Bioaugmented System Research Articles

Bioaugmentation with a novel alkali-tolerant Pseudomonas strain for alkaline phenol wastewater treatment in sequencing batch reactor

A novel alkali-tolerant strain JY-2, which could utilize phenol as sole source of carbon and energy, was isolated from activated sludge. It was identified as Pseudomonas sp. by 16S rDNA sequencing analysis. The appropriate conditions for strain growth and phenol biodegradation were as follows: pH 8.0–10.0 and temperature 23–30°C. With initial phenol concentrations of 225, 400, 550 and 750 mg/l, the degradation efficiencies were 94.9, 93.3, 89.3 and 48.2% within 40 h at pH 10.0 and 30°C, respectively. The alkaline phenol-containing wastewater treatment augmented with strain JY-2 in sequencing batch reactor (SBR) system was investigated, which suggested that the bioaugmented (BA) system exhibited the better performance for adjusting high pH to neutral value than the non-bioaugmented (non-BA) one. Also, the BA system showed strong abilities for phenol degradation and maintaining good sedimentation coefficient (SV30). The microbial community dynamics of both sequencing batch reactor (SBR) systems were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) technique, which showed substantial changes between the two systems. This study suggests that it is feasible to treat alkaline phenol-containing wastewater augmented with strain JY-2.

Application of bioaugmentation in the rapid start-up and stable operation of biological processes for municipal wastewater treatment at low temperatures

To accelerate the start-up of biological municipal wastewater treatment processes at low temperatures and ensure their daily stable performances, bioaugmentation was adopted with the addition of specialized mixed bacteria. As a result, three types of biological processes were successfully started within 15days and showed stable and efficient daily performances. Results of PCR–DGGE analysis demonstrated the long-term predominance of the bioaugmented specialized bacteria in the bioaugmented systems. And results of BIOLOG analysis showed that the bacterial community structure and catabolic capability of the biological systems varied with wastewater temperature variations. However, the stability of the bacterial community under normal operating conditions and adaptability to perturbations enabled the long-term stable and efficient performances of the biological systems. In conclusion, bioaugmentation was successful for rapid start-up and stable performances of three typical biological municipal wastewater treatment processes at low temperatures.

Dynamics of augmented soil system containing biphenyl with Dyellaginsengisoli LA-4

One high efficient biphenyl-degrading strain Dyella ginsengisoli LA-4 was inoculated into biphenyl-contaminated soil for bioaugmentation in this study. The results showed that bioaugmentation could accelerate the startup period of the biphenyl bioremediation process compared with the non-augmented one. PCR-DGGE fingerprints demonstrated that both of the diversity and pattern of microbial community were affected by the addition of strain LA-4 and biphenyl. Biphenyl-utilizing populations gradually increased and become the dominant species. The introduced strain LA-4 could be persistent and co-exist well with the indigenous populations. However, both of the strain LA-4 and indigenous microorganisms in the bioaugmented system would be partially inhibited by Zn 2+ and Ni 2+. This study suggests that it is feasible and potentially useful to remediate biphenyl-contaminated soil using bioaugmentation with D. ginsengisoli LA-4.

Decolorization of Brilliant Scarlet GR enhanced by bioaugmentation and redox mediators under high-salt conditions

The synergistic effects of bioaugmentation and redox mediator catalysis on Brilliant Scarlet GR decolorization under high-salt conditions were investigated. Two start-up models were tested in simulated anaerobic sequencing batch reactors (AnSBRs). The results showed that the start-up was shortened by bioaugmentation with salt-tolerant strain TL, and the biodecolorization was promoted by adding anthraquinone after acclimatization. The start-up time of non-bioaugmented and bioaugmented activated sludge systems were about 9 d and 5 d, respectively. It was proved that anthraquinone could not enhance the process in the start-up stage because it would inhibit some potential azo-reducing microorganisms in the non-acclimatized activated sludge. When anthraquinone was added to the acclimatized bioaugmented system, the maximal decolorization rate was 700–800 mg L −1 d −1. PCR–DGGE fingerprints demonstrated that both the diversity and pattern of microbial community were affected by the addition of bioaugmentation strain and anthraquinone. All the dominant bands were confirmed as anaerobic bacteria.

