Feasibility studies of in situ bioremediation of nylon- 6 oligomer waste contaminated soil

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1 Current Research in Microbiology and Biotechnology Vol. 2, No. 3 (2014): Research Article Open Access ISSN: Feasibility studies of in situ bioremediation of nylon 6 oligomer waste contaminated soil Nandita N Baxi* Department of Microbiology and Biotechnology Centre, Faculty of Science, M S University of Baroda, Vadodara, , India * Corresponding author: Nandita N Baxi; nanditabaxi@yahoo.com Received: 27 April 2014 Accepted: 14 May 2014 Online: 23 May 2014 ABSTRACT Solid oligomeric waste of a nylon6 production plant dumped in an open heap on soil near the factory site was solubilisable in boiling water. The waste was found to consist of several components using high performance liquid chromatography and thin layer chromatography. The components consistently present were the linear monomer, 6aminocaproic acid (6ACA), its dimer and trimer, the cyclic monomer, caprolactam and its dimer, trimer, tetramer, pentamer. The monomers were identified by comparison of retention time / retention factor with that of authentic standards but the identity of oligomers was more clearly resolved using TLC as compared to HPLC. Progressive acid hydrolysis of the oligomeric waste revealed that all the cyclic and linear oligomers and cyclic monomer were eventually converted to 6ACA, though cyclic dimer was most acid resistant. When the oligomeric waste was incubated in fertile soil for 30days 6ACA and the linear oligomers were degraded. However caprolactam was degraded only marginally and the cyclic oligomers were not degraded. Alcaligenes faecalis was a soil bacterial isolate which grew in medium containing only caprolactam or only 6ACA as sole source of carbon and nitrogen, as present in native or acid hydrolysed solid waste respectively. Alcaligenes faecalis also grew in synthetic medium containing only the components of oligomeric waste as nutrients. When the soil contaminated with oligomeric waste was augmented with Alcaligenes faecalis, the bioremediation of the contaminated soil was found to be successful because caprolactam, 6ACA and linear oligomers were markedly degraded in 15days. The cells of Alcaligenes faecalis isolated from such bioremediated soil also showed the ability to solubilise insoluble phosphate thus indicating that such cells also had typical plant growth promoting metabolic potential. The other bacteria isolated from bioremediated soil showed normal colony morphology and detectable protease, amylase activity as is found in case of usual soil isolates with normal metabolic physiology. Keywords: Alcaligenes faecalis, 6amino caproic acid, bioremediation, oligomeric waste of nylon6 plant INTRODUCTION Unreacted monomers and incompletely polymerised oligomers obtained during the manufacture of nylon6 form solid waste and such waste is usually dumped in soil near the production plant till further disposal. The monomers are caprolactam and its linear form 6 aminocaproic acid (6ACA) and its linear and cyclic oligomers. Caprolactam, a xenobiotic compound, is a pollutant and is toxic to a variety of living forms and is mutagenic at high concentration. [1]. Thus caprolactamcontaining wastes / wastewaters are toxic. ACA is a synthetic aminoacid and may have toxic effects as a protease inhibitor. Apart from toxicity, the wastes of nylon6 industries thus contain considerable quantity of organic matter. If released into the environment and surface waters without treatment, such wastes / effluents would prove to be organic pollutants exert a high biological oxygen demand (BOD) for their stabilization to the extent possible. The use of such industrial wastes as nutrient feedstock has been practiced in the fermentation industry [2]. On one hand the load of pollution of the environment and the cost of treatment of wastes are also decreased. However there are only a few reports of the use of toxic industrial wastes as nutrients in fermentation media [3] or in microbial processes. In the present study as compared to biodegradation by native soil microflora, bioremediation (biostimulation and bioaugmentation techniques) using caprolactamdegrading bacteria Alcaligenes faecalis lead to degradation of caprolactam and 6ACA and its linear oligomers, when waste was incubated in either sterile or fertile soil augmented with caprolactamdegrading bacteria thus indicating a 378

