Isolation and selection of native microorganisms for the aerobic treatment of simulated dairy wastewaters

Milk fat/protein degrading microorganisms were isolated from different locations of a dairy wastewater treatment system with the goal of developing an inoculum for bioaugmentation strategies. Eight isolates, identified by 16S rRNA gene sequence analysis as belonging to the genera Bacillus, Pseudomonas, and Acinetobacter, were tested for their ability to remove COD and protein from a milk-based medium (3000 mg/L COD) and compared to a commercial bioaugmentation inoculum. The Acinetobacter isolate exhibited a pellet-type growth in liquid culture, a property that could potentially aid in the separation of microbes and liquid phase following treatment. Based on the individual degradation capacity and growth behavior of the isolates, three microorganisms were further selected and tested together. This consortium exhibited a COD removal similar to the commercial inoculum (57% and 63%, respectively), but higher protein (consortium: 93%; commercial inoculum: 54%), and fat removals (consortium: 75%; commercial inoculum: 38%).     

Kinetic properties of a commercial and a native inoculum for aerobic milk fat degradation

The aerobic fat biodegradation potential and growth characteristics of a commercial and a native inoculum (activated sludge from a dairy wastewater treatment pond), were evaluated. Batch tests were conducted with a medium based on butter oil, as the sole source of carbon, and mineral salts. Residual fat, biomass and CO(2) production were measured. Overall fat removal values were above 78% for both inocula. The growth kinetics of the commercial and native inocula followed Haldane and Monod models respectively. Both inocula showed a similar behaviour when butter oil concentration was under 360 mg/l; at higher values, the difference between the growth rates increased as a consequence of the inhibition exhibited by the commercial inoculum. The selection of an inoculum for bioaugmentation of bioreactors in the wastewater treatment requires a comprehensive knowledge of their degradation ability and tolerance to fluctuating compounds and of the operational conditions that will be utilized.     

Effects of pH amendment on metal working fluid wastewater biological treatment using a defined bacterial consortium

The aim of this study was to determine whether pH amendment of a highly alkaline metal working fluid (MWF) wastewater would improve biological treatment in a bioreactor system following introduction of a bacterial inoculum (comprised of the following strains: Agrobacterium radiobacter, Comamonas testosteroni, Methylobacterium mesophilicum, Microbacterium esteraromaticum, and Microbacterium saperdae). The pH values tested were 6, 7, 8, and 9. Three replicate batch mode bioreactors inoculated with the bacterial inoculum (plus an abiotic control bioreactor) were operated for each of the four pH conditions. After 14 days, the final mean chemical oxygen demand (COD) reduction at pH 9 was 50 +/- 1.4%; at pH 8, 58 +/- 1.4%; pH 7, 65 +/- 1.0%; and pH 6, 75 +/- 2.7% of the initial COD (approximately 10,000 mg L(-1)), respectively. Interestingly, within 5 days, the pH in all inoculated bioreactors progressed toward pH 8. However, all abiotic control bioreactors remained at the pH at which they were amended. The fate of the inoculum, determined by denaturing gradient gel electrophoresis (DGGE) and by cluster analysis of the resulting DGGE profiles, revealed that the inocula survived throughout operation of all pH-amended bioreactors. Length-heterogeneity polymerase chain reaction (PCR) was used to track the population dynamics of individual strains. After 7 days of operation, M. esteraromaticum was the most abundant population in all bioreactors, regardless of pH. From our findings, it appears necessary to adjust the MWF wastewater from pH 9 to between 6 and 7, to achieve optimal biological treatment rates.     

