4 Effect of pH and temperature on perchlorate degradation using succinate as sole carbon source by mixed consortium primarily Burkholderia sp. 4.12 (b) Reduction of perchlorate at different initial concentrations of phenol present in synthetic wastewater by mixed consortium mainly Pseudoxanthomonas sp.
Introduction
Literature review and aim of the study
Materials and Methods
Result and Discussion
Conclusions and scope of future study
LITERATURE REVIEW
Perchlorate anion
- Redox properties of chlorine compounds
- Sources of perchlorate in the environment
- Groundwater mobilization of perchlorate
- Fate of perchlorate in the environment
Studies have shown that foods are a source of perchlorate in the United States (Sanchez et al., 2005). A commonly investigated mechanism for the natural attenuation of perchlorate in the subsurface environment is anaerobic microbial degradation.
Health effects of perchlorate
Perchlorate is used by some bacteria as an electron acceptor for cellular respiration and is completely broken down into chloride ions. The studies have shown that perchlorate is found in plant tissues that incorporate a weakening mechanism (Yu et al., 2004).
Permssible limits of perchlorate in drinking water
Additional research is needed to determine the contribution of sources of perchlorate other than drinking water. This requires more progress in the field of analytical methods to extend current approaches to other media of perchlorate pollution.
Perchlorate treatment technologies
- Membrane-based technologies
- Anion exchange technology
- Precipitation
- Chemical and electrochemical reduction
- Biological treatment method
- Perchlorate bioreduction pathway
- Use of different carbon sources for microbial perchlorate bioreduction
- Bioprocess for perchlorate remediation
In this technique, the low solubility of the HNitClO4 (ion pair of protonated nitro cation and the perchlorate anion) was used. Photoactivation of the perchlorate by UV or laser irradiation can promote an intramolecular redox reaction (probably by oxygen atom transfer).
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Literature Review 27
Literature Review 28
Literature Review 29
Material
- Chemicals and reagents
- Glassware, apparatus and instruments
All chemicals and reagents used in the present study were either of analytical reagent (AR) or laboratory reagent (LR) grade. Double distilled water or ultrapure water was used for the preparation of all chemical reagents. All glassware used in this study was borosilicate and kept immersed overnight in chromic acid solution (2.25 gm K2Cr2O7 and 20 ml concentrated H2SO4 in 1L distilled water) followed by washing with water tap and then distilled water.
For drying, the cleaned glassware was kept in an oven at 110°C for 2–4 hours before being used in the experiment.
MATERIALS AND METHODS
Sludge biomass and growth media
Sludge from the wastewater treatment plant of Indian Institute of Technology Guwahati was collected and stored in a 2 liter glass aspirator bottle containing approximately 1.5 liters of culture medium with acetate as carbon source. The culture medium prepared in distilled water contained the following: Sodium perchlorate, 1.00 g/L, Sodium acetate, 1.00 g/L Nitrogen gas was supplied to the medium from a nitrogen gas cylinder to drive off any dissolved oxygen present.
Packed bed reactor (PBR)
For sampling, an outlet port with a length of 7.5 cm was provided below the top of the reactor. An inverted T-junction with a long silicon tube at the top was connected to the effluent port of the reactor to facilitate the release of biogas formed in the effluent tube. The biogas formed in the reactor was allowed to pass through the conical top of the reactor, a liquid trap and finally through a gas trap as shown in fig. 3.1.
An actual photograph of the reactor setup in the laboratory is shown in Fig 3.2.
Experimental Methodologies
- Seed culture development
- Inoculum preparation
- Culture condition
- Acclimatization
- Batch studies to evaluate the effect of different C-sources on perchlorate bioreduction
- Batch study on perchlorate bioreduction by the mixed consortium predominantly Burkholderia sp. at different initial succinate concentration
- Enrichment, Isolation and identification of predominant perchlorate reducing strains
- Growing of isolated bacterial strains
- Optimization of the culture conditions for perchlorate bioreduction by the mixed consortium predominantly Burkholderia sp
- Optimization of the culture conditions for perchlorate bioreduction by the mixed consortium predominantly Pseudoxenthomonas sp
- Kinetics of perchlorate bioreduction by the mixed consortium
- Batch study on perchlorate bioreduction by the mixed culture predominantly Pseudoxanthomonas sp. at different initial phenol concentration
- Batch study on perchlorate biodegradation by the mixed consortium predominantly Pseudoxanthomonas sp. from industrial wastewater
- Effect of co-pollutants on perchlorate bioreduction by the mixed consortium The capability of the seed sludge, grown in succinate or phenol as carbon sources, on
- Perchlorate bioreduction in continuous PBR by the mixed consortium
An amount of 0.5-L sludge and 1.0-L medium was added to a 2.0-L conical flask, incubated at 28oC in an incubator so that the final concentration of the biomass in the flask was 200 mg/L. The potential of the enriched mixed consortium to use different organic acids (such as succinic acid, acetic acid, oxalic acid, formic acid and citric acid) and phenol as sole source of carbon in degrading perchlorate was analyzed in a series of batch shake flasks. Increasing concentrations of phenol were added in batch shake flasks with a fixed amount of perchlorate (500 mg/L) to investigate the capacity of the consortium to withstand phenol concentration.
