperatures

The simultaneous removal of 4-chlorophenol (4-CP) and phenol in lab-scale sequencing batch reactors at different temperatures has been studied. Phenol feed concentration was fixed at 525 mg/L and 4-CP concentration was increased from 105 to 2100 mg/L at a constant hydraulic residence time (HRT) of 10.5 d. Complete phenol and 4-CP biodegradation was achieved during the aerobic stage working with 4-CP concentrations up to 1470 mg/L in the feed. Both 4-CP and phenol specific initial removal rates were strongly affected by 4-CP feed concentration and temperature. Only at the highest temperature tested (35 °C) it was possible to increase the maximum assimilative 4-CP concentration by the biological sludge up to 2100 mg/L, and a significant reduction of the ecotoxicity of the effluents was observed. 4-chlorocatechol (4-CC) was identified as the major intermediate in the aerobic cometabolic 4-CP degradation, being the ecotoxicity of that species substantially lower than that of 4-CP.     

Comparative studies on immobilized laccase behaviour in packed-bed and batch reactors

Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized covalently via glutaraldehyde to cellulose-based carrier Granocel. Laccase was partially purified by membrane concentration and diafiltration followed by precipitation with acetone. Five-fold increase in the measured activity of immobilized enzyme was obtained when six times purer laccase was used for immobilization. For the best preparation, with very high activity of 2053 U per 1 mL of the carrier, thermal- and pH-stability, and activity profiles were determined. Experiments carried out in a batch reactor showed that k_cat/K_m for immobilized enzyme (0.65) is three times lower than the value obtained for the native laccase (2.19) whereas k_cat/K_m estimated from continuous reactor (1.50) is notably closer to that for the native enzyme. Continuous process probably reflects more precisely kinetics of the reaction accompanied by simultaneous product precipitation on the carrier’s surface. Operational stability of immobilized laccase was tested in continuous mode operation with ABTS, guaiacol and trichlorophenol as substrates and showed that packed-bed reactor is unprofitable system for laccase immobilized on Granocel carrier due to the high bed compaction. However, excellent stability of the preparation was noted under 20 successive runs in the well mixed tank reactor and better ability towards trichlorophenol biotransformation was observed in the case of immobilized laccase.     

Atrazine biodegradation by a bacterial community immobilized in two types of packed-bed biofilm reactors

Through selective enrichment of atrazine-metabolizing microorganisms, a microbial community was selected from agricultural soil. Bacterial isolates, identified by their closest similarity with 16S rDNA sequences stored in NCBI GeneBank, belonged to the genera: Massilia, Stenotrophomonas, Klebsiella, Sphingomonas, Ochrobactrum, Arthrobacter, Microbacterium, Xanthomonas and Ornithinimicrobium. From these strains, only the first six used atrazine as nitrogen and carbon source. The microbial community attached to a non-porous support was evaluated for its atrazine biodegradation rate and removal efficiency under aerobic conditions in two types of packed-bed biofilm reactors fed with a mineral salt medium containing glucose plus atrazine, or atrazine as the sole carbon and nitrogen source. Removal efficiencies near 100% were obtained at loading rates up to 10 mg l⁻¹ h⁻¹. After long periods of continuous operation, the richness of microbial species in biofilm reactors diminished to only three bacterial strains; Stenotrophomonas sp., Ochrobactrum sp. and Arthrobacter sp. By PCR analysis of their DNA, the presence of atzABC genes codifying for the enzymes of the upper catabolic pathway of atrazine, was confirmed in the three strains. The gene atzD that encodes for the cyanuric acid amidohydrolase enzyme was detected only in Stenotrophomonas sp.     

