ELECTRONIC SUPPLIMENTARY INFORMATION. The calorimetric experiments were carried out in an isothermal real time reaction calorimeter

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1 ESI-1 Materials and methods ELECTRNIC SUPPLIMENTARY INFRMATIN Biocalorimeter The calorimetric experiments were carried out in an isothermal real time reaction calorimeter (Bio RC1e- RTcal, Mettler-Toledo AG, Switzerland) made up of a two liter jacketed reactor. The calorimetric system ESI Figure-S1 (Mettler-Toledo) was equipped with ph, turbidity and inbuilt D probes. The inbuilt D probe was used to monitor the variations in dissolved oxygen (D) and the heat generated from the biological reaction was monitored and controlled by icontrol RC1e 4.0 software. The details of the calorimetric principle and measurement have been described elsewhere [21]. xygen uptake rate (UR) Dissolved oxygen (D) sensor (Mettler-Toledo model M 700) equipped in the biocalorimeter was initially calibrated in the growth medium by purging with pure oxygen and nitrogen prior to inoculation. A minimum D value of 2 ppm was always maintained to ensure aerobic conditions throughout the reaction. xygen uptake rate (UR) was calculated using the dynamic method [28]. Biomass Growth of S. lentus was monitored simultaneous by spectrophotometric methods and gravimetric analysis. Samples withdrawn at regular time intervals from the calorimeter were analyzed for growth of S. lentus by recording the absorbance in a UV-Spectrophotometer (Shimadzu, Kyoto, Japan UV- 210 PC) at 600 nm. In the gravimetric method, samples were centrifuged at 10,000

2 rpm for 5 min and cells separated from the supernatant. The harvested cells were freed from soluble salts, nutrients and waste products by washing them thrice with sterile water, and their dry weights recorded after 24 h of drying at 80 o C. Fourier Transform Infrared Spectroscopy (FTIR) Analysis FT-IR analysis of the degraded samples was carried out using ABB MB3000 Spectrometer. The culture medium containing the degradation products was centrifuged and the 5 µl of supernatant was sandwiched between two plates of high purity Potassium Bromide (KBr) salt. This sample was placed in the Infrared Spectra beams and the spectrum recorded. High Performance Liquid Chromatography (HPLC) Analysis HPLC analysis was performed (Shimadzu Model CT -10 AVP) to monitor the progress of degradation. The bacterial culture medium along with the degradation products was centrifuged and filtered through 0.2µm filters and the supernatant extracted thrice with equal volume of ethyl acetate. The extract was dried over Na 2 S 4, concentrated in a rotary evaporator and an equal amount of HPLC grade methanol was added to the sample. About 25µl of this filtrate was subjected to HPLC analysis using Gemini C-6 phenyl mobile phase column with a solvent system consisting of methanol and water Gas Chromatography Mass spectrometry (GCMS) Analysis In this study, GC-MS used to characterize the degradation products of the dye acid blue 113. Samples withdrawn from the shaker flask and biocalorimetric experiments were centrifuged. The supernatant was extracted thrice with equal volumes of ethyl acetate. The extract was dried over Na 2 S 4 and concentrated in a rotary evaporator. It was then subjected to GC-MS analysis in a Perkin Elmer Autosystem XL GC with Turbomass MS spectrometer after dissolving it in 1 ml of

3 ethyl acetate. Identification of metabolites was done by matching the fragmentation pattern with the NIST chemistry web book [31]. Principle of DNS method DNS method was widely used for estimating the soluble reducing sugars either directly or indirectly during biotransformations. In the present study, lactate anion of the choline lactate ionic liquid was tracked using a slightly modified DNS method. DNS method usually involved the oxidation of keto/aldehyde functional group (such as glucose/fructose) containing compounds while simultaneously it was reduced to 3-amino-5 nitrosalicylic acid under alkaline conditions. In the modified DNS method, the carboxylic group of lactate is oxidized while DNS is reduced under alkaline conditions. As the choline lactate IL was moderately basic in nature, the reduction of DNS was possible and observed by the formation of 3-amino-5 nitrosalicylic acid which resulted in the development of red color and UV absorption at 540 nm. It was further observed that the intensity of the color was dependent on the concentration of choline lactate. Reduction 3, 5-dinitrosalicylic acid 3-amino, 5-nitrosalicylic acid A standard plot was generated for various concentrations of ionic liquid. To estimate the ionic liquid concentration during the biocalorimetric experiments, samples were drawn from the calorimeter (BioRC1e) at periodic time intervals and centrifuged to separate the cells. The supernatant was used for the modified DNS analysis. Chemical oxygen demand

