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Abstract Biodegradation of THF is a process that involves a microbial system that would remove originally from the effluent. Occupational Safety & Health Administration (OSHA), U S Department of Labor and The FDArecommended limit of THF exposure as a residual solvent in pharmaceuticals is 7.2 mg/day, with a concentration limit of 720 ppm by 16/03/2004. Currently, the pharmaceuticals industries are used the THF fills that role in many others where strong coordination is desirable, and the precise properties of ethereal solvents such as these (alone and in mixtures and at various temperatures) allows for fine-tuning modern chemical reactions. In this present work on biodegradation or removal of Tetrahydrofuran using isolated strain from municipal sewage effluent is performed using a packed bed column. The continuous degradation of THF using a Biofilm supported by Sugar cane baggase is performed. After startup the column was run continuously for two days. Synthetic effluent was prepared with THF concentration of about 1% was used for the entire study. The treated effluent was analyzed by GC – FID for THF degradation. A single cycle of thirty two days provided degradation rate of about 84%. This provides further scope for experimentation with varying flow rates and higher Retention Time. Key words: Degradation, THF, Packed bed reactor, municipal sewage effluent, Biofilm.

Corresponding author: KVCET, G.S.T.Road, Karpaga Vinayaga Nagar, Chinna Kolambakkam, Palayanoor Post, Madurantakam TK., Kanchipuram-603308. Tel. : +91 4427565486 T/F Email: 1. Introduction Cyclic ethers are used to a large extend in many industrial process as solvents or as chemical bulk products for different synthesis. Due to their high water solubility and vapor pressure these compounds occur in the ground water and in the atmosphere as pollutants1. Tetrahydrofuran (THF) is common cyclic aliphatic ether used as a bulk solvent in chemical and pharmaceutical industries. The THF discharged into the environment is expected to be mainly distributed in the water phase because of its high hydrophobicity. In some rivers THF was estimated to be derived from the domestic effluents including sewage treatment plants. The Environmental Protection Agency (EPA) has listed THF as a carcinogen in its fourth annual report on carcinogens2. EPA has also issued a drinking water health advisory with an estimated lifetime cancer risk of 1 in 10000 for drinking water concentration of 300µg/l. Very recently the solvent stabilizer THF has emerged as an important ground water contaminant through out the world3. The solvent character Of THF and the inhibitory effect on cytochrome p450 dependent enzymes might cause human health problems. Because of their high vapor pressure and water solubility significant amount will be released into the environment and causes pollution4. When it is released into the soil along with the effluent, it is leached into the soil and causes ground water pollution. The use of this contaminated ground water causes various health problems like skin irritation, gastrointestinal problems and respiratory problems 5, 6. Prolonged exposure to this environment can even lead to lung and brain cancer and hence it is a potential carcinogen.

1.1 Histroy of THF Tetrahydrofuran (THF) is a colorless, water-miscible organic liquid with low-viscosity at "room" (standard) temperature and pressure (and across a further range of temperatures). It is a heterocyclic compound with a chemical formula C4H8O (shown in fig.1) and is the fully hydrogenated analog of the aromatic organic compound furan. It is one of the most polar of the organic functional class of ethers, and has a relatively low freeze point, and so is a commonly used modern organic chemical laboratory solvent across a range of temperatures. THF has a smell similar to its chemical cousin, diethyl ether, but is a much less potent anesthetic than diethyl ether, and is quite dangerous if inhaled or ingested.