2 Nandita N Baxi / Curr Res Microbiol Biotechnol. 2014, 2(3): potential for in situ bioremediation process for soil contaminated with such solid waste. Such bacteria also showed plant growth promoting potential (phosphate solubilisation activity). MATERIALS AND METHODS Solubilisation of solid waste The solid waste was solubilised in hot water or the basal synthetic medium at 80 C or above. After this the waste remained in solution even at ambient temperature as used for incubation. Such waste was inoculated into soil or media and incubated under conditions indicated in the text. Detection of components of solubilised solid waste Thin layer chromatography (TLC) for detection of caprolactam and cyclic oligomers: Caprolactam and its cyclic oligomers were resolved on TLC plates coated with silica gel G (E. Merck, India) as the adsorbent using a solvent system composed of n butanol: acetic acid: water (12:3:5) and detected by spraying Dragendorff s reagent followed by 10 % sulphuric acid. Dragendorff s reagent was prepared as follows: (a) 1.0 g bismuth subnitrate was dissolved in a small amount of concentrated hydrochloric acid precipitated with aqueous ammonia. The precipitate was filtered and dissolved again in a small amount of concentrated hydrochloric acid and then 3.0 g of potassium iodide was added. The whole mixture was diluted in 100 ml distilled water. (b) 25 g potassium iodide was dissolved in 100 ml distilled water. (c) 70 % aqueous acetic acid. The reagents a, b and c were mixed in the volume ratio 5: 5: 40. For analysis of 6ACA and its linear oligomers: 6ACA and its linear oligomers were resolved by TLC/ or paper chromatography and detected using ninhydrin reagent. Acid hydrolysis of solubilised solid waste Acid hydrolysis of the solid waste was carried out using 2M and 4M sulphuric acid at either 95 C or 130 C for varying time periods ranging from 0.5 h to 15 h, neutralized with calcium carbonate and the precipitate obtained was removed by centrifugation at 10,000 x g (Heraeus microcentrifuge) for 20 min. The supernatant (hydrolysate) was analyzed for its constituents. Estimation of relative proportion of constituents High Pressure Liquid Chromatography (HPLC) was used for the quantitative estimation of major components of native solid waste as described by Kulkarni and Kanekar [4] using Varian Chromatograph equipped with an UV detector (205nm) and a RP C18 column, and 20% acetonitrile as the eluent. Identification of the peaks of caprolactam and 6ACA was done by comparison of their retention times (Rt) with those of peaks obtained using authentic caprolactam and 6ACA. Caprolactam content was also estimated spectrophotometrically [5]. 6ACA and its linear oligomers were quantitatively estimated using the modified ninhydrin method. 2.0 ml aliquots of standard (0.05 to 0.2 mm. 6ACA) or samples (containing 6ACA and its linear oligomers) were mixed with 2.0 ml of buffered ninhydrinreagent [2% (w/v) ninhydrin and 3% (w/v) hydrindantin in 3:1 mixture of methyl cellosolve and 4 N sodium acetate buffer having ph 5.5], covered with marble and heated for exactly 15 min in boiling water bath after which the reaction mixture was diluted with 3.0 ml of 50 % ethanol, cooled to room temperature and the absorbance was read at 570 nm. Bacteria and cultivation 6ACA and caprolactamdegrading bacterial isolate used in this study was Alcaligenes faecalis [6]. Media The synthetic basal medium used in this study contained (g/l) KH 2PO 4 0.2, K 2HPO 4 0.6, NaCl 0.3, MgSO 4.7H 2O 0.2, CaCl 2.H 2O 0.1, FeCl and ph was adjusted to 7.2 ±0.2. Caprolactam, or 6ACA or the solid waste of nylon6 plant was supplied as sole sources of carbon and nitrogen in the basal synthetic medium and such medium is referred to as solid waste medium and media were then autoclaved at 15 psi for 15 min Bioremediation of the soil containing components of the solid waste Biomass of caprolactamdegrading bacteria Alcaligenes faecalis was obtained by growth in the broth culture using solubilised solid waste medium. After growth the cells were separated from the medium by centrifugation of the medium at 10,000 x g for 20 minutes. The biomass of cells thus obtained was used for inoculation of fertile or sterilized soil (10 8 cfu Alcaligenes faecalis /g soil). The fertile soil was ascertained to have indeed a high viable count (10 11 cfu/g). 