Biodegradation of pyridine by an isolated bacterial consortium/strain and bio-augmentation of strain into activated sludge to enhance pyridine biodegradation
Pyridine and pyridine based products are of major concern as environmental pollutants due to their recalcitrant, persistent, toxic and teratogenic nature. In this study, we describe biodegradation of pyridine by an isolated consortium/strain under aerobic condition. Batch experiment results reveal that at lower initial pyridine concentrations (1–20 mg l−1), almost complete degradation was observed whereas at higher concentration (30–50 mg l−1), the degradation efficiency was dropped significantly. This may be due to inhibitory effect of pyridine at higher concentrations. The value of decay and yield coefficient was also determined. Furthermore, the bio-augmentation of isolated consortium/strain into the activated sludge consortium in different quantity has been also done and the effect of bio-augmentation on degradation has been studied. The results reveal that as the quantity of bio-augmentation increases, the degradation of pyridine increases. At 25% bio-augmentation, complete degradation of 20 mg l−1 of pyridine can be achieved within 96 h of incubation. Thus, the study concluded that the bio-augmentation of the isolated consortium/strain into the sludge enhances the pyridine degradation efficiency of the biomass.
KeywordsBio-augmentation Biokinetic Activated sludge N-heterocyclic compound Pyridine Growth kinetics
The rapid expansion and increasing sophistication of the chemical industries in the past century and particularly over the last 30 years has meant that there has been increasing amount of complexity of toxic waste effluents. Xenobiotic compounds are generally man made synthetic aromatic compounds having a persistent toxic nature and are characteristically difficult to degrade in the environment. Biological degradation of xenobiotic compounds is considered to be one of the challenging tasks. Biological degradation unlike physicochemical methods is a natural process in which microorganisms make use of organic pollutants present for its growth and other cellular processes.
Aromatic heterocyclic compounds are of major concern as environmental pollutants due to their recalcitrant, persistent, toxic, and teratogenic nature. Heterocyclic compounds are generated by many industries. Most of these chemicals are toxic to human health (Sims and O’Loughlin 1989; Liu et al. 1998; Padoley et al. 2006; Mudliar et al. 2008). Pyridine, one of the important N-heterocyclic compounds, occurs in the environment as a result of oil shale retorting, coal gasification, and pesticide use (Sims and Sommers 1985; Stuermer et al. 1982; Liu et al. 1998) and has potential application in manufacturing of dyes, explosives, pesticides, and pharmaceuticals (Kaiser et al. 1996; Liu et al. 1998).
The presence of pyridine in the environment creates severe health hazards because pyridine is toxic, teratogenic, and at higher concentration results in weakness and ataxia (Browning 1965; Padoley et al. 2006; Mudliar et al. 2008). The amounts of pyridine bases produced worldwide were estimated as: pyridine-26,000 t/year, 2-methylpyridine-8,000 t/year, 3-methyl pyridine- 9,000 t/year, 4-methylpyridine−1500 t/year), 5-ethyl-2-methylpyridine- 8,000 t/year (Shimizu et al. 1993). Thus the production of the pyridine based compound in large quantity is severe concern for the environmentalist. Hence, there is an urgent need to develop a bioremediation system for the remediation of pyridine based compounds from environment.
Various physico-chemical methods such as adsorption, chemical oxidation or incineration are available for the treatment of waste emissions containing pyridine and its derivatives (Devinny et al. 1999). However, these processes are energy intensive, require high capital and operating costs and also generate secondary waste streams (Mudliar et al. 2008). Alternatively, biological treatment methods can provide a better option in view of their low capital and operating cost and the treatment results in formation of innocuous products.
Various studies showed that microbes under aerobic and anaerobic conditions are able to utilize pyridine and its derivatives (Gupta and Shukla 1975; Sims and O’Loughlin 1989; Kaiser et al. 1996; Fetzner et al. 1998; Leenheer and Stuber 1981; Mohan et al. 2003; Mudliar et al. 2008).