Fig.1 Structure of THF 1.2 Microbial Degradation The microbial degradation of the analogous aromatic compound furan is well established. For a long time this compound is classified as not readily biodegradable and several attempts to isolate pure or mixed culture capable to grow on THF was failed. Degradation studies using activated sludge from different sources have concluded that THF is not readily biodegradable7. But later studies showed that the THF is degradable and the degradation appeared to be depending on the inoculum source, temperature and inoculation time. The first organism growing on THF as sole carbon source was identified by sole carbon source, Rhodococcus strain 219, was isolated and identified as Rhodococcus ruber8. However, THF concentrations higher than 10 mM led to an increased lag phase and doubling time and a significantly decreased yield of strain 219. A pure culture of an actinomycete designated as strain 1190 (ATCC 55486) belonging to the family Pseudonocardiaceae was isolated from a 1, 4-dioxane contaminated sludge using a variety of cyclic and linear ethers as sole carbon and energy source9. A modification of the conventional activated sludge process where the clarifier is replaced by a membrane system for separating the biomass solids from the treated effluent stream was proposed later10, 11. Retention of all microorganisms results in high biomass concentrations in the bioreactor, which allow the system to treat high-strength wastewater resulting in a system with a relatively small footprint. This technology has been successfully applied to treat a variety of high-strength wastes including high molecular weight compounds, organic wastes containing Surfactants, fermentation wastewater, phenol, contaminated gas streams and industrial waste12. Retention of soluble high molecular weight compounds keeps slowly degradable compounds in the bioreactor while non-degradable compounds are discharged with the sludge 13. The system is capable of handling fluctuations in waste influent concentrations due to the high biomass concentration in the reactor14. High biomass concentrations can lead to decreased oxygen transfer rates and a decline in permeate flow, both of which can adversely affect system performance15. The small size, coupled with the ability to treat high-strength wastes, make this method an attractive option for pretreating waste prior to conventional treatment. 1.3 Applications Tetrahydrofuran finds many applications as a solvent, reaction medium or as a starting material for many syntheses in the pharmaceutical and chemical industries. It is used as reaction medium primarily by pharmaceutical industries, for example in Grignard syntheses and lithium aluminum hydride reductions. In fact it is the only medium in which many Grignard compounds can be obtained. By virtue of its good solvent power for alkali metals, THF is a useful reaction medium for the production of organometallic compounds. Anionic polymerization can be carried out in THF as well.

It is widely used as an extractant in pharmaceutical industries. Some drugs, for example alkaloids can be obtained in the pure form from their natural or synthetic precursors by extraction with THF. Also impurities like fat, wax or substances with a high molecular mass can be removed from compounds that are insoluble In THF. It is also used as a starting material for a number of syntheses in pharmaceutical industries. Tetrahydrofuran is the starting material for a number of syntheses. For instance, ethers with a high molecular mass and chains of various lengths can be obtained by polymerization. The oxygen in the pentagonal ring can be replaced by nitrogen or sulfur to yield other five-membered heterocyclic such as tetrahydrothiophene or pyrrolidine. The ring can be cleaved with halocarbons, e.g., 1,4-dihalobutane, or by oxidation (succinaldehyde and butyrolactone). The solvent character finds many applications in the chemical Industries. PVC or polyurethene spread coating system mainly contains THF alone or blending with other solvents. The abrasive resistance of the video tapes can be improved by applying coating derived from PVC in which the solvent phase is a blend of THF and toluene. The mechanical strength, impermeability to water vapor, heatsealability and printability of cellophane can be improved by applying coatings formulated from poly(vinylidene chloride) (PVDC), in which the solvent phase is a blend of THF and toluene. By virtue of its excellent solvent power,THF allows the preparation of highly concentrated solutions of various polymers. These solutions can be diluted to the appropriate final concentration by adding thinners. Although THF evaporates rapidly, films formed from its solutions have very minimal tendency toward blushing during drying. The solvent character of THF enables us to use it in hydroboration reactions to synthesize primary alcohols. Its lower melting point makes it useful for lower temperature reactions. Tetrahydrofuran is suitable for the production of PVC adhesives for rigid PVC. It is used as a swelling agent for bonding plasticized PVC film and for fabricating PVC pipe systems. In both cases, the evaporation rate can be regulated by blending the THF with another solvent such as cyclohexanone16. The objective of our present study is to improve the efficiency of biodegradation of tetrahydrofuran using Pseudonocardia hydrocarbonoxydans (MTCC * 3435). To optimize the microbial growth of organism in batch biodegradation of THF in shake and flask method and to design a packed bed reactor column with biofilm. 2. MATERIALS AND METHODS 2.1 Source of Microorganisms The aerated municipal sewage effluent (microbial sample) collected from Chennai Metropolitan Water Supply and Sewage Board’s Domestic Effluent Treatment Plant situated at Koyambedu. 2.2 Media and Culture conditions for THF degrading strain The strains are isolated from aerated municipal sewage effluent (microbial sample). Streptomyces Medium - THF for cultivation of THF degrading strains SM - THF medium contains The culture medium has the following composition per liter: 4.0g Glucose, 10.0g Malt extract, 4.0 g yeast extract, 2.0g CaCO3,along with 0.25 g THF, pH was adjusted to 7.2 with KOH. The medium was sterilized at 121°C under 15 psi for 15 min. 2.3 Biofilter Different packing materials such as wood chips, Sugar cane baggases and compost were used as a support for Actinomycetes biofilm in THF removal studies. The packing materials were sterilized before packing and the liquid innoculum was added in the column to develop a biofilm. The innoculum was allowed to stand for 8 days each in the case of woodchips packing, Sugar cane baggases and compost packing. The biofilm growth was observed in the column. Separate columns made of acrylic polymer were used for woodchips, Sugar cane baggases and compost packings. The column dimensions for woodchips