Bioaugmentation with the nicotine-degrading bacterium Pseudomonas sp. HF-1 in a sequencing batch reactor treating tobacco wastewater: Degradation study and analysis of its mechanisms

The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented in an SBR system that is used to treat tobacco wastewater. Compared to the non-bioaugmented (non-BA) system, the bioaugmented (BA) system exhibited considerably stronger pollution disposal abilities, with 100% nicotine degradation and more than 84% chemical oxygen demand (COD) removal within 12 h. Nicotine degradation had a significant effect on COD removal in SBRs ( r = 0.928, p < 0.01). The mechanisms of bioaugmentation were systematically investigated using a combination of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR–DGGE) and a toxicity assay (protein carbonyl (PC) and DNA–protein crosslinking (DPC)). DGGE fingerprint profiles showed that the number of bands and the Shannon–Wiener index decreased at a nicotine load of 250 mg/L compared to a 40–130 mg/L nicotine load in the non-BA system. However, a stepwise increase in the Shannon–Wiener index was found during all periods in the BA system. A comparison of sequences excised from DGGE gels demonstrated significant differences in the dominant microbial species between the two SBRs. This result suggested that bioaugmentation of strain HF-1 could select cooperators for treating complicated tobacco wastewater. The PC content and the DPC coefficient increased significantly at levels higher than 80 mg/L in the non-BA system; nevertheless, no increase was observed in the BA system during the stepwise nicotine load. This indicated that bioaugmentation of strain HF-1 resulted in the maintenance of high treatment activity by minimizing the nicotine toxicity for other microbes in the BA system. In conclusion, the rapid nicotine degradation of strain HF-1 performed a vital function in SBR by influencing the microbial community structure, dynamics and activity of the activated sludge system.

Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii for simultaneous selenate and nitrate removal

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 µg Se × L−1) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30°C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) × L−1 day−1. Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 µg Se × L−1 was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) analysis revealed spherical bioprecipitates of ≤2 µm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. To validate the bioaugmentation success under microbial competition, gel cubes with immobilized S. barnesii cells were added to an Upflow Anaerobic Sludge Bed (UASB) reactor, resulting in earlier selenate (24 hydraulic retention times (HRTs)) and sulfate (44 HRTs) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. S. barnesii was efficiently immobilized inside the UASB bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that S. barnesii was present in both the sludge bed and the effluent. This demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. Eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-1915-x) contains supplementary material, which is available to authorized users.

Bioremediation of an aged hydrocarbon-contaminated soil by a combined system of biostimulation–bioaugmentation with filamentous fungi

This paper presents a study of the effect of a combined biostimulation–bioaugmentation treatment applied to a silty-loam soil polluted with 60,600 mg kg −1 of a complex mixture of total petroleum hydrocarbons (TPH), which comprises 40% aliphatic hydrocarbons (AH) and 21% polycyclic aromatic hydrocarbons (PAH). The bioaugmentation was performed with Rhizopus sp., Penicillium funiculosum and Aspergillus sydowii strains isolated from two aged soils contaminated with 60,600 and 500,000 mg of TPH per kilogram of dried soil. The native fungi were able to grow in a complex solid mixture of hydrocarbons of high molecular weight, after previous acclimatization in liquid culture. The three fungi mentioned above were able to remove, respectively, 36%, 30% and 17% more PAH in comparison with biostimulation alone. In the bioaugmented systems with Rhizopus sp. and A. sydowii, a positive correlation of respirometric activity (CO 2 production) with hydrocarbon removal was obtained ( R 2=0.75; p( F)=0.001 and R 2=0.78; p( F)=0.001, respectively); in contrast, P. funiculosum did not show any correlation. An interesting finding from this work is that two of these species of fungi had not previously been reported as being PAH-degrading.

Bioaugmented sulfur-oxidizing denitrification system with Alcaligenes defragrans B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100-1,000 mg NO3-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO3-N/m(3) per day. At nitrate loading rate of more than 0.5 kg NO3-N/m(3) per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO3-N/m(3) per day.

Biological Enhancement of Tetrachloroethene Dissolution and Associated Microbial Community Changes

A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating that there was at least a 3-fold enhancement of PCE dissolution atthis time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.