10g fertile soil taken in sterile 250ml flasks was inoculated with 70mg solubilised solid waste (native or acid hydrolysed, at equivalent concentration). Soil not inoculated with A. faecalis was kept as control. Intermittent addition of sterile water was done to maintain the system sufficiently moist. At the end of 15 and s of incubation, 10.0 ml of sterile water was added to the entire system contained in sterile static 250 ml flasks, the contents were vigorously vortexed and the aqueous extract was used to determine the presence and concentration of residual caprolactam, 6 ACA and its linear and cyclic oligomers. Application potential of enriched bacteria The soil incubated for 30days in presence of solid waste was used to isolate specific bacteria using solid waste medium (50 ml broth broth in 250 ml shake flasks). Also Alcaligenes faecalis was reisolated from the soil using colony morphology and known methicillin resistance as the identification marker. The bacteria were checked for possible application oriented physiological properties. To demonstrate production of protease or amylase respectively synthetic basal medium containing agar was supplemented with skimmed milk/ starch (1%) respectively. Presence of clear zones near region of bacterial growth, against the opaque medium indicated enzyme activity. Phosphate 379

3 Nandita N Baxi / Curr Res Microbiol Biotechnol. 2014, 2(3): solubilisation was demonstrated using Pikovskaya s medium which is opaque due to presence of insoluble tricalcium phosphate and presence of clear zone around region of bacterial growth indicated solubilisation of phosphate. Quantitative inorganic phosphate estimation was done using Fiske and Subbarow method. RESULTS AND DISCUSSION The solid waste of nylon6 plant was an uncharacterized solid mass. However some constituents were consistently present (Figure 1a). The major component of the solid waste medium was caprolactam (3.4 g /l) and it was resolved at around minutes. 6ACA was found to be present in solid waste of nylon6 plant. As compared to caprolactam, 6 ACA was found to be resolved (Rt 6.7 min) as a very minor peak in the untreated sample and there was increase in amount after acid hydrolysis. Due to its extremely low molar absorbance coefficient [4] 6ACA was poorly resolved. The peak at Rt minutes was due to some organic component contributed by the plastic tubes used for storage and processing of the samples (boiling of the samples to redissolve the components of the samples which precipitated during storage at 4 C) prior to HPLC analysis. This peak was present even in basal synthetic medium not containing any organic constituents. Terpthalate ester used as elasticizer in microfuge tubes has been reported to contribute a peak during HPLC analysis [7]. Several other peaks at Rt , , minutes found to be present were cyclic dimer, trimer, tetramer and after acid hydrolysis only cyclic dimer was present. However, owing to the nonavailability of authentic standards of the various linear and cyclic oligomers of 6ACA, these components were identified using information available in literature, [8, 4]. A B Figure 1. HPLC profile of components present in solid waste of nylon6 production plant. HPLC (Varian Chromatograph) was done using a RP C18 column using 20% acetonitrile as the eluent, and the detection of resolved components was done using UV detector at 205 nm. A and B represent samples before and after acid hydrolysis respectively Figure 2. Chromatographic analysis of products obtained during the acid hydrolysis of solid oligomeric waste Partial hydrolysis of the solid waste was carried out using 2 M sulphuric acid at 130 C for 6.75 h. Samples were withdrawn at different time intervals and the supernatants obtained after neutralization with calcium carbonate and centrifugation at 10,

