Enhanced micropollutant biodegradation and assessment of nitrous oxide concentration reduction in wastewater treated by acclimatized sludge bioaugmentation.

This research investigated the micropollutant biodegradation and nitrous oxide (N2O) concentration reduction in high strength wastewater treated by two-stage activated sludge (AS) systems with (bioaugmented) and without (non-bioaugmented) acclimatized sludge bioaugmentation. The bioaugmented and non-bioaugmented systems were operated in parallel for 228 days, with three levels of concentrations of organics, nitrogen, and micropollutants in the influent: conditions 1 (low), 2 (moderate), and 3 (high). The results showed that, under condition 1, both systems efficiently removed the organic and nitrogen compounds. However, the bioaugmented system was more effective in the micropollutant biodegradation and N2O concentration reduction than the non-bioaugmented one. Under condition 2, the nitrogen and micropollutant biodegradation efficiency of the non-bioaugmented system slightly decreased, while the N2O concentration declined in the bioaugmented system. Under condition 3, the treatment performance and N2O concentration abatement were substantially lowered as the compounds concentration increased. Further analysis also showed that the acclimatized sludge bioaugmentation increased the bacterial diversity in the system. In essence, the acclimatized sludge bioaugmentation strategy was highly effective for the influent with low compounds concentration, achieving the organics and nitrogen removal efficiencies of 92-97%, relative to 71-97% of the non-bioaugmented system. The micropollutant treatment efficiency of the bioaugmented system under condition 1 was 75-92%, indicating significant improvement in the treatment performance (p < 0.05), compared with 60-79% of the non-bioaugmented system.

Use of aged sludge bioaugmentation in two-stage activated sludge system to enhance the biodegradation of toxic organic compounds in high strength wastewater.

This research investigates the toxic organic compounds biodegradation efficiency of two-stage activated sludge systems with (bioaugmented) and without aged sludge bioaugmentation (non-bioaugmented). The influent was a mixture of leachate and agriculture wastewater (1:1, v/v), used as the representative high strength wastewater. The bioaugmented and non-bioaugmented systems were operated in parallel, with three levels (low, moderate, and high) of concentrations of organics, nitrogen, and toxic organic compounds in the influent (conditions 1, 2, and 3). The results showed that both systems could efficiently degrade the organic compounds. Nevertheless, the toxic organic compounds biodegradation efficiency of the bioaugmented system was higher than that of the non-bioaugmented one. The bioaugmentation enhanced the overall removal efficiency under conditions 1 and 2. However, the bioaugmented system became less effective under condition 3. Further analysis indicated that the bacterial groups essential to the toxic organic compounds biodegradation were abundant in the aged sludge, including heterotrophic bacteria, heterotrophic nitrifying bacteria, and nitrifying bacteria. The abundance of the effective bacteria improved the biodegradation and wastewater treatment performance of the bioaugmented system. In essence, the aged sludge bioaugmentation is a viable and eco-friendly solution to improving the treatment efficiency of the biological activated sludge system, despite limited biodegradation efficiency in an elevated compounds-concentration environment.

Effects of hydraulic retention time and carbon to nitrogen ratio on micro-pollutant biodegradation in membrane bioreactor for leachate treatment.

This research investigated the biodegradation of the micro-pollutants in leachate by the membrane bioreactor (MBR) system under six treatment conditions, comprising two C/N ratios (6, 10) and three hydraulic retention time (HRT) durations (6, 12, 24h). The experimental results indicated that the C/N 6 environment was more advantageous to the bacterial growth. The bacterial communities residing in the sludge were those of heterotrophic bacteria (HB), heterotrophic nitrifying bacteria (HNB) and ammonia oxidizing bacteria (AOB). It was found that HB and HNB produced phenol hydroxylase (PH), esterase (EST), phthalate dioxygenase (PDO) and laccase (LAC) and also enhanced the biodegradation rate constants (k) in the system. At the same time, AOB promoted the production of HB and HNB. The findings also revealed that the 12h HRT was the optimal condition with regard to the highest growth of the bacteria responsible for the biodegradation of phenols and phthalates. Meanwhile, the longer HRT duration (i.e. 24h) was required to effectively bio-degrade carbamazepine (CBZ), N,N-diethyl-m-toluamide (DEET) and diclofenac (DCF).