Development and characteristics of phosphorus-accumulating microbial granules in sequencing batch reactors

Phosphorus (P)-accumulating microbial granules were developed at different substrate P/chemical oxygen demand (COD) ratios in the range of 1/100 to 10/100 by weight in sequencing batch reactors. The soluble COD and PO4-P profiles showed that the granules had typical P-accumulating characteristics, with concomitant uptake of soluble organic carbon and the release of phosphate in the anaerobic stage, followed by rapid phosphate uptake in the aerobic stage. The size of P-accumulating granules exhibited a decreasing trend with the increase in substrate P/COD ratio, while the structure of the granules became more compact and denser as the substrate P/COD ratio increased. The P uptake by granules fell within the range of 1.9% to 9.3% by weight, which is comparable with uptake obtained in conventional enhanced biological phosphorus removal (EBPR) processes. It was further found that low aerobic respirometric activity of granules in terms of specific oxygen utilization rate favors P uptake by granules. The results presented would be useful for the further development of a novel granule-based EBPR technology.     

Growth kinetics of aerobic granules developed in sequencing batch reactors

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.     

Hydrocarbon biodegradation in oxygen-limited sequential batch reactors by consortium from weathered, oil-contaminated soil

We studied the use of sequential batch reactors under oxygen limitation to improve and maintain consortium ability to biodegrade hydrocarbons. Air-agitated tubular reactors (2.5 L) were operated for 20 sequential 21-day cycles. Maya crude oil-paraffin mixture (13,000 mg/L) was used as the sole carbon source. The reactors were inoculated with a consortium from the rhizosphere of Cyperus laxus, a native plant that grows naturally in weathered, contaminated soil. Oxygen limitation was induced in the tubular reactor by maintaining low oxygen transfer coefficients (k(L)a < 20.6 h(-1)). The extent and biodegradation rates increased significantly up to the fourth cycle, maintaining values of about 66.33% and 460 mg x L(-1) x d(-1), respectively. Thereafter, sequential batch reactor operation exhibited a pattern with a constant general trend of biodegradation. The effect of oxygen limitation on consortium activity led to a low biomass yield and non-soluble metabolite (0.45 g SS/g hydrocarbons consumed). The average number of hydrocarbon-degrading microorganisms increased from 6.5 x 10(7) (cycles 1-3) to 2.2 x 10(8) (cycles 4-20). Five bacterial strains were identified: Achromobacter (Alcaligenes) xylosoxidans, Bacillus cereus, Bacillus subtilis, Brevibacterium luteum, and Pseudomonas pseudoalcaligenes. Asphaltene-free total petroleum hydrocarbons, extracted from a weathered, contaminated soil, were also biodegraded (97.1 mg x L(-1) x d(-1)) and mineralized (210.48 mg CO2 x L(-1) x d(-1)) by the enriched consortium without inhibition. Our results indicate that sequential batch reactors under oxygen limitation can be used to produce consortia with high and constant biodegradation ability for industrial applications of bioremediation.     

Microbial adaptation to biodegradation of tert-butyl alcohol in a sequencing batch reactor

This study demonstrates the utility of the sequencing batch reactor (SBR) to adapt microorganisms towards biological removal of tert-butyl alcohol (TBA). The reactor was inoculated with activated sludge and fed with TBA as the sole carbon source. Start-of-cycle TBA concentrations were initially set at 100 mgL(-1) with a cycle time of 24 h and a volumetric exchange ratio of 50% to maintain a TBA loading rate of not more than 100 mgL(-1)d(-1). Step increases in TBA loading rates up to 600 mgL(-1)d(-1) were achieved by first raising the start-of-cycle TBA concentration to 150 mgL(-1) on day 90 and subsequently by reducing the cycle time from 24 to 12, 8 and 6h on days 100, 121 and 199, respectively. This acclimation strategy favored the retention of increasingly higher densities of well-adapted microbial populations in the reactor. The increases in TBA loading produced better settling biomass and higher biomass concentrations with higher specific TBA biodegradation rates. Effluent TBA concentrations were consistently below the detection limit of 25 microgL(-1). The use of progressively shorter cycle times created selection pressures that fostered the self-immobilization of the reactor microorganisms into aerobic granules which first appeared on day 125. Specific TBA biodegradation rates in the granules followed the Haldane model for substrate inhibition, and peaked at 13.8 mgTBAgVSS(-1)h(-1) at a TBA concentration of 300 mgL(-1). Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes from granules sampled between days 220 and 247 confirmed the existence of a highly stable microbial community with members belonging to the alpha, beta and delta subdivisions of Proteobacteria and the Cytophaga-Flavobacteria-Bacteroides (CFB) group.     