Enhancement of domestic wastewater treatment under long sewer line condition in a laboratory set-up by Aspergillus niger bioaugmentation

The effect of Aspergillus niger bioaugmentation on COD and protein removal in domestic wastewater under sewage conditions was assessed. The sewer simulating bioreactor was running at a hydraulic retention time of 17 h, 20°C and pH 7.8 under aerobic condition. When A. niger was bioaugmented, 45 % to 72 % of COD was removed compared to 28 % to 48 % removal of COD in the control at the same period. An overall protein removal of 66 % resulted when A. niger was bioaugmented compared to 29.7 % in the control. Regarding enzymatic activities, we observed that as long as the bioaugmented system biomass concentration was higher than the control, the enzymatic activities were also higher. This research is an initial investigation on wastewater transformation under transitory conditions by A. niger and demonstrated the capacity of A. niger to remove both COD and protein under actual conditions. A. niger bioaugmentation under sewer conditions could be an alternative for wastewater treatment with a valorisation of fungal waste biomass.

The effect of a secondary chlorophenol presence on the removal of 2,4-dichlorophenol (2,4-DCP) in an activated sludge system bioaugmented with 2,4-DCP degrading special culture

Bioaugmentation is often used to improve the removal of some recalcitrant pollutants in the contaminated water or soils. In this study, 2,4-dichlorophenol (2,4-DCP) degrading special microorganisms, which was cultured in our lab, was augmented in an activated sludge (AS) system to enhance the degradation of 2,4-DCP. The effect of a secondary chlorophenol, 4-monochlorophenol (4-MCP), which was present with 2,4-DCP, on the removal of 2,4-DCP in the bioaugmented system was investigated through batch and fed-batch tests. Results showed that the first addition of 4-MCP in feed inhibited the degradation of 2,4-DCP, and the inhibition increased with the increase of 4-MCP initial concentration. However, for the second and third fed-batch run, the system receiving the mixed components of 4-MCP and 2,4-DCP demonstrated higher 2,4-DCP removal rate than the system receiving 2,4-DCP alone. In the system with mixed components, 2,4-DCP was always degraded first, and when it nearly reached complete removal, 4-MCP degradation began. A double-stage degradation pattern was observed.

Enhancement of 2,4-dichlorophenol degradation in conventional activated sludge systems bioaugmented with mixed special culture

Efficient and reliable removal of recalcitrant compounds, which are present in wastewater intermittently, is critically important to prevent toxicity discharge and system upset, especially for those biotreatment systems with poor anti-shock loading capacity such as conventional activated sludge (CAS) systems. In this study, 2,4-dichlorophenol (2,4-DCP) was chosen as a model recalcitrant substance. 2,4-DCP degrading mixed culture was bioaugmented to a CAS system in terms of enhancing 2,4-DCP removal and maintaining system stability under shock loading conditions. The effects of bioaugmentation on the performance of CAS systems after single inoculation were investigated under long-term continuous operation, during which four times shock loading occurred. Results showed that the two bioaugmented CAS systems, which were inoculated with 5% and 15% 2,4-DCP degrading mixed culture, respectively, demonstrated and maintained stronger ability to degrade 2,4-DCP than the non-supplemented control one during the first three shock loading periods within the first month after the first inoculation. For the fourth 2,4-DCP shock loading which occurred 100 days after inoculation, the advantages demonstrated by the bioaugmented systems were greatly reduced compared to those of the previous three runs. In addition, ribosomal intergenic spacer analysis method was used to track the supplemented special culture and assay the effect of bioaugmentation on the changes of microbial community structure.

Bioaugmentation of the phyllosphere for the removal of toluene from indoor air.

The removal of airborne toluene by means of the phyllosphere of Azalea indica augmented with a toluene-degrading enrichment culture of Pseudomonas putida TVA8 was studied. The 95% disappearance time [DT95%; the time in which an initial toluene concentration of 90 ppmv (339 mg.m(3)) was removed in a batch experiment] was 75 h for Azalea plants. Under the same experimental conditions, DT95% of inoculated Azalea plants decreased remarkably to about 27 h. Subsequent additions of toluene further increased the removal efficiency of the bioaugmented system (DT95% decreased by a factor of four). A decrease in DT95% was also recorded after repeated incubations of non-inoculated plants, but the toluene-removal rate was remarkably low, compared with the inoculated plants. Hence, inoculation of the leaf surface appeared essential for obtaining rapid removal rates. It was not possible to obtain comparable and sustained removal of airborne toluene by inoculating artificial plant surfaces. This is, to our knowledge, the first report on bioaugmentation of the leaf surface of plants to remove gaseous pollutants from air. The results presented are promising and could be of great practical importance in the field of indoor air pollution control.