Diversity of purple nonsulfur bacteria in shrimp ponds with varying mercury levels.

This research aimed to study the diversity of purple nonsulfur bacteria (PNSB) and to investigate the effect of Hg concentrations in shrimp ponds on PNSB diversity. Amplification of the pufM gene was detected in 13 and 10 samples of water and sediment collected from 16 shrimp ponds in Southern Thailand. In addition to PNSB, other anoxygenic phototrophic bacteria (APB) were also observed; purple sulfur bacteria (PSB) and aerobic anoxygenic phototrophic bacteria (AAPB) although most of them could not be identified. Among identified groups; AAPB, PSB and PNSB in the samples of water and sediment were 25.71, 11.43 and 8.57%; and 27.78, 11.11 and 22.22%, respectively. In both sample types, Roseobacter denitrificans (AAPB) was the most dominant species followed by Halorhodospira halophila (PSB). In addition two genera, observed most frequently in the sediment samples were a group of PNSB (Rhodovulum kholense, Rhodospirillum centenum and Rhodobium marinum). The UPGMA dendrograms showed 7 and 6 clustered groups in the water and sediment samples, respectively. There was no relationship between the clustered groups and the total Hg (HgT) concentrations in the water and sediment samples used (<0.002-0.03 µg/L and 35.40-391.60 µg/kg dry weight) for studying the biodiversity. It can be concluded that there was no effect of the various Hg levels on the diversity of detected APB species; particularly the PNSB in the shrimp ponds.

Comparative biochemical methane potential of paragrass using an unacclimated and an acclimated microbial consortium.

The effect of inoculum sources on the anaerobic digestion of paragrass was investigated. Two types of sludge were used as the inoculums: an anaerobic sludge obtained from a domestic wastewater treatment plant (OS) and a sludge acclimated to fibrous substrates in raw palm oil mill effluent (AMC). Microbial activity assays showed that the AMC had hydrolytic and acetogenic activities two times greater than the activities of the OS. In addition, the production of methane from acetate by the AMC occurred without a lag phase, while it took 8 days for the OS to start producing methane from the same substrate. The biochemical methane potential after 80 days digestion was 316 ml STP/g VS(added) using the AMC, and 277 ml STP/g VS(added) using the OS. The methane potential of the paragrass was estimated to be 3337 Nm(3) CH4/ha a.

Simultaneous treatment of raw palm oil mill effluent and biodegradation of palm fiber in a high-rate CSTR.

A high-rate continuous stirred tank reactor (CSTR) was used to produce biogas from raw palm oil mill effluent (POME) at 55°C at a highest organic loading rate (OLR) of 19 g COD/ld. Physical and chemical pretreatments were not performed on the raw POME. In order to promote retention of suspended solids, the CSTR was installed with a deflector at its upper section. The average methane yield was 0.27 l/g COD, and the biogas production rate per reactor volume was 6.23 l/l d, and the tCOD removal efficiency was 82%. The hydrolysis rate of cellulose, hemicelluloses and lignin was 6.7, 3.0 and 1.9 g/d, respectively. The results of denaturing gradient gel electrophoresis (DGGE) suggested that the dominant hydrolytic bacteria responsible for the biodegradation of the palm fiber and residual oil were Clostridium sp., while the dominant methanogens were Methanothermobacter sp.