Modeling and optimization of granulation process of activated sludge in sequencing batch reactors

Aerobic granulation is a promising process for wastewater treatment, but this granulation process is very complicated and is affected by many factors. Thus, a mathematical model to quantitatively describe such a granulation process is highly desired. In this work, by taking into account all of key steps including biomass growth, increase in particle size and density, detachment, breakage and sedimentation, an one‐dimensional mathematic model was developed to simulate the granulation process of activated sludge in a sequencing batch reactor (SBR). Discretization methodology was applied by dividing operational time, sedimentation process, size fractions and slices into discretized calculation elements. Model verification and prediction for aerobic granulation process were conducted under four different conditions. Four parameters indicative of granulation progression, including mean radius, biomass discharge ratio, total number, and bioparticle size distribution, were predicted well with the model. An optimum controlling strategy, automatically adjusted of settling time, was also proposed based on this model. Moreover, aerobic granules with a density higher than 120 g VSS/L and radius in a range of 0.4–1.0 mm were predicted to have both high settling velocity and substrate utilization rate, and the corresponding optimum operating conditions were be determined. Experimental results demonstrate that the developed model is appropriate for simulating the formation of aerobic granules in SBRs. These results are useful for designing and optimizing the cultivation and operation of aerobic granule process. Biotechnol. Bioeng. 2013; 110: 1312–1322. © 2012 Wiley Periodicals, Inc.     

Decolorization of reactive dyes by the white rot fungus Trametes versicolor in sequencing batch reactors

The white rot fungus Trametes versicolor was shown to be capable of decolorizing three reactive dyes in a sequencing batch process, using glucose as the carbon and energy source over an extended period without supplementation of new mycelium. Decolorization activity was related to the expression of extracellular peroxidases and could be continuously reactivated by sheering the suspended pellets. Pure culture experiments were carried out simultaneously in agitated Erlenmeyer flasks and in completely stirred tank reactors with two azo dyes, C.I. Reactive Black 5 and C.I. Reactive Red 198 as well as the anthraquinone dye C.I. Reactive Blue 19 (Brilliant Blue R). Results show high and stable degrees of decolorization of 91%-99% in both systems, which could be repeated without decrease in activity over time. Under nonsterile conditions only five cycles of decolorization could be achieved. An increasing bacterial population suppressed fungal growth and the formation of peroxidases. Copyright John Wiley & Sons, Inc.     

Causes and control of filamentous growth in aerobic granular sludge sequencing batch reactors

Poor long-term stability of aerobic granules developed in sequencing batch reactors (SBRs) remains a limitation to widespread use of aerobic granulation in treating wastewater. Filamentous growth has been commonly reported in aerobic granular sludge SBR. This review attempts to address the instability problem of aerobic granular sludge SBR from the perspective of filamentous growth in the system. The possible causes of filamentous growth are identified, including long retention times of solids, low substrate concentration in the liquid phase, high substrate gradient within the granule, dissolved oxygen deficiency in the granule, nutrient deficiency inside granule, temperature shift and flow patterns. Because of cyclic operation of aerobic granular sludge SBR and peculiarities of aerobic granules, various stresses can be present simultaneously and can result in progressive development of filamentous growth in aerobic granular sludge SBR. Overgrowth of filamentous bacteria under stress conditions appears to be a major cause of instability of aerobic granular sludge SBR. Specific recommendations are made for controlling filamentous growth.     