4 Chemical xygen Demand (CD) analysis was performed by the closed reflux method and the measurement made calorimetrically, as per APHA guidelines [32]. Dye decolourization measurement The residual colour in the control sample and treated samples were analyzed by measuring the absorbance at 546 nm wavelength (absorbance maxima of Acid blue 113) using a UV visible spectrophotometer (Shimadzu, Kyoto, Japan UV- 210 PC). The absorbance values were correlated to the calibration graph. The percentage of dye degradation was then calculated as follows: ci c f % Degradation= (1) c i where c i is initial concentration and c f is final concentration. Azoreductase assay Azoreductase activity was assayed by the Zimmermann method [27] using Acid blue 113 as the dye substrate. The assay mixture contained 0.8 ml of 100 mm phosphate buffer (ph 7.0) with 0.2mM of the dye Acid blue 113, 0.1 ml of 1mM NADH and 0.1 ml of enzyme in 1ml of reaction mixture. The reaction mixture without NADH was preincubated for 4 min and the reaction started by the addition of NADH. Dye decolorization was followed by monitoring the decrease in colour intensity at 565 nm at room temperature. ne unit (U) of Azoreductase activity was defined as the amount of enzyme required to reduce 1 µm of dye min 1 ml 1 under the assay conditions. Bioenergetics

5 Though power-time profiling depicts the metabolic shifts occurring during a growth process, quantitative information on relative consumption of substrates Y Q/S (kj heat evolved per gram of glucose consumed), energy changes associated with biomass growth Y Q/X (kj heat evolved per gram cell dry weight formed), oxycalorific coefficient Y Q/ (heat generated in kj/mol of oxygen consumed), and the heat changes associated with reduction in CD, Y Q/CD ( kj heat evolved per mg of CD reduction) are evaluated by calculating cumulative heat production values by integrating the power-time curve [21].

6 ESI Table S1 Heat yields due to biomass growth, substrate consumption and oxygen uptake by S. lentus S.No Glucose concentration (g/l) Biomass growth Y Q/ x (kj/g) Substrate consumption Y Q/S (kj/g) xy calorific coefficient Y Q/ (kj/mol)

7 ESI Figure-S1: Schematic representation of BioRC

8 C CHLINE LACTATE H N + H 0th HUR - CHLINE METABLISM [] Pyruvate Dehydrogenation 3ATP C - CYTSL LACTATE METABLISM Betaine aldehyde N + C2 3ATP 2H Acetyl-CoA CoA-SH Betaine H De-methylation H N + H 3 ATP xaloacetate H Dehydrogenation Condensation - Citrate - - H 4th HUR Product identified at 4th hour using FTIR and 13C NMR. Dimethyl glycine De-methylation N - Malate - MITCHNDRIA 3ATP Isomerization H 2 H H - - Sarcosine H N H - Fumarate Hydration - CITRIC ACID CYCLE - Iso citrate De-methylation Glycine H NH 2 2ATP Dehydrogenation 1 ATP xidative decarboxylation Product identified at 12th hour. H Succinate - CoA-SH Succinyl CoA C 2 3 ATP - C C - alpha ketoglutarate 12th HUR Serine H NH 2 + H 2 alpha-amino acid TRANSAMINATIN alpha-keto acid urea cycle alpha-ketoglutarate L-Glutamate XIDATIVE DEAMINATIN NH 3 C 2 40th HUR Urea ESI Figure-S2 Classical subdivision of choline lactate Metabolism by Staphylococcuss lentus

9 3.5 Phase Phase 3 40hr Power (w) hr phase 1 12hr :17:00 16:37:00 24:57:00 33:17:00 41:37:00 Time (h) Heat production due to ionic liquid consumption Heat production due to glucose consumption 50:28:00 ESI Figure-S3: Comparitive power curves for the growth of Staphylococcus lentus in presence of glucose (5 g/l) and choline lactate (2 g/l) media.

10 % of degrdation % 1 % 2 % 3 % 4 % 5