Bioaugmentation of latex rubber sheet wastewater treatment with stimulated indigenous purple nonsulfur bacteria by fermented pineapple extract

Background Treating latex rubber sheet wastewater often leads to the generation of a rotten-egg odor from toxic H2S. To increase the treatment efficiency and eliminate H2S, purple nonsulfur bacteria (PNSB), prepared by supplementing non-sterile rubber sheet wastewater (RAW) with fermented pineapple extract (FPE), were used to treat this wastewater under microaerobic light conditions. The following 3 independent variables: chemical oxygen demand (COD), initial pH and FPE dose were investigated using the Box-Behnken design to find optimal conditions for stimulating the growth of indigenous PNSB (PNSBsi). Results The addition of 2.0% FPE into RAW, which had a COD of 2000 mg L- 1 and an initial pH of 7.0, significantly decreased oxidation reduction potential (ORP) value and stimulated PNSBsi to reach a maximum of 7.8 log cfu mL- 1 within 2 d. Consequently, these PNSBsi, used as inoculants, were investigated for their ability to treat the wastewater under microaerobic light conditions. A central composite design was used to determine the optimal conditions for the wastewater treatment. These proved to be 7% PNSBsi, 0.8% FPE and 4 d retention time and this combination resulted in a reduction of 91% for COD, 75% for suspended solids, 61% for total sulfide while H2S was not detected. Results of abiotic control and treatment sets indicated that H2S was produced by heterotrophic bacteria and it was then effectively deactivated by PNSBsi. Conclusions The stimulation of PNSB growth by FPE under light condition was to lower ORP, and PNSBsi proved to be effective for treating the wastewater.

Enhancement of biogas production from swine manure by a lignocellulolytic microbial consortium.

Anaerobic digestion of lignocellulosic wastes is limited by inefficient hydrolysis of recalcitrant substrates, leading to low biogas yield. In this study, the potential of a lignocellulolytic microbial consortium (LMC) for enhancing biogas production from fibre-rich swine manure (SM) was assessed. Biochemical methane potential assay showed that inoculation of structurally stable LMC to anaerobic digestion led to increase biogas production under mesophilic and thermophilic conditions. The greatest enhancement was observed at 37°C with a LMC/SM ratio of 1.5:1 mg VSS/g VS leading to biogas and methane yields of 355 and 180 ml/g VS(added) respectively, equivalent to 40% and 55% increases compared with the control. The LMC was shown to increase the efficiency of total solid, chemical oxygen demand removal and degradation of cellulose and hemicelluloses (1.87 and 1.65-fold, respectively). The LMC-supplemented process was stable over a 90 d biogas production period. This work demonstrates the potential of LMC for enhancing biogas from lignocellulosic wastes.

Comparative mesophilic and thermophilic anaerobic digestion of palm oil mill effluent using upflow anaerobic sludge blanket.

The effects of organic loading rate and operating temperature on the microbial diversity and performances of upflow anaerobic sludge blanket (UASB) reactors treating palm oil mill effluent (POME) were investigated. The following two UASB reactors were run in parallel for comparison: (1) under a mesophilic condition (37 degrees C) and (2) under a mesophilic condition in transition to a thermophilic condition (57 degrees C). A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis showed that the microbial population profiles significantly changed with the organic loading rate (OLR) and the temperature transition from the mesophilic to the thermophilic condition. Significant biomass washout was observed for the mesophilic UASB when operating at a high organic loading rate (OLR) of 9.5 g chemical oxygen demand (COD)/L.d. In contrast, the thermophilic UASB can be operated at this OLR and at a temperature of 57 degrees C with satisfactory COD removal and biogas production. The PCR-based DGGE analysis suggested that the thermophilic temperature of 57 degrees C was suitable for a number of hydrolytic, acidogenic, and acetogenic bacteria.

Microbial communities and their performances in anaerobic hybrid sludge bed-fixed film reactor for treatment of palm oil mill effluent under various organic pollutant concentrations.

The anaerobic hybrid reactor consisting of sludge and packed zones was operated with organic pollutant loading rates from 6.2 to 8.2 g COD/L day, composed mainly of suspended solids (SS) and oil and grease (O&G) concentrations between 5.2 to 10.2 and 0.9 to 1.9 g/L, respectively. The overall process performance in terms of chemical oxygen demands (COD), SS, and O&G removals was 73, 63, and 56%, respectively. When the organic pollutant concentrations were increased, the resultant methane potentials were higher, and the methane yield increased to 0.30 L CH4/g COD(removed). It was observed these effects on the microbial population and activity in the sludge and packed zones. The eubacterial population and activity in the sludge zone increased to 6.4 × 109 copies rDNA/g VSS and 1.65 g COD/g VSS day, respectively, whereas those in the packed zone were lower. The predominant hydrolytic and fermentative bacteria were Pseudomonas, Clostridium, and Bacteroidetes. In addition, the archaeal population and activity in the packed zone were increased from to 9.1 × 107 copies rDNA/g VSS and 0.34 g COD-CH4/g VSS day, respectively, whereas those in the sludge zone were not much changed. The most represented species of methanogens were the acetoclastic Methanosaeta, the hydrogenotrophic Methanobacterium sp., and the hydrogenotrophic Methanomicrobiaceae.