But for designing a system for bioremediation of pyridine using an isolated strain, the studies on applicability of the isolated consortium/strain into the activated sludge should be tested but unfortunately, there is not much study related with the bio-agumentation of isolated consortium/strain into the activated sludge. Hence, in this study, we describe the biodegradation of pyridine by the isolated consortium/strain and its applicability in the activated sludge for the treatment of pyridine containing wastewater. The bio-augmentation of isolated consortium/strain into the activated sludge consortium in different quantity has been done and the effect of bio-augmentation on degradation has also been studied.
Materials and methods
The pyridine degrading microorganism/consortium has been isolated from pesticide contaminated dumpsite and was maintained on the plates containing pyridine as sole carbon source by periodic sub- culturing. The culture was stored at 4°C.
Media and conditions
The minimal mineral media used for the study was composed of (mg per liters of deionized water) KH2PO4-175, K2HPO4-570, Na2HPO4-668, NaHCO3-20, NH4NO3-185.5, and MgSO4-225 in the media. Media was sterilized by autoclaving at 121°C and 15 lb/in2 for 20 min. All components were autoclaved separately to avoid formation of precipitate. After cooling, the solutions were mixed under sterile conditions. The study was conducted at 30°C in an incubator with shaking at 50 rpm.
Batch studies on biodegradation of pyridine using isolated consortium/strain
In this study, the whole cells were used as enzyme system for pyridine degradation. The cell biomass was obtained by growing the bacterial culture in the incubator at 30°C. The culture was grown for fifteen days in minimal mineral media supplemented with addition of 10 mg l−1 of pyridine everyday. The cells were harvested at 4°C by centrifugation, suspended with minimal mineral media and pyridine of concentrations 1, 2, 5, 10, 20, 30, 40, and 50 mg l−1 in different flasks for biokinetic study. The samples at particular time interval were withdrawn and analyzed for the bacterial growth via optical density (O.D) and mixed liquor volatile suspended solids (MLVSS) and pyridine degradation via colorimetric method (Mohan et al. 2003). The control experiments was carried out using autoclaved biomass at all initial concentrations to determine the loss in the pyridine concentration due to bio-sorption and also the control experiment without the biomass was performed to determine any type of abiotic degradation.
Batch studies on effect of bio-augmentation of isolated consortium/strain into the activated sludge biomass on pyridine degradation efficiency
In the study, the isolated consortium/strain is bioagumented with the biomass of activated sludge (procured from an activated sludge plant treating wastewater from pharmaceutical industry) in different quantity and the effect of bio-augmentation on degradation was studied. The studies were done with addition of different quantity of isolated consortium/strain to the biomass, ranging from 0 to 25% (by wt.). The study was conducted such that the overall biomass quantity remains same in the system. The study was carried out at an initial pyridine concentration of 20 mg l−1. To determine the effect of bio-augmentation on pyridine degradation, the pyridine concentration was analyzed at different time intervals.
Liquid sample for chemical analysis were taken with sterile pipette for analysis of substrate, the samples were centrifuged and the supernatant was stored at 4°C until analyzed. Cell growth was monitored by measuring the OD of the culture broth samples at 620 nm and MLVSS as per APHA (1998). The pyridine present in the aqueous phase was monitored colorimetrically at 450 nm as mentioned by Mohan et al. (2003).
Results and discussions
Enrichment and isolation
During the acclimatization process certain enzymes in the bacteria are induced so that they are available for taking part in metabolism reaction. This is much more important when dealing with toxic compounds such as pyridine, and these too at high concentrations. In this study, it was envisaged to degrade pyridine using an isolated bacterial consortium/strain at the initial pyridine concentration up to 50 mg l−1. To initiate the acclimatization procedure 5 mg l−1 of pyridine as carbon source along with the media described earlier was used for growth.