Dissolved oxygen in the medium was removed by flushing with oxygen-free nitrogen gas for 5 min in each of the flasks.
Analytical methodologies
The absorbance values were expressed as dry cell weight using a calibration curve plotted between the biomass optical density (OD) versus mixed liquid suspended solids (MLSS). For determination of biomass concentration as MLSS, 10 ml samples at different optical density ranging from 0.1-0.8 OD600 were centrifuged for 10 minutes at 10000g in ambient temperature (Remi C-24-BL Mumbai, India) in a pre-weighed 15 ml centrifuge tube (Tarson, India). Ion meter equipped with perchlorate sensitive electrode (Thermoscientific, Singapore) was also used for determination of perchlorate anion.
Standards were made based on the likely concentration of the samples, the range of standards was determined.
RESULTS AND DISCUSSION
Acclimatization of perchlorate reducing microbial consortium
Effect of different C-sources on perchlorate bioreduction by mixed consortium
The likely reaction mechanisms of perchlorate degradation by the mixed consortium using these five organic acids are given below. The influence of the succinate concentration on the degradation of perchlorate was further investigated by adding different amounts of succinate ranging from 300 to 1000 mg/l. With the increase of the initial concentration, the removal efficiency (%) of perchlorate was increased, and with an initial succinate concentration of 1000 mg/L, the mixed consortium was able to degrade the total amount of perchlorate within 10 days.
The degradation rate constant (kd) of perchlorate increased from 0.15 per day to 0.29 per day as the initial succinate concentration increased from 300 to 1000 mg/L.
Isolation and identification of perchlorate reducing strains from mixed consortium
The sequences were submitted to Genbank and the accession numbers for strain AG and AG2 were given as HM104637 and JX860406. Gene analysis by online BLAST tool indicates that the isolates contain sequences specific to the members of the family Proteobacteria. The high bootstrap support of the tree shown in derived from the 16S rDNA analysis showed that strain AG is a typical member of the genus Burkholderia sp.
The results of biochemical tests performed on both strains were listed in Table 4.1, which confirmed the identification of both strains.
Results and Discussion 70
Results and Discussion 71
Results and Discussion 72
Optimization of the culture conditions for the bioreduction of perchlorate using succiante as sole carbon source by the mixed consortium predominantly Burkholderia sp
It was found that temperature, inoculum age, carbon to ḍ ratio are more significant according to the results obtained by Plackett-Burman method of screening using succinate as a sole C source for reducing ḍ to a To analyze the relative importance of each factor more systematically, an analysis of variance (ANOVA) was applied to the data. The main purpose of ANOVA is to extract from the results how much variation each factor causes. It is also reported that 28oC is the optimum culture condition for identified PRB (সি reducing bacteria) responsible for স and chlorate respiration growing in anaerobic condition.
The inoculum age was found to be optimal, as 3 days in the case of degradation using succinate, which falls within the early log phase. The observation of isolated dominant strains is also similar to the previously reported studies of ส degrading bacteria (PRB), where ส has been found reduced in early and mid log phase.
Optimization of the culture conditions for the bioreduction of perchlorate using phenol as sole carbon source by the mixed consortium predominantly Pseudoxanthomonas sp
Perchlorate bioreduction in batch system using succinate as sole carbon source by mixed consortium predominantly Burkholderia sp
The reduction of ღ using succinate as the sole C source was found to follow more appropriate zero-order kinetics compared to first order except for sets where the initial ღ concentration was 100 mg/L. The rate of C reduction was found to be increased with increasing C concentration from 100 to 500 mg/L and then decreased with further increase in initial C concentration up to 800 mg/L (Table 4.10). The results indicated a probable inhibitory effect of initial C concentration on its reduction, which has not been reported to date and requires further investigation emphasizing metabolic aspects of PRB (perchlorate-reducing bacteria).