Biodegradation of 2,4-dichlorophenol in sequencing batch reactors augmented with immobilized mixed culture

2,4-Dichlorophenol (2,4-DCP) degrading mixed culture was immobilized in polyvinyl alcohol jel beads and supplemented to sequencing batch reactors (SBR) to treat 2,4-DCP containing wastewater. Impacts of bioaugmentation level on the performance of bioaugmented systems were studied. Results show that inoculum size affected the start-up time of the SBR systems. For the non-augmented SBR system, nine days was needed for the system to start-up, whereas it only took six, four, three and two days for the SBRs with 1.9%, 3.7%, 5.6% and 9.3% immobilized culture, respectively. In addition, bioaugmented SBR systems demonstrated stronger capacity to cope with high 2,4-DCP shock loading than the control system. The control SBR failed to treat 2,4-DCP at 166 mg/l in influent, while the SBR with 1.9% inoculation could successfully cope with 2,4-DCP at 166 mg/l, but failed at 250 mg/l, and the SBR with 3.7%, 5.6% and 9.3% immobilized culture could successfully degrade 250 mg/l 2,4-DCP in feed. Furthermore, the contributions to the removal of 2,4-DCP by the introduced and indigenous culture in an augmented SBR system at various operation stages were investigated. It was found that augmented culture played the primary role in degrading 2,4-DCP at the beginning of system start-up, but after one-month operation, both the indigenous and the introduced culture posed strong ability to degrade 2,4-DCP.

Preparation and characteristics of resting cells of bioluminescent Pseudomonas putida BLU

A bioluminescent strain, Pseudomonas putida BLU, which was created by inserting the lux gene into the chromosome of P. putida mt-2 (TOL), a well-known degrader for benzene, toluene and xylene (BTX), produces luminescence depending on both cell concentration and intracellular metabolic activity, including the intracellular level of reduced riboflavin phosphate (FMNH 2). To estimate the cell concentration of a BTX degrader by the rapid detection of bioluminescence in a bioaugmentation system, the effect of metabolic activity on the output of bioluminescence needs to be considered. For this purpose, the preparation of resting cells of P. putida BLU, defined as incapable of growing but having metabolic activity, was tried using different surfactants. Resting cells formed on addition of n-dodecyltrimethylammonium bromide (DTAB, a cationic surfactant) at a concentration below the critical micellar concentration (CMC) to the bacterial culture, and exhibited morphological change. The P. putida BLU showed a decrease in bioluminescence output to a constant level and stopped growing immediately after the addition of DTAB, while maintaining activity for oxygen consumption. When the bacterial culture treated with DTAB was washed with a 0.9% saline solution, the growth and bioluminescence of the resting cells recovered to the normal level. The bioluminescence output (LUX) of the resting cells prepared at different time points from the bacterial culture correlated only with the cell concentration ( X), LUX∝ X 1.0, suggesting that generating resting cells enables the cell concentration to be simply and rapidly quantified by measuring the luminescence.

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.

Biodegradation kinetics and fate modelling of pentachlorophenol in bioaugmented activated sludge reactors

The growth and biodegradation kinetics of Mycobacterium chlorophenolicus comb nov. was studied in a series of batch experiments in the laboratory to identify and quantify the significant factors for its bioreactor performance. A mathematical model was formulated taking into account the degradation of primary and secondary substrate, as well as the growth of nonspecific and specific biomass. Using model parameters determined by the batch tests, the model was validated by simulations of a continuous flow experiment with bioaugmentation of the PCP-degrading culture in 2 activated sludge systems. A novel technique using immunofluorescence microscopy for quantifying the pure culture made it possible to evaluate the specific PCP degradation rates related to the PCP-degrading culture. The comparisons showed an excellent agreement for the total PCP removal, although there were some over- and underestimations of sorption and biodegradation as individual removal processes. The specific first order biodegradation rate constants were about 3 times higher in the bioaugmented systems that in batch experiments with the added culture alone. Even rough estimates for specific biodegradation rate constants may give meaningful predictions of the fate of specific organic compounds in wastewater treatment processes. For practical engineering and management purposes this may in many cases be adequate. The study highlighted the importance of reporting operating conditions in experiments from which biodegradation kinetic parameters such as Y, μ m and K S were derived.