After 48 h significant growth was observed; the media turned milky. Then, 10 mg l−1 of pyridine from stock solution was added. Thereafter, 10 mg l−1 of pyridine was added periodically after 24 h along with media. After continuous addition of pyridine for about 2 weeks, more than 99% degradation was observed. Before starting the kinetics studies, a last enrichment was done by growing this acclimatized culture on 25 mg l−1 concentration of pyridine.
Morphological and bio-chemical characteristics of isolated consortium/strain
The isolate showed fluorescent pigmented colonies and was found out to be gram negative. The detailed rapid biochemical test (Otto and Pickett 1976) were performed and it was found that the isolate showed positive tests for Citrate, Lactate, Malonate, Fructose, D-Glucose, Acetamide, Cellobiose, Butyrate, Propionate, Glutamine, Galactose, Glycerol, Betaine, D-Ribose, and D-Mannose. The isolate showed negative test for the Nicotinate, Tartrate, Nicotinamide, m-Inositol, Sorbitol, Phenylalanine, and Sucrose.
Effect of initial concentration of pyridine on the degradation efficiency of isolated consortium/strain
Various researchers reported that batch tests need to be design with appropriate biomass and substrate concentrations for analyzing the abilities of the bacterial strains for the biodegradation (Bielefeldt and Stensel 1999). Hence batch flask experiments were conducted to examine the effect of various initial concentration of pyridine on pyridine degradation efficiency by isolated consortia/strain. The measurements of substrate concentration and bacterial growth were followed till the substrate concentration became steady. The initial concentration of pyridine was varied in the range between 1 and 50 mg l−1. The control experiments was carried out using autoclaved biomass at all initial concentrations to determine the loss in the pyridine concentration due to biosorption and also the control experiment without the biomass was performed to determine, if any other type of abiotic degradation is occurring or not.
The degradation of pyridine was observed more than 99% at initial pyridine concentrations between 1 and 20 mg l−1 and about 52% at initial pyridine concentration of 50 mg l−1, after 96 h of incubation. However, no significant removal in pyridine was observed in the control flask containing autoclaved biomass. The removal due to biosorption was about 1–3% at different initial pyridine concentrations. About 1–3% pyridine removal was observed due to abiotic activities in the other control flask without biomass. Hence from both control experiment data it can be concluded that about 1–3% pyridine was lost due to volatilization and biosorption was negligible as the loss in pyridine concentration in the control flask containing autoclaved biomass is same as volatilization.
During the experiments the pH in each flask dropped from 7 to about 6.2. The decrease in pH during the experiment may be due to acid formation because of the biotransformation of pyridine to acids. It is assumed that pyridine is degraded after removal of N atom from the cleavage ring. The N atom is removed by the enzymatic reaction and remaining ring subsequently converts to acid and thereafter to gases like CO2, etc. (Rhee et al. 1997). Subsequently the dissolved oxygen concentration was also monitored after the experiment and the DO concentration was dropped from about 3.2 to 0.69 mg/L. This drop in the DO concentration may be due to the consumption of the DO by the microbes in the batch reactor and limitation of air transfer through the cotton plugs used to cover the flask in the shaker.
Evaluation of growth kinetics of isolated consortium/strain
Endogenous data or decay coefficient
Effect of bio-augmentation of isolated consortium/strain on biodegradation of pyridine
The isolated bacterial consortium/strain was found to be capable of degrading pyridine under aerobic conditions. The result reveals that the degradation at low initial pyridine concentrations (1–20 mg l−1) is higher than that at the high concentrations (30–50 mg l−1). This may be due to toxicity and inhibitory effect of pyridine at higher concentrations. Based on μ versus S curve, maximum specific growth rate was found out to be 0.0212 h−1. The yield coefficient and the decay coefficient were found to be 0.155 g of biomass/g of pyridine and 0.0055 h−1, respectively. Furthermore, the bio-augmentation of the isolated bacterial consortium/strain showed a significant improvement in the pyridine degradation efficiency of the activated sludge. The 25% bio-augmentation of isolated consortium/strain in activated sludge results in almost complete degradation of pyridine (20 mg l−1) within 96 h of incubation.
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