Comparison of PCR-DGGE and selective plating methods for monitoring the dynamics of a mixed culture population in synthetic brewery wastewater

Enrichment of an activated sludge inoculum in synthetic brewery wastewater, which included glucose, maltose, and ethanol, was conducted in batch experiments to identify the dominant microbes present, to determine methodologies capable of monitoring the mixed culture population dynamics, and to determine the consortium's substrate degradation behavior. These results and methodologies were subsequently used in the determination of the population dynamics of suspended and attached microorganisms in a sequencing batch system in the second part of this research work. The three-membered microbial community comprised two bacterial and one fungal species that were identified as Acinetobacter sp., Enterobacter sp., and Candida sp. PCR-DGGE and plating on selective media were used to track the population dynamics of the consortium during the degradation of different substrates in synthetic wastewater containing glucose, maltose, and ethanol. Enterobacter sp. could degrade glucose and maltose but not ethanol, whereas Acinetobacter and Candida could degrade all three carbon sources. In buffered batch mixed culture experiments, Enterobacter was the predominant bacterium until the sugar concentrations decreased to levels that enabled Acinetobacter and Candida to degrade ethanol. PCR-DGGE was effective for detecting the dominant species, but culture-based methods were more accurate for monitoring the population dynamics of these microorganisms during growth in the wastewater medium.     

Development of a microbial consortium for dairy wastewater treatment

The wastewater from the dairy industries usually contains high concentrations of contaminants and, since the volume generated is also high, the total contaminant load is very significant. Among the available options for treatment, biological degradation looks like the most promising one. Furthermore, the supplementation of the native microbial populations with external microorganisms with high specific degradation rates (bio-augmentation) has demonstrated to improve the performance of treatment. The main objective of this research was to select a combination of bacteria to improve the aerobic treatment of dairy processing wastewater. For this purpose, eleven fat/protein-degrading microorganisms belonging to the genera Bacillus, Serratia, Lactococcus, Enterococcus, Stenotrophomonas, Klebsiella and Escherichia, were evaluated as potential degrading bacteria using a Plackett-Burman design. Assays were carried out to select the strains that most significantly influenced the degradation of wastewater and biomass yield, in terms of COD removal. A simulated dairy industry effluent was used as culture medium. Four strains were selected as potential members of the microbial consortium: Lactococcus garvieae, Bacillus thuringiensis, Escherichia coli and Stenotrophomonas sp. The optimal operation temperature and pH range of the selected consortium were 32°C and 6 ～ 8, respectively. The degradation percentages reached with the selected consortium were 80.67 and 83.44% at 24 and 48 h, respectively. The selected consortium significantly improved the degradation of the dairy wastewater, and the degradation degree achieved by this consortium was higher than by using the strains individually.     

Inhibition of Biogas Production by Alkyl Benzene Sulfonates (LAS) in a Screening Test for Anaerobic Biodegradability

The effect of the inoculum source on the digestion of linear alkylbenzene sulfonates (LAS) under anaerobic conditions has been investigated. The potential for primary and ultimate LAS biodegradation of anaerobic sludge samples obtained from wastewater treatment plants (WWTPs) of different geographical locations was studied applying a batch test system. It was found that only 4–22% of the LAS added to the batch anaerobic digesters was primarily transformed suggesting a poor primary degradation of the LAS molecule in anaerobic discontinuous systems. Regarding ultimate biodegradation, the addition of LAS to the batch anaerobic digesters caused a reduction on the extent of biogas production. Significant differences in the inhibition extent of the biogas production were observed (4–26%) depending on the sludge used as inoculum. Effect of the surfactant on the anaerobic microorganisms was correlated with its concentration in the aqueous phase. Sorption of LAS on anaerobic sludge affects its toxicity by depletion of the available fraction of the surfactant. LAS content on sludge was related to the total amount of calcium and magnesium extractable ions. The presence of divalent cations promote the association of LAS with anaerobic sludge reducing its bioavailability and the extent of its inhibitory effect on the biogas production.     

