Chapter 3

TWO LIQUID PHASE PARTITIONING BIOREACTOR SYSTEM FOR BIODEGRADATION OF PYRENE BY MYCOBACTERIUM FREDERIKSBERGENSE
Biswanath Mahanty, Kannan Pakshirajan & Veeranki Venkata Dasu
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, India

ABSTRACT Biodegradation of pyrene by Mycobacterium frederiksbergense was evaluated in a two liquid phase partitioning bioreactor (TPPB) containing 20% v/v silicone oil in an aqueous phase medium. At an initial concentration of 400 mg l1 in silicone oil, the mycobacterium could completely degrade pyrene within 6 d with a rate of 140 mg l1 d1. When compared to the results of slurry phase and surfactant aided systems, the TPPB system was found superior not only in terms of pyrene removal efficiency and rate but also in terms of cost effectiveness of the process.

INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) produced via natural and anthropogenic sources during the incomplete combustion of solid and liquid fuels in industrial activities are harmful to environment and human health.1 However, due to low aqueous solubility/bioavailability, bioremediation of PAHs is largely limited. Two-phase partitioning bioreactors (TPPBs) have gained considerable attention, in which PAHs dissolved in an organic phase partitions into the aqueous phase based on equilibrium considerations and real-time demand of the microorganisms.2,3 In this present study, an efficient TPPB system was investigated for pyrene biodegradation by M. frederiksbergense. Its performance in terms of pyrene degradation rate and efficiency, and cost benefit over slurry phase and surfactant aided systems was also compared.

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MATERIALS AND METHODS CHEMICALS AND REAGENTS Analytical grade pyrene and silicone oil used in the study were purchased from Sigma-aldrich Chemicals, India, and Loba Chemie, India, respectively. Non-ionic surfactant (Tween 80) and media components were purchased from Himedia laboratory, India. MICROORGANISM AND CULTURE MEDIA M. frederiksbergense NRRL B-24126 was obtained from USDA, USA. While nutrient Broth was used for its routine growth and maintenance, Bushnell Hass media was used in the pyrene biodegradation experiments. SCREENING OF NON AQUEOUS PHASE LIQUID FOR USE IN TPPB SYSTEM For selecting a suitable non aqueous phase liquid (NAPL) that is biocompatible with the microorganism in TPPB system, isopropyl myristate, n-hexadecane and silicone oil were considered. Five 125 ml flasks each containing 40 ml of BH medium, 5 ml of M. frederiksbergense, 5 ml of any of the solvents and 2 ml of soybean oil were taken; an additional flask containing all the ingredients except the solvents served as the positive control in this experiment. All the flasks were incubated at 28C for 3 d, and at the end the biomass in the flasks were determined by the dry weight method. PYRENE BIODEGRADATION EXPERIMENTS TPPB system All pyrene biodegradation experiments in the TPPB system were carried out in a 3 l autoclavable glass fermenter (Applikon). Both the aqueous (1 L BH media) and organic phases (pyrene in silicone oil, 250 ml) were loaded together in the vessel, autoclaved in toto and allowed to cool down to room temperature. Thereafter 50 ml of overnight grown M. frederiksbergense culture was added as the inoculum, and the bioreactor operated at temperature 28C, pH 7.0, aeration rate 1.5 vvm and agitation rate 600 rpm. During its operation, duplicate samples of 1.5 ml each were withdrawn from the fermenter for 15 d and remaining pyrene in the silicone oil samples was extracted with methanol to monitor its degradation in the system. Slurry phase system Pyrene biodegradation experiments in this system were carried out using the same fermenter mentioned in experiments under TPPB system, but by taking