are 100 cm in height and 10 cm internal diameter, fitted with three sampling ports all along the length of the column, with total holdup of 6.28 l. 2.4 Biofilter Startup and Operation Inorder to evaluate the biofilter removal efficiency, continuous experiments were carried out at different inlet THF concentrations by changing the influent flow stream as shown below.

Fig No 3.1 Biofilter setup

2.5 Analytical procedure Influent and effluent THF concentrations were determined using Gas chromatography (NUCON) equipped with Flame ionization detector and WCOT (fused silica) coated with DB 1; 1μm thickness; 30 meters in length; inner diameter 0.25 mm. The temperature of injector, oven and detector were maintained at 200°C Column heater: Initially heat for 5 minutes at 70°C; then heat up at 20°C/min. to 180°C, 250°C respectively, Dry nitrogen was used as a carrier gas. Carrier gas: Helium Evaluation: Percentage weight (noctane as internal standard) mixture was used for ignition. Periodically, the samples were collected using 1µ l air – tight syringe (Hamilton, Bonaduz, Swiss) and 0.6µ l was injected for the analysis. THF concentrations were quantified by comparision with standards. 3. RESULTS AND DISCUSSION 3.1 Isolation of Strains In the study of isolation of strains, 1µl of aerated municipal sewage effluent collected from Chennai Metropolitan Water Supply and Sewage Board’s Domestic Effluent Treatment Plant situated at Koyambedu, which is added into each one of the five 100 ml conical flasks of liquid SM medium. This is allowed to ferment for 3 days at 28°C. The solid SM medium is prepared by using agar. SM solid agar plate is then coated with THF solutions. Fermented liquid SM medium is sprayed on THF coated agar plate. This agar plate is incubated at 28°C for three days. Colonies of THF degrading actinomycetes are obtained by plating the enrichment cultures on THF coated agar plates.

3.2 Screening of Strains The THF degrading strain was isolated from the aerated municipal sewage effluent. The screening test is conducted with THF containing .The degrading strain (Fig No. 3.1) was isolated, which has the capability to degrade the THF containing sample.

Fig No. 3.1 THF degrading Strain in slant

Fig No. 3.2 THF degrading Strain in petriplate The feed and two days old sample results are shown in Graph No. 1 & 2. GC – FID detects THF compounds at each one minute. For example in Graph No. 1 it indicates the THF at Retentions time 1.447 th minute. The peaks and total area (319.8734mV.s) indicates the amount of THF present in feed and also the Graph No.2 it indicates the THF at Retentions time 1.333rd minute. The peaks and total area (15.6738mV.s) indicates the reduction of THF in the degraded sample.

Graph No.1 THF Contain Feed sample

Graph No.2 THF sample after two days of degradation 3.3 Optimization Studies for THF degrading strain 3.3.1 Effect on Temperature To estimate the optimum temperature for maximum growth of mixed culture, media at five different temperature values was prepared. Four different test tubes were taken and marked from as 22 oC, 28oC, 34oC, 40oC and 46oC. 7ml media was poured into each test tube. Following this media was inoculated with mixed microbial sample. The tube marked 4oC was kept in the cold freezer of refrigerator. The tube marked 4oC was kept in the compartment of the refrigerator. The tube marked 28 oC was kept in open room. The tube marked 35oC was kept in an incubator. The samples were then incubated for 24 hrs. After 24hrs the spectrophotometric analysis of the media was performed for 5 days at 578 nm. The results were then tabulated. Table No 1 Effect of Temperature on cellmass Production for the THF degrading strain (Initial glucose concentration: 1% (w/v); Fermentation period: 96h) Temperature 22 28 34 40 46 Cellmass (g/l) 1.06 3.14 1.4 1.02 0.68