Measurement and mathematical modelling of competition between fast- and slow-growing ordinary heterotrophic organisms in low and high substrate-loaded systems

Ordinary heterotrophic organisms (OHO) of an activated sludge wastewater treatment system showed an atypical growth behaviour when they are inoculated to batch aerobic growth tests with a high substrate-loaded condition. For example, the OHO maximum specific growth rates on readily biodegradable substrates (μ _H) increased with a high ratio of substrate concentration to OHO active biomass concentration (So/Xo), although they were assumed to be constant in a conventional microbial growth kinetic model with a single OHO population group. We, therefore, set a hypothesis in that the change of OHO maximum specific growth rates in the batch test condition is caused by turnover of fast-growing OHO population against slow-growing OHO population. And, a competitive microbial growth kinetic model of the fast- and slow-growing OHO population groups was developed and validated with model-data fitting analysis for the batch test results. The competitive microbial growth kinetic model of process selection, rather than that of kinetic selection, was capable of simulating microbial growth kinetics in high substrate-loaded dynamic systems (i.e., batch tests) and low substrate-loaded steady-state systems (i.e., continuously operated wastewater treatment systems), better than the conventional non-competitive growth kinetic model.     

Treatment of high strength distillery wastewater (cherry stillage) by integrated aerobic biological oxidation and ozonation

The performance of integrated aerobic digestion and ozonation for the treatment of high strength distillery wastewater (i.e., cherry stillage) is reported. Experiments were conducted in laboratory batch systems operating in draw and fill mode. For the biological step, activated sludge from a municipal wastewater treatment facility was used as inoculum, showing a high degree of activity to distillery wastewater. Thus, BOD and COD overall conversions of 95% and 82% were achieved, respectively. However, polyphenol content and absorbance at 254 nm (A(254)) could not be reduced more than 35% and 15%, respectively, by means of single biological oxidation. By considering COD as substrate, the aerobic digestion process followed a Contois' model kinetics, from which the maximum specific growth rate of microorganisms (mu(max)) and the inhibition factor, beta, were then evaluated at different conditions of temperature and pH. In the combined process, the effect of a post-ozonation stage was studied. The main goals achieved by the ozonation step were the removal of polyphenols and A(254). Therefore, ozonation was shown to be an appropriate technology to aid aerobic biological oxidation in the treatment of cherry stillage.     

Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria

Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria. In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.     

Local soil,but not commercial AMF inoculum,increases native and non‐native grass growth at a mine restoration site

Soil communities are often degraded in mined sites, and facilitating the recovery of soil mutualists such as arbuscular mycorrhizal fungi (AMF) may assist with the restoration of native plants. At a grassland mine restoration site, I compared a commercial AMF inoculum with soil collected from beneath native grasses as a source of inoculum, as well as a control treatment. Field plots were broadcast‐inoculated and seeded with native grasses, and biomass of native and non‐native species was measured in three consecutive years. In addition, greenhouse‐grown seedlings of a native bunchgrass (Stipa pulchra) were inoculated with similar treatments, transplanted into the field, and assessed after 18 months. When broadcast inoculation was used, the local soil inoculum tended to increase non‐native grass biomass, and marginally decreased non‐native forb biomass in the second year of study, but did not significantly affect native grass biomass. Broadcast commercial inoculum had no detectable effects on biomass of any plant group. Stipa pulchra transplants had greater N content and mycorrhizal colonization, and marginally higher shoot mass and K content, when pre‐inoculated with local soil (relative to controls). Pre‐inoculation with commercial AMF increased AMF colonization of the S. pulchra transplants, but did not significantly affect biomass or nutrient content. The findings indicate that at this site, the use of local soil as an inoculum had greater effects on native and non‐native plants than the commercial product used. In order to substantially increase native grass performance, inoculation of transplanted plugs may be one potential strategy.     

Nitrate-removal activity of a biofilm attached to a perlite carrier under continuous aeration conditions

The nitrate-removal activity of a biofilm attached to a perlite carrier from an aerobic bioreactor used for treating dairy farm wastewater was examined by batch experiments under continuous aeration conditions. Despite aeration, the biofilm removed nitrate at a rate of 114.4 mg-N/kg-perlite/h from wastewater containing cow milk and manure. In a clone library analysis of the biofilm, bacteria showing high similarity to the denitrifying bacteria Thauera spp. were detected.     