Two liquid phase bioreactor for biodegradation of pyrene 15

pyrene stock solution (25 g l1 in acetone) in the empty vessel to give final pyrene content of 50 mg l1, which is based on the final aqueous phase volume. Other experimental conditions in this system remained the same as that of the TPPB system. Samples (1 ml each) from this slurry phase reactor were extracted twice with equal volumes of ethyl acetate before final analysis of pyrene. Surfactant aided system Based on our earlier study,4 using Tween 80 as the surfactant , experiments in this system were carried out using the earlier mentioned 3 l fermenter with 1 l BH media containing pyrene (25 mg l1) that was solubilised by the surfactant. Again, the experimental conditions were maintained the same in this system as those of the two other systems. ANALYTICAL METHODS Pyrene analysis Pyrene concentrations in all the samples, with or without pretreatment as mentioned before, were quantified using synchronous fluorescence spectroscopy using a FluoroMax-3 (HORIBA Jobin Yvon, USA) fluorescence spectrometer with the detection condition of = 36.0 nm, excitation peak maximum = 335 nm. COST BENEFIT ANALYSIS OF THE THREE DIFFERENT SYSTEMS Cost analysis of the three biodegradation systems investigated in this work was performed considering the costs involved in the preparation of inoculum and media, and power required for running fermenter and shake incubator in the experiments. These are presented in Tables 1 and 2, respectively.

Table 1 Preparation costs of inoculums and media used in the biodegradation experiments. For inoculums (50 ml) Chemical/power input Nutrient broth Power consumption in shaker (12 h) BH media Trace element solution Total cost (in USD)
*All costs are represented in USD.

For media (1000 ml) Amount 3.27 g 2 ml Cost* 0.30 0.01 0.31

Amount 0.65 g 41.4 unit

Cost* 0.04 3.90 3.94

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Table 2 Power requirements for running fermenter and shaker incubator in the experiments. System Fermenter Unit/Components (Power in kW) Bioconsole (1.6), Controller (0.07), Agitator (0.04), Chiller unit (0.9), Air compressor (1.5) Complete system (3.45) Total power (kW) 4.11 Cost/h 0.38

Shaker incubator

3.45

0.07

Cost in USD; 1 USD = 48.00 Indian rupees and Electricity charges are based on LT-commercial tariff of AERC.

RESULTS AND DISCUSSION Selection of NAPL for use in the TPPB system Solvent selection experiments were performed to choose a candidate solvent that is biocompatible but not bioavailable to M. frederiksbergense. Figure 1 presents the results in terms of relative metabolic activity (RMA) of the mycobacterium in presence of the different solvents, which clearly reveals that all except IPM (RMA = 1.7) were found non-bioavailable. It is also apparent from Fig. 1 that relative metabolic activity in presence of silicone oil was comparable to that due to hexadecane. Hence, based on these results and economic considerations, silicone oil was selected as the suitable solvent in the study. Silicone oil has traditionally been considered highly stable, hydrophobic and chemically resistant to oxidative attack, and has been employed successfully in several other TPPB studies3,5,6, as well. PYRENE BIODEGRADATION EXPERIMENTS TPPB system Pyrene biodegradation experiments employing the TPPB system was carried out at its initial concentration of 400 mg l1. Pyrene degradation profiles, shown in figure 2, reveal that pyrene was completely degraded in 6 d with a rate of 139 mg l1 d1 without any lag phase in this TPPB system. MacLeod and Daugulis reported complete degradation of pyrene (1 g) within 4 d at a rate of 138 mg l1 d1 by Mycobacterium PYR-1in a TPPB system containing bis(ethylhexyl) sebacate.7 In another study, Vandermeer and Daugulis observed only 26% degradation of pyrene after 120 h of TPPB operation by a defined Sphingomonas consortium using a very high initial pyrene concentration of 1 g l1 in dodecane.8 These results clearly indicate the superiority of the NAPL silicone oil M. frederiksbergense pair in biodegradation of pyrene over other TPPB systems reported in the literature.

Two liquid phase bioreactor for biodegradation of pyrene

17

2.0 1.8 1.6

Relative metabolic activity

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

il il l) tro no no on ea ea yb yb l (c o oi So +S +S an il + ne be IPM eo ca oy n S de ico xa Sil He b oy ea no il

IPM

ic Sil

on

il ne eo ca de xa He

Figure 1 Results of solvent selection experiments.