4 Nandita N Baxi / Curr Res Microbiol Biotechnol. 2014, 2(3): x g for 20 min were used for chromatographic analysis. Sample aliquotes were either spotted on Whatman filter paper (number 3) or on TLC silica gel plate. A: Chromatogram showing 6ACA and its linear oligomers. The chromatogram was developed using the solvent system composed of npropanol: ethyl acetate: ammonia: water (6:1:1:3) and the resolved components were detected using Ninhydrin reagent. 1: ACA, Rf : linear dimer, Rf : linear trimer, Rf B: Chromatogram showing caprolactam and its cyclic oligomers. The chromatogram was developed using the solvent system composed of nbutanol: acetic acid: water (12:3:5) and the resolved components were detected using Dragendorff's reagent. 1: caprolactam Rf, : cyclic dimer Rf, : cyclic trimer, Rf : cyclic tetramer, Rf : cyclic pentamer, Rf 0.56 For further analysis, the solid oligomeric waste was hydrolyzed, using 2 M sulphuric acid at 95 C for 15 h or 130 C for 6.75 h. After hydrolysis for 6.75 h, the hydrolyzed sample was analysed (Figure 1b). The only major component in the hydrolysate was identified as 6ACA. The other component present in residual amounts was the cyclic dimer and it was in a minor quantity as compared to ACA. However in HPLC profile it appears to be that cyclic dimer is more in amount than ACA because ACA is a poorly resolved molecule in HPLC as compared to TLC (Figure 2). The TLC clearly shows that ACA is in high proportion as compared to cyclic dimer in hydrolysed sample. These results clearly indicate that the usually accepted general view that HPLC analysis is more accurate than TLC is not true in case of mixture of linear, cyclic, polar, non polar compounds and general TLC technique results are more reliable. Caprolactam and all other linear and cyclic oligomers were completely hydrolysed by acid and the end product of hydrolysis of the cyclic as well as linear oligomers was 6ACA. However, an 18fold increase in the amount of 6ACAafter acid hydrolysis as compared to initial amount in the unhydrolyzed sample indicated that probably all the oligomers were converted into 6ACA.The absence of other peaks that were initially present indicated that caprolactam and all other components were completely hydrolysed. A decrease in the amount of cyclic dimer (3.6 fold) as compared to the unhydrolyzed sample, indicated that the cyclic dimer was less succeptible to acid hydrolysis as compared to the other oligomers. Thus, 6ACA was formed as the end product of hydrolysis of caprolactam and the oligomers of nylon6. Herman [9] also reported the amide groups of caprolactam cyclic dimer were more resistant to hydrolysis as compared to those present in other cyclic lactam oligomers. This was due to the fact that in the cyclic dimer, the two CONHgroups of caprolactam assumed a position opposite to each other, favouring the formation of two intramolecular hydrogen bonds, making the compound less reactive. In situ bioremediation of solid waste of nylon6 plant in soil using Alcaligenes faecalis The solid oligomeric waste of nylon6 plant emits harmful gases during incineration and thus is dumped on land for disposal. Dumping of solid waste on land often results in deterioration of the land causing it to become barren [10]. In the present study since the components of solid oligomeric waste of nylon6 plant such as caprolactam, 6ACA and linear oligomers could be utilized by A. faecalis as sole nutrient source in basal synthetic medium, further its feasibility as a soil bioremediation agent was tested. The bioremediation techniques of landfarming (traditional/contained) and biostimulation using inorganic nutrients and bioaugmentation using specific cultures are well known in literature. However though wastewater bioremediation reports are available [6, 11] no in situ bioremediation report is available for solid waste of nylon6 plant till date. This study envisages that at the site of the nylon6 manufacturing plant itself where the solid waste is inevitably produced; it can be mixed with soil priorly enriched with a bioremediating culture such as A. faecalis. The external addition of inorganic nutrients can be made if required. Since A. faecalis was isolated from soil and had no complex growth requirements, it was better suited to thrive and function in the soil environment. Bioremediation of soil artificially contaminated (mixed) with solubilised solid waste was carried out. The viable count of bacteria of fertile mixed with solid waste was cfu/g of soil in control experiment and such soil was inoculated in experimental case with A. faecalis (10 7 cfu/g soil). Control and experimental soil incubated in sterile flasks were analysed at fixed time after incubation: 0h, 15days, 30days (Table 1). Though earlier workers [12] have isolated caprolactam degrading bacteria from dump yard, they have not investigated its potential for solid oligomeric waste degradation. In the present work, in order to achieve increased degradation of the solid waste of the nylon6 production plant, the solid oligomeric