Enhanced remediation of black liquor by activated sludge bioaugmented with a novel exogenous microorganism culture

Black liquor (BL) is a notoriously difficult wastewater to treat due to the economic and efficiency limitations of physiochemical methods and intrinsic difficulties with bioremediation strategies caused by the high pH (10–13) and lignin content. This study investigated the feasibility of a novel bioaugmentation strategy for BL treatment, which uses a mixed microorganism culture of lignocellulose-degrading microorganisms isolated from degraded bamboo slips. Black liquor treatment was assessed in terms of chemical oxygen demand (COD) and color removal with a sequencing batch reactor organic loading rate of 9 kg COD/L·day under highly alkaline conditions (pH?10). Results revealed that bioaugmented activated sludge treatment of BL with special mixed microorganisms significantly enhanced the removal efficiency of COD, color, and lignin from the wastewater up to 64.8, 50.5, and 53.2 %, respectively. Gel permeation chromatography profiles showed that the bioaugmentation system could successfully degrade high molecular lignin fragments in black liquor. This work confirms bioaugmentation as a feasible alternative strategy for enhanced biological treatment of wastewater with high lignin content and high organic load rate under strongly alkaline conditions.     

Enhancing denitrification using a carbon supplement generated from the wet oxidation of waste activated sludge

This study compared the effect of four pure carbon supplements on biological denitrification to a liquor derived as a by-product from the wet oxidation (WO) of waste activated sludge. Sequencing batch reactors were used to acclimate sludge biomass, which was used in batch assays. Acetate, WO liquor and ethanol-supplementation generated the fastest denitrification rates. Acetate and WO liquor were efficiently utilised by all acclimated biomass types, while poor rates were achieved with methanol and formate. When comparing an inoculum from an ethanol-supplemented and non-supplemented wastewater treatment plant (WWTP), the ethanol-acclimated sludge obtained superior denitrification rates when supplemented with ethanol. Similarly high nitrate removal rates were achieved with both sludge types with acetate and WO liquor supplementation, indicating that WO liquors could achieve excellent rates of nitrate removal. The performance of the WO liquor was attributed to the variety of organic carbon substrates (particularly acetic acid) present within the liquor.     

分子生态学方法在微生物肥料质量监测中的应用

应用PCR—DGGE技术对某微生物肥料的质量进行了跟踪监测,并结合分离培养和克隆文库分析对其菌种组成进行了检测。结果表明,同一生产批次3个不同包装样品的细菌和真菌DGGE（denaturing gradient gel eleetrophoresis）图谱相似性为80％-100％,3个不同生产批次之间DGGE图谱相似性为80％-88％,表明该微生物肥料的菌种组成的稳定性较好。但分离培养和克隆文库分析结果显示样品的菌种组成与产品标签说明之间存在较大差异。对于产品标注的6种微生物组成,只有Lactobacillus属的微生物可与之对应,其他检测到的Bacillus、Monascus、Brevibacillus、Psudomonas和Penicillium属的微生物并未包括在产品说明中。研究表明用分子生态学方法可以比较客观准确的对微生物肥料质量进行评估和峪测。     

Nitrate-Removal Activity of a Biofilm Attached to a Perlite Carrier under Continuous Aeration Conditions

The nitrate-removal activity of a biofilm attached to a perlite carrier from an aerobic bioreactor used for treating dairy farm wastewater was examined by batch experiments under continuous aeration conditions. Despite aeration, the biofilm removed nitrate at a rate of 114.4 mg-N/kg-perlite/h from wastewater containing cow milk and manure. In a clone library analysis of the biofilm, bacteria showing high similarity to the denitrifying bacteria Thauera spp. were detected.     