500 50
-1

Slurry phase system Tween 80 aided system TPPB system

400

Pyrene concentration, mg l

40 300 30

200 20

10

100

0 0 50 100 150 200

Time, h

Figure 2 Pyrene biodegradation profiles obtained using slurry phase, surfactant aided and TPPB systems.

Slurry phase and Tween 80 aided systems Results of pyrene biodegradation experiments in slurry phase system containing an initial concentration of 50 mg l1 revealed complete degradation of the compound within 200 h (Fig. 2) with an overall degradation rate of 6 mg l1 d1.

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Table 3 Overall cost comparison of pyrene degradation employing the three different systems. Cost involved for (in USD) Cost per mg of pyrene removed (in USD) 1.10 0.73 0.62

System Slurry phase Surfactant aided TPPB

Inoculums Basic media Pyrene preparation preparation delivery 3.94 3.94 3.94 0.46 0.31 0.46 0.04 11.40

Power 77.51 13.95 46.51

Total cost 81.91 18.24 62.30

Amount of pyrene removed (mg) 75 25 100

On the other hand, pyrene degradation profile with Tween 80 indicated near complete degradation within 50 h without any lag period with a degradation rate of 17.7 mg l1 d1. It should be noted here that in both these two systems degradation of pyrene by the mycobacterium at initial concentrations above 50 mg l1 were not investigated owing to either of the two limitations associated with such high concentration levels in these systems: no change in effective aqueous concentration of pyrene (slurry phase system) or huge requirement of surfactant (surfactant aided system). The chosen pyrene concentration of 25 mg l1 in the present Tween 80 aided system was also based on our earlier study.4 COST BENEFIT ANALYSIS COMPARISON As described earlier, cost benefit analysis was conducted considering the costs for preparing inoculum and media ingredients, and for power required to run fermenter and shaker incubator in these experiments. Table 3 comparatively illustrates the combined cost for the three systems, which clearly indicate that the TPPB system was the most economical among the three, and in which case only $0.62 was required for biodegrading a unit weight of pyrene. REFERENCES
1 Mastral AM and Callen MS, A review on polycyclic aromatic hydrocarbon (PAH) emissions from energy generation. Environ Sci Technol 34:30513057 (2000). 2 Daugulis AJ, Two-phase partitioning bioreactors: a new technology platform for destroying xenobiotics. Trends Biotechnol 19:457462 (2001). 3 Mahanty B, Pakshirajan K and Dasu VV, Biodegradation of pyrene by Mycobacterium frederiksbergense in a two-phase partitioning bioreactor system. Bioresour Technol 99:26942698 (2008).

Two liquid phase bioreactor for biodegradation of pyrene 19

4 Mahanty B, Sarma SJ and Pakshirajan K, Evaluation of different surfactants for use in pyrene biodegradation by Mycobacterium frederiksbergense. Int J Chem Sci 5:15051512 (2007). 5 Vanneck P, Beeckman M, De Saeyer N, DHaene S and Verstraete W, Biodegradation of polycyclic aromatic hydrocarbons in a two-liquid-phase system. In Bioremediation of recalcitrant organics, ed by Hinchee RE, Anderson DB and Hoeppel RE. Battelle Press, Columbus, pp. 5562 (1995). 6 Muoz R, Guieysse B and Mattiasson B, Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors. Appl Microbiol Biotechnol 61:261267 (2003). 7 MacLeod CT and Daugulis AJ, Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent. Appl Microbiol Biotechnol 62:291296 (2003). 8 Vandermeer KD and Daugulis AJ, Enhanced degradation of a mixture of polycyclic aromatic hydrocarbons by a defined microbial consortium in a two-phase partitioning bioreactor. Biodegradation 18:211221 (2007).