白腐真菌对酸性橙7的脱色降解

偶氮染料广泛应用于纺织、造纸和包装等行业,因其具有三致性、结构稳定且难降解,已成为染料废水处理的研究热点之一。本研究以白腐真菌作为脱色菌株,考察了不同白腐真菌对偶氮类染料酸性橙7(acid orange 7,AO7)的脱色降解,探讨了AO7染料的浓度、pH、温度以及脱色时间对染料脱色率的影响,同时应用紫外-可见光谱吸收法、红外光谱吸收法、高效液相色谱法和气相色谱-质谱法对AO7的降解产物进行分析,并对其产物进行植物毒性实验,以推断AO7可能的降解途径及其降解产物的毒性。结果表明:在pH 4.5、28℃条件下,刺芹侧耳(Pleurotus eryngii)和杂色云芝(Trametes versicolor)的混合菌丝脱色降解100 mg/L AO7,24 h脱色率可达93.46%。推测AO7可能的生物降解途径:AO7偶氮键断裂生成对氨基苯磺酸和1-氨基-2-萘酚;接着对氨基苯磺酸脱去磺酸基,生成对苯二酚;同时1-氨基-2-萘酚开环生成邻苯二甲酸和对羟基苯甲醛,之后进一步降解生成苯甲酸;最后对苯二酚和苯甲酸继续氧化成其他小分子中间体、H2O和CO_(2)。植物毒性实验表明,P.eryngii和T.versicolor混合菌丝对AO7脱毒效果较好。以上研究为探究白腐真菌在工业废水中降解偶氮类染料的应用奠定基础。     

Nitrogen removal from aquaculture pond water by heterotrophic nitrogen assimilation in lab-scale sequencing batch reactors

The potential use of sequencing batch reactors (SBRs) as an alternative bio-flocs technology (BFT) approach in aquaculture was explored. One SBR was dosed with glycerol and one with acetate for the decrease of the nitrogen concentration in simulated aquaculture water by microbial assimilation. At an optimal C/N ratio between 10 and 15, the nitrogen removal efficiency reached up to 98% (=110 mg N L(-1) reactor day(-1)) for both SBRs. The estimated biomass productivity reached 0.62-0.94 g C L(-1)r eactor day(-1) for the glycerol SBR and 0.54-0.82 g C L(-1) reactor day(-1) for the acetate SBR. The floc protein content, indicating biomass quality, reached up to 57% if grown on glycerol. With acetate, it attained a value of 61%. The highest average poly-beta-hydroxybutyrate (PHB) content was 16% on a dry weight basis for the acetate biomass.     

The effect of temperature on slaughterhouse wastewater treatment in anaerobic sequencing batch reactors

High strength slaughterhouse wastewater was treated in four 42 l anaerobic sequencing batch reactors (ASBRs) operated at 30 degrees C, 25 degrees C and 20 degrees C. The wastewater contained between 30% and 53% of its chemical oxygen demand (COD) as suspended solids (SS). The ASBRs could easily support volumetric organic loading rates (OLRs) of 4.93, 2.94 and 2.75 kg/m3/d (biomass OLRs of 0.44, 0.42 and 0.14 g/g volatile SS (VSS)/d) at 30 degrees C, 25 degrees C, and 20 degrees C, respectively. At all operating temperatures, the total COD (TCOD) and soluble COD (SCOD) were reduced by over 92%, while average SS removal varied between 80% and 96%. Over the experimental period, 90.8%, 88.7% and 84.2% of the COD removed was transformed into methane at 30 degrees C, 25 degrees C and 20 degrees C, respectively. The decrease in the conversion of the COD removed into methane as operating temperature was lowered, may be partly explained by a lower degradation of influent SS as temperature was reduced. The reactors showed a high average methanogenic activity of 0.37, 0.34 and 0.12 g CH4-COD/gVSS/d (22.4, 12.7 and 11.8 l/d) at 30 degrees C, 25 degrees C and 20 degrees C, respectively. The average methane content in the biogas increased from 74.7% to 78.2% as temperature was lowered from 30 degrees C to 20 degrees C.     