Biodegradation of azo and anthraquinone dyes in continuous systems

The purpose is to develop a complete microbiological model system for the treatment of wastewater from textile mills in developing countries. Artificial wastewater was treated by microorganisms growing on wood shavings from Norway spruce during unsterile conditions. The microorganisms were inoculated from forest residues. Mixtures of the azo dyes Reactive Black 5 and Reactive Red 2 were degraded in batch as well as continuous experiments. Reactive Red 2 mixed with the anthraquinone dye Reactive Blue 4 was also treated in the continuous system. The system consisted of three reservoirs - the first two with an anaerobic environment and the third with an aerobic. The dye concentrations were 200 mg l⁻¹ of each dye in the continuous system and the retention time was approximately 4 days and 20 h per reservoir. Samples from the process were analysed with spectrophotometer and LC/MS to monitor the degradation process. 86-90% of the colour was removed after a treatment of 4 days and 23 h in the continuous process. Two metabolites were found in the outlets of reactors one and two, but they were degraded to below the detection limit in the aerobic reactor.     

Comparison of microbial community composition and activity in sulfate-reducing batch systems remediating mine drainage

Five microbial inocula were evaluated in batch tests for the ability to remediate mine drainage (MD). Dairy manure (DM), anaerobic digester sludge, substrate from the Luttrell (LUTR) and Peerless Jenny King (PJK) sulfate-reducing permeable reactive zones (SR-PRZs) and material from an MD-treatment column that had been inoculated with material from a previous MD-treatment column were compared in terms of sulfate and metal removal and pH neutralization. The microbial communities were characterized at 0, 2, 4, 9, and 14 weeks using denaturing gradient gel electrophoresis and quantitative polymerase chain reaction to quantify all bacteria and the sulfate-reducing bacteria of the genus Desulfovibrio. The cultures inoculated with the LUTR, PJK, and DM materials demonstrated significantly higher rates of sulfate and metal removal, and contained all the microorganisms associated with the desired functions of SR-PRZs (i.e., polysaccharide degradation, fermentation, and sulfate reduction) as well as a relatively high proportion of Desulfovibrio spp. These results demonstrate that inoculum influences performance and also provide insights into key aspects of inoculum composition that impact performance. This is the first systematic biomolecular examination of the relationship between microbial community composition and MD remediation capabilities.     

Modification of norfloxacin by a Microbacterium sp. strain isolated from a wastewater treatment plant

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, "M. nematophilum" (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.     

Feasibility of methanogenic digestion applied to a low pH acetic acid solution

This paper discusses the methanogenic digestion of a synthetic acetic acid wastewater inoculated using a mixed culture obtained from an anaerobic digester at a municipal wastewater treatment facility. Experiments were conducted in 500mL batch reactors containing an unbuffered acetic acid solution. Test conditions compared methane production and acetic acid degradation at both acidic (pH 4.5) and neutral (pH 7.0) initial system conditions. Results showed that methane production increased by 30% when the initial pH was decreased from 7.0 to 4.5.     

Anaerobic ammonium oxidation (Anammox) in immobilized activated sludge biofilms during the treatment of weak wastewater

This work studied the formation of molecular nitrogen by the microbial population of immobilized activated sludge of the domestic wastewater treatment plants (WWTP) that employ the technology developed by ZAO ECOS Company. The technology includes physicochemical water pretreatment and treated water recycling. A hard flexible fibrous brush carrier is used for the immobilization of microorganisms. The presence of both aerobic and anaerobic microorganisms and functioning of the methanogenic microbial community was shown in the biofilms developing on the carrier fibers and in suspended sludge. The high efficiency of nitrogen removal at a low C/N ratio was established to be due to the conjugated nitrification, denitrification, and anammox processes, whose functioning was demonstrated by laboratory cultivation methods and by studying the processes in batch and continuous reactors. Fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH) revealed bacteria belonging to the order Planctomycetales, particularly their anammox group. This work is the first evidence of the important role of the anammox process in the combined system of physicochemical and biological treatment of weak wastewater (BCDEAMOX).