waste prior to biological treatment was partially hydrolyzed, using 2 M sulphuric acid at 95 C for 15 h for 6.75 h and such waste was also mixed with soil and incubated using control and inoculated experimental set up. In case of soil (nonsterile) mixed with solid waste, and inoculated with A. faecalis, majority of the caprolactam of solid waste was degraded when analyzed after 15 days. The supplementation of soil with inorganic salts only marginally enhanced caprolactam degradation, indicating that the nutrients available in the soil were sufficient for the biodegradation of caprolactam. In case of soil (nonsterile) not inoculated with A. faecalis, only marginal (10 to 15%) decrease in caprolactam content of the solid waste was observed, even after s and even the supplementation of inorganic salts did not enhance the degradation. Low level of degradation of caprolactam observed during incubation of solid waste 381

5 Nandita N Baxi / Curr Res Microbiol Biotechnol. 2014, 2(3): in garden soil, even in the absence of added bacteria could be due the presence of microorganisms which either present in low numbers or had poor ability to degrade caprolactam, although during screening for potent caprolactamdegrading microorganisms (using caprolactammedium), soil from a variety of different locations, except from caprolactam or nylon6 manufacturing units, did not yield caprolactamdegrading microorganisms. Table 1. Bioremediation of solid waste of nylon6 plant using garden soil a Soil +solid waste with and without supplementation of inorganic salts Presence of linear monomer: 6ACA b 15 day Native soil Soil inoculated with Alcaligenes faecalis Sterile Nonsterile Sterile Nonsterile Presence of linear oligomers b 15 day (+) Presence of cyclic oligomers b + Presence of cyclic monomer caprolactam b + Decrease in caprolactam (%) after s c Decrease in Ninhydrin positive material (%) after 0 0 aa bioremediation system (ph 7.0) consisting of 10 g of either fertile garden soil (10 11 cfu/g) or sterile garden soil artificially contaminated (mixed) with the solubilized solid waste (70 mg) and water with or without supplementation of inorganic salts and with or without inoculation of solid wasteadapted A. faecalis G (10 8 cfu/g soil) was incubated in sterile 50 ml flasks, under static conditions, at 30 ( 2) o C for s. Intermittent addition of sterile water was done to maintain the system sufficiently moist. At the end of 0, 15 and s of incubation, the entire content of the system was mixed with 10 ml of sterile water and vigorously vortexed. The aqueous extract was centrifuged at 10,000 x g for 20 min and the supernatant was used for various analysis. banalyzed by chromatography and detected using ninhydrin and Dragendorff s reagent. Key: original, + decreased, (+ ) highly decreased, absent c The relative decrease in caprolactam estimated spectrophotometrically The degradation of caprolactam of solid waste in nonsterile soil inoculated with A. faecalis was marked (90%) but not higher than that in sterile soil inoculated with A. faecalis. This once again indicated that the inherent microbial flora of the garden soil was not capable of bringing about any significant degradation of caprolactam. Unlike caprolactam, the linearized form of caprolactam i.e. 6ACA, and also its linear oligomers were found to be degraded in nonsterile soil not inoculated with A. faecalis. This indicated the presence of inherent microbial flora in the soil capable of utilization of 6 ACA and its linear oligomers. Even when the ph of the system (soil + solid waste) was 8.9 ie not adjusted to 7.1, soil microflora was able to degrade 6ACA and its linear oligomers. Linear oligomers of 6ACA have a structure similar to that of linear peptides found in biological systems and thus could probably be acted upon by the proteases/ peptidases/ amidohydrolases produced by native microorganisms present in soil. The hydrolysis of 6aminocaproyl6aminocaproic acid i.e. linear dimer of 6ACA by trypsin (EC ) has been reported [13]. However, as expected, the specific activity of trypsin for the hydrolysis of linear dimer was found to be five times lower than the specific activity of crude extract of Corynebacterium aurantiacum B2, a caprolactamdegrading strain which also utilized some oligomers. In literature, only one microorganism, Corynebacterium aurantiacum [13] has been reported to be capable of degrading caprolactam as well as