Perchlorate bioreduction in batch system using phenol as sole carbon source by mixed consortium predominantly Pseudoxanthomonas sp
While it took 10 days to completely reduce all ส for the initial concentration of 200 and 300 mg/L. The rate of perchlorate degradation was determined according to the zero-order and first-order reaction for each initial concentration. The R2 values show that the first-order kinetics fit better than the zero-order considering all initial concentrations because with all initial concentrations ሆ, the R2 values for the first order were above 90% (Table 4.10).
This result differs from the previous experiment, where the zero-order reaction was found to be followed for σ reduction with succinate as the sole C source.
Degradation of phenol at different initial concentrations by mixed consortium predominantly Pseudoxanthomonas sp. in synthetic and industrial wastewater
The total degradation of phenol was faster in industrial wastewater collected from an oil refinery. Within 120 days of the experiment, 100% removal of phenol was observed in the case of wastewater collected from a refinery where 140 days were required for complete phenol removal for synthetic wastewater. І was degraded in a significant amount in both cases, viz. using waste water from refineries and also synthetic waste water (Fig.
In lower initial concentration of phenol (50 mg/L) only 60% of ฆ was removed, while at the highest phenol concentration (600 mg/L) 95%.
Effect of co-pollutants on perchlorate bioreduction using succinate as sole carbon source by the mixed consortium predominantly Burkholderia sp
In the presence of phosphate, both anions are simultaneously taken up to a significant extent by the enriched mixed culture (Fig 4.13 (b)). In contrast to previous cases, the consortium was able to degrade the total amount of perchlorate (~100%) while only ~33% of chlorate was reduced (after 6 days of incubation). Therefore, the observed effective degradation of ฆ by the mixed microbial culture in the study in the presence of chlorate is unlikely.
Simultaneous bioreduction of nitrate and perchlorate by mixed consortium predominantly Burkholderia sp
COD of the media was also measured to know the utilization of carbon sources during the degradation of both pollutants. From the results of varying the initial concentration with the initial concentration, it was observed that the degradation of nitrate was inhibited when the ratio of nitrate was more than 1:10. The usage profile of the carbon source (succinate) by the mixed consortium with different ratios of perchlorate and nitrate was almost the same in all cases.
Extra c source was added fresh after depletion of the c source in the media, where perchlorate was almost unused.
Effect of co-pollutants on perchlorate bioreduction by mixed consortium predominantly Pseudoxanthomonas sp.using phenol as sole source of carbon
Therefore, observed effective degradation of perchlorate with uptake of chlorate by the mixed microbial culture in the study is not unlikely. Although in the presence of phosphate, the culture utilized both the anions (perchlorate and phosphate) simultaneously at a significant rate by the enriched mixed culture (Fig. The reduced degradation of perchlorate in the presence of other co-pollutants can be analyzed by the use of phenol in each system.
From the results, it was found that the mixed consortium almost completely utilized the phenol after 6 days in each case, which affected the degradation of the perchlorate.
Start-up period for perchloarte reduction in PBR (packed bed reactor)
Perchlorate bioreduction in continuous system succinate as sole alternative carbon source by the mixed consortium predominantly Burkholderia sp
When the HRT was reduced to 2 days, steady state occurred after 11 days of reactor operation. It was observed that the effluent concentration of perchlorate, phenol and succinate increased and then decreased within 10 days of changing the loading rate. While the phenol concentration was increased to 400 mg/l, the removal efficiency was found to be 10 to 15% for both perchlorate and phenol (Fig. 4.20).
The phenol concentration was reduced to 350 mg/L and the removal efficiency increased within 3 to 4 days, reaching a steady state for both perchlorate and phenol removal.
Effluen erchlorate = 400
Perchlorate bioreduction in continuous system (PBR) phenol as sole C-source by the mixed consortium predominantly Pseudoxanthomonas sp
When the HRT was changed from 10 to 7 days, the removal efficiency of perchlorate and COD decreased to 15. As in PBR 1, the reactors took 3 to 4 days to reach steady state each time the HRT was changed. When HRT was changed from 4 days to 3 days, steady state was reached after 10 days of surgery.
HRT was increased to 18 hours and steady state was reached within 10 days of operation.
CONCLUSIONS AND SCOPES OF FUTURE STUDY
Conclusion
According to the ANOVA results, the C/P ratio showed the greatest variance in the case of perchlorate reduction with predominantly Burkholderiasp. 9 Feasibility of using sequential simultaneous degradation of perchlorate and phenol in wastewater in anaerobic systems. Treatment of perchlorate- and nitrate-contaminated groundwater in an autotrophic packed-bed gas-phase bioreactor.
Simultaneous removal of perchlorate and nitrate from drinking water using the ion exchange membrane bioreactor concept.