Identification of newly zeaxanthin-producing bacteria isolated from sponges in the Gulf of Thailand and their zeaxanthin production.

Sponge-associated bacteria have been found to produce a variety of bioactive compounds including natural pigments. Here, we report the molecular identification of zeaxanthin-producing sponge-associated bacteria isolated from sponges in the Gulf of Thailand and the effect of environmental factors on zeaxanthin production from a bacterium. Three colorful sponge-associated bacteria (CHOB06-6, KODA19-6, and MAKB08-4) were identified based on the 16S rDNA profile. The 16S rDNA sequence-based analyses revealed that CHOB 06-6 and MAKB 08-4 were the closest relatives to Sphingomonas phyllosphaerae FA2(T), and KODA19-6 was a relative of Shingomonas (Blastomonas) natatoria DSM 3183(T). After all bacteria were cultivated in a modified Zobell medium, S. natatoria KODA19-6 was found to produce the highest zeaxanthin at 0.62 mg/l. pH and temperature considerably affected its zeaxanthin production. Its optimal condition for zeaxanthin production was found at a pH of 7 and 30 °C. The bacterium had a maximum specific growth rate (µ(max)) of 0.06 1/h with zeaxanthin productivity (Q(p)) of 6.27 µg/l·h. Therefore, this newly zeaxanthin-producing bacterium has a potential to produce natural zeaxanthin for the food, feed, pharmaceutical, and cosmetic industries.

UASB performance and microbial adaptation during a transition from mesophilic to thermophilic treatment of palm oil mill effluent.

The treatment of palm oil mill effluent (POME) by an upflow anaerobic sludge bed (UASB) at organic loading rates (OLR) between 2.2 and 9.5 g COD l(-1) day(-1) was achieved by acclimatizing the mesophilic (37 °C) microbial seed to the thermophilic temperature (57 °C) by a series of stepwise temperature shifts. The UASB produced up to 13.2 l biogas d(-1) with methane content on an average of 76%. The COD removal efficiency ranged between 76 and 86%. Microbial diversity of granules from the UASB reactor was also investigated. The PCR-based DGGE analysis showed that the bacterial population profiles significantly changed with the temperature transition from mesophilic to thermophilic conditions. In addition, the results suggested that even though the thermophilic temperature of 57 °C was suitable for a number of hydrolytic, acidogenic and acetogenic bacteria, it may not be suitable for some Methanosaeta species acclimatized from 37 °C. Specifically, the bands associated with Methanosaeta thermophila PT and Methanosaeta harundinacea can be detected during the four consecutive operation phases of 37 °C, 42 °C, 47 °C and 52 °C, but their corresponding bands were found to fade out at 57 °C. The DGGE analysis predicted that the temperature transition can result in significant methanogenic biomass washout at 57 °C.

Effect of chitosan on UASB treating POME during a transition from mesophilic to thermophilic conditions.

The effects of chitosan addition on treatment of palm oil mill effluent were investigated using two lab-scale upflow anaerobic sludge bed (UASB) reactors: (1) with chitosan addition at the dosage of 2 mg chitosan per g volatile suspended solids on the first day of the operation (R1), (2) without chitosan addition (the control, R2). The reactors were inoculated with mesophilic anaerobic sludge which was acclimatized to a thermophilic condition with a stepwise temperature increase of 5 °C from 37 to 57 °C. The OLR ranged from 2.23 to 9.47 kg COD m(-3) day(-1). The difference in biogas production rate increased from non-significant to 18% different. The effluent volatile suspended solids of R1 was 65 mg l(-1) lower than that of R2 on Day 123. 16S rRNA targeted denaturing gradient gel electrophoresis (DGGE) fingerprints of microbial community indicated that some methanogens in the genus Methanosaeta can be detected in R1 but not in R2.