most of the linear and cyclic oligomers of 6ACA (except cyclic dimer) but the supplementation of yeast extract was an absolute requirement for the growth of this organism on caprolactam or the oligomers. As in contrast, the isolate used in this study had no requirement of yeast extract/ growth factors for degradation of ACA, caprolactam and linear oligomers. However detailed experiments to enhance degradation of cyclic oligomers by A. faecalis are required to be carried out and it is likely that on prolonged incubation or in presence of identified factors available from soil, the degradation of cyclic oligomers might also occur. Properties of bacteria isolated from bioremediated soil after degradation of solid waste of nylon6 plant The inoculated culture A. faecalis was reisolated from bioremediated soil after s. The authenticity was established by colony morphology and known resistance to methicillin. Such cells were inoculated on Pikovskaya s agar medium containing insoluble inorganic phosphate and after growth of cells a clear zone obtained near the bacterial growth indicated solubilisation of phosphate by A. faecalis. This property is a plant growth promoting parameter known for few 382

6 Nandita N Baxi / Curr Res Microbiol Biotechnol. 2014, 2(3): soils/ rhizospheric isolates. Thus growth of A. faecalis in soil will have beneficial outcome as soil has insoluble phosphate. In broth, the solubilised phosphate obtained was 200 micromolar. The other bacteria isolated from the bioremediated soil showed amylase and protease activity as shown by usual soil isolates if their physiology is normal. This indicated that bioremediated soil probably did not contain further toxic compounds. Table 2. Specific physiology of bacteria isolated from bioremediated soil. Activity detected Protease a Amylase b Insoluble Phosphate solubilisation c Isolate1 + + Isolate 2 Alcaligenes faecalis + + (200 micromole/ 100ml) Production of protease a or amylase b respectively synthetic basal medium containing agar was supplemented with skimmed milk/ starch (1%) respectively. CONCLUSION The degradation of the toxic waste of nylon6 plant by bioaugmentation of Alcaligenes faecalis in medium (in flasks or reactors) or in soil was attained upto 90 percent for caprolactam, 6ACA and linear oligomers. For degradation of cyclic oligomers more incubation time may be required. Further the use of the biomass of A. faecalis generated by growth on such waste for insoluble phosphate solubilisation ability was also demonstrated as a typical plant growth promoting parameter. Acknowledgements This study was partially funded by the Department of Biotechnology, India. REFERENCES 1. Shama G, Wase DAJ (1981) The biodegradation of caprolactam and some related compounds. A review. Int Biodeterior Bull 17: Stanbury PF, Whitaker A, Hall SJ (1997) Effluent treatment. In: Principles of Fermentation Technology, 2nd edn. Aditya Books P Ltd., N Delhi, India, pp Einsel A (1983) Biomass from higher nalkanes. In: Dellweg H ed Biotechnology, vol 3. Verlag Chemie, Weinheim, pp Kulkarni RS, Kanekar PP (1997) Simultaneous detection of caprolactam and 6amino caproic acid by HPLC. Process Control and Quality 9: Bergmann F (1952) Colorimetric determination of amides as hydroxamic acids. Anal Chem 24: Baxi NN, Shah AK (2002) caprolactam degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon6 production plant. Biotech Lett 24: Maki H, Masuda N, Fujiwara Y et al. (1994) Degradation of alkyl ethoxylates by Pseudomonas sp. Appl and Environ Microbiol 60: Bonifaci l, Frezzoti D, Cavalca G et al. (1991) Analysis of caprolactam and its yclic oilgomers by high performance liquid chromatography. J Chromato 585: Hermans PH (1956) Intramolecular hydrogen bonds in certain cyclic polyamides. Nature 177: Shornborn W (1986) Historical developments. In : Rehm HJ and Reed G eds. Biotechnology vol8 VCH Weinheim pp Kulkarni RS, Kanekar PP (1998) Bioremediation of caprolactam from nylon6 wastewater by use of Pseudomonas aeruginosa MCM B407. Curr Microbiol 37: Sanuth HA, Yadav A, Fagade OE et al, (2013) caprolactam utilization by Proteus and Bordetella species isolated from solid waste dump sites in Lagos state, Nigeria. Ind J Microbiol 53: Fukumura T (1966) Bacterial breakdown of caprolactam and its cyclic oligomers. Plant Cell Physiol 7: ; AIZEON Publishers; All Rights Reserved This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ***** 383