Biodegradation of oil sands process affected water in sequencing batch reactors and microbial community analysis by high-throughput pyrosequencing

Two sequencing batch reactors (SBR) were constructed and filled with different inocula of activated sludge (AS) and mature fine tailings (MFT) to treat oil sands process-affected water (OSPW). The COD was reduced by 82% in the AS-SBR and 43% in the MFT-SBR during phase I using 10% OSPW and 90% synthetic wastewater as reactor feed. However, COD removal reached 12% and 20% in the AS-SBR and the MFT-SBR, respectively, when 100% raw OSPW was fed into the reactors. Maximum removal of acid-extractable organics (AEO) was 8.7% and 16.6% in the AS-SBR and the MFT-SBR, respectively with a hydraulic retention time of one day. Pyrosequencing analysis revealed that Proteobacteria was the dominant phylum and beta- and gamma-Proteobacteria were dominant classes in both reactors. Evidence of a microbial community change was observed when influent raw OSPW was switched from 50 to 100%. More significant changes in the AS-SBR community were detected.     

Influence of starvation time on formation and stability of aerobic granules in sequencing batch reactors

Three sequencing batch reactors, R1, R2 and R3, with a 1.5-h, 4-h and 8-h cycle time, respectively, were used to cultivate aerobic granules with the same synthetic wastewater containing 1000 mg l(-1) COD. As the initial COD concentrations in the cycles were the same, three different cycle times led to three different starvation times in repeated cycles of the three reactors. It was found that 63 cycles were needed to form granules with the longest starvation time in R3 while it took 256 cycles in R1 with the shortest starvation time. However, as far as the formation time was concerned, granules were formed on day 16 with 1.5-h cycle time while on day 21 with 8-h cycle time, which indicated that a shorter cycle time with a shorter starvation time speeded up the granulation. This was mainly due to the stronger hydraulic selection pressure at shorter cycle time. However, it was found that granules formed with cycle time of 1.5h were unstable. Fluffy granules with poor settling ability were observed in R1 in the 4th month, which led to the collapse of R1 after 160-day of operation. Granules in R2 and R3 showed good stability during the long-term operation. Therefore, a reasonable starvation time was necessary to maintain the long-term stability of aerobic granules.     

Structure and stability of aerobic granules cultivated under different shear force in sequencing batch reactors

The cultivation of stable aerobic granules as well as granular structure and stability in sequencing batch reactors under different shear force were investigated in this study. Four column sequencing batch reactors (R1–R4) were operated under various shear force, in terms of superficial upflow air velocity of 0.8, 1.6, 2.4, and 3.2 cm s⁻¹, respectively. Aerobic granules were formed in all reactors in the experiment. It was found that the magnitude of shear force has an important impact on the granule stability. At shear force of 2.4 and 3.2 cm s⁻¹, granules can maintain a robust structure and have the potential of long-term operation. Granules developed in low shear force (R1, 0.8 cm s⁻¹ and R2, 1.6 cm s⁻¹) deteriorated to large-sized filamentous granules with irregular shape, loose structure and resulted in poor performance and operation instability. Granules cultivated under high shear force (R3, 2.4 cm s⁻¹ and R4, 3.2 cm s⁻¹) stabilized to clear outer morphology, dense and compact structure, and with good performance in 120 days operation. Fractal dimension (D_f) represents the internal structure of granules and can be used as an important indicator to describe the structure and stability of granules. Due to the combined effects of shear force and growth force, the mature granules developed in R3 and R4 also displayed certain differences in granular structure and characteristics.     

Correspondence between community structure and function during succession in phenol- and phenol-plus-trichloroethene-fed sequencing batch reactors

The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors. One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day. After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg(-1) day(-1) for 2 years. In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg(-1) day(-1). Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function. In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates. Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate). Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high. Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates. In contrast, the community under no stress exhibited a dynamic structure and dynamic function.     

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.