Nitrogen removal of anammox cultures under different enrichment conditions.

Anammox bacteria in sludge from an anoxic tank of a municipal wastewater treatment plant at Nongkhaem, Bangkok, Thailand were enriched in two sequencing batch reactors (SBRs; SBR-1 and SBR-2), under different conditions. SBR-1 was open to the atmosphere, while SBR-2 was closed and flushed with a mixture of 95% argon and 5% CO(2) during the fill period in order to provide strict anaerobic conditions. The specific nitrogen removal rates of SBR-1 and SBR-2 were 0.43 g N/g VSS-d and 2.59 g N/g VSS-d, respectively. Denaturing gradient gel electrophoresis (DGGE) analyses showed differences in band patterns among the Nongkhaem sludge and the two enrichment cultures. Based on fluorescent in situ hybridization (FISH), the anammox bacteria in both systems were either "Candidatus Brocadia anammoxidans" or "Candidatus Kuenenia stuttgartiensis". The results from this study demonstrate the potential of alternative anammox systems for nitrogen removal and provide information on the microbial communities of anammox cultures under different enrichment conditions.

Molecular monitoring of microbial population dynamics during operational periods of anaerobic hybrid reactor treating cassava starch wastewater.

This study characterized the microbial community and population dynamics in an anaerobic hybrid reactor (AHR) treating cassava starch wastewater. Methanogens and nonmethanogens were followed during the start-up and operation of the reactor, and linked to operational and performance data. Biomass samples taken from the sludge bed and packed bed zones of the AHR at intervals throughout the operational period were examined by 16S rRNA fluorescence in situ hybridization (FISH). The start-up seed and the reactor biomass were sampled during the feeding of the wastewater with a chemical oxygen demand (COD) value of 8 g L(-1) and a hydraulic retention time (HRT) of 8 days. These samples were characterized by the predominance of cells with long-rod morphology similar to Methanosaeta spp. Following a sharp operational change, accomplished by increasing the COD concentration of the organic influent from 8 to 10 g L(-1) and reducing the HRT from 8 to 5 days, there was a doubling of the organic loading rate, a reduction of the COD removal efficiency, as well as decreased methane content in the biogas and an accumulation of total volatile acids in the reactor. Moreover, this operational change resulted in a significant population shift from long-rod Methanosaeta-like cells to tetrad-forming Methanosarcina-like cells. The distributions of microbial populations involved in different zones of the AHR were determined. The results showed that nonmethanogens became the predominant population in both sludge and the packed bed zone. However, the percentage of methanogens in the packed bed zone was higher than that in the sludge bed zone. This higher percentage of methanogens was likely caused by the fact that the packed bed zone provided a suitable environmental condition with an appropriate nutrient availability for methanogen growth.

Use of an alternative Archaea-specific probe for methanogen detection.

An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted by performing whole cell hybridization using target and nontarget organisms. Anaerobic sludge samples were examined by in situ hybridization for methanogenic populations. The relative abundance of methanogens was monitored with epifluorescence microscopy. Individual cells could be visualized with strong fluorescence signals after hybridization with the newly developed probe.

Use of fluorochrome-labeled rRNA targeted oligonucleotide probe and tyramide signal amplification to improve sensitivity of fluorescence in situ hybridization.

A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe.

Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91.

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chiC, with the same transcriptional direction, and two unlinked genes, chiP and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, ChiB and ChiC exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while ChiP and ChiQ belonged to family 20. ChiP and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; ChiB bound chitin but had no chitinase activity; ChiC possessed strong chitinase activity and also bound chitin. Production of ChiC in S91 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of ChiC proteins in a chitinase-negative mutant. In Escherichia coli, ChiC appeared to be expressed from its own promoter.