Tem perature vs Cellm ass 3.5 3 Cellmass in g/l 2.5 2 Series1 1.5 1 0.5 0 0 10 20 30 40 50 Tem perature C

Graph.No.3 Effect of Temperature on Cell growth production for THF degrading strain 3.3.2 Effect of Initial pH The optimum pH for the THF degrading strain is studied by conducting the experiments at five different pH values 4 – 9, keeping the temperature at 28°C and initial glucose concentration at 1% (W/V). The results are shown in Table. 1 and Graph No.3 As the initial pH increases the cellmass concentration also increases and it reaches maximum for the initial pH is found to be 7. In neutral pH the production of

cellmass is high because the maximum cellmass is obtained at that initial pH. Hence an initial pH of 7 is used for further studies using this strain. Table No 2 Effect of Initial pH on cellmass Production for the THF degrading strain (Temperature: 28°C; Initial glucose concentration: 1% (w/v); Fermentation period: 96h) pH 4 5 6 7 8
pH vs Cellmas s

Cellmass (g/l) 1.06 1.14 1.4 3.32 0.68

3.5 3 Cellmass in g/l 2.5 2 1.5 1 0.5 0 0 2 4 pH 6 8 10

Graph.No.4 Effect on Initial pH on Cell growth production for THF degrading strain 3.3.3 Effect of Biofilter on Production of Cellmass The effect of Biofilter on production of cellmass was carried out in THF degrading strain in batch degradation. The biofilter such as wood chips, sugar cane baggases and Compost material were sterilized and then taken in conical flasks with SM – THF medium and the flasks were incubated for 8 days. The growth of organism was noted in all the flasks. Among them the flask with sugar cane baggases showed the highest cellmass production (Table. No 3). Table No 3 Effect of Biofilter on cellmass Production for the THF degrading strain (Temperature: 28°C; Initial glucose concentration: 1% (w/v); Fermentation period: 8 days pH: 7) Biofilter Wood Chips Sugar cane Baggases Compost Material Cellmass (g/l) 1.12 3.48 1.45

Biofilter vs Cellm ass 4 3.5 3 Cellmass in g/l 2.5 2 1.5 1 0.5 0 Wood Chips Sugar cane Baggases Biofilter Compost Material

Graph No 5 Effect of Biofilter on cellmass Production for the THF degrading strain 3.3.4 Continuous Biodegradation The biodegradation was carried out in bioreactor setup in optimized conditions for THF degrading strain. The organisms were cultured inside the column in the presence of sugar cane baggasses. . Effluent stream with 1% THF concentration was prepared synthetic and used as the inlet stream. The inlet flow rate was set at 32 ml/min. Then the column was allowed to run continuously for two days for a single cycle. The treated effluent obtained was then analyzed for THF by GC – FID Analysis. Performance of the biofilter is presented as removal efficiency, which may not be an important parameter characterizing the biofilter performance because it can be increased either by lengthening or by broadening the biofilter column. In order to study the mass transfer effects across the column packing, time course of THF removal was assayed by collecting the samples from different port I, port II and port III in order to check the equilibrium for maximum removal efficiency. The maximum removal efficiency of 83.14% was obtained for the sugar cane baggases packing at port III (Graph No.7 & 8).

Graph No.7 THF Contain Feed sample for Continuous Biodegradation reactor

Graph No.8 THF degraded sample using THF degrading strain from Continuous Biodegradation reactor Conclusion A fermentation time of 96 hr is required to obtain maximum degradation in case of THF degrading utilizing glucose. The optimum Temperature for strain is found to be 28°C. For the optimum temperature of THF degrading strain utilizing glucose at a fermentation time of 696hr and the optimum pH was found to be 7 at 28°C. The optimum temperature and pH of THF degrading strain was incubated for 28 - 40 days in conical flask with different biofilters such as wood chips, sugar cane baggases and compost materials. Among this group the maximum degradation was obtained from sugar cane baggases. The cell mass production of the THF degrading strain was higher. The optimized environmental conditions of THF degrading strain was incubated in continuous biodegradation reactor with sugar cane baggasess and the efficiency of degradation was noted around 69%. This provides further scope for experimentation with varying flow rates and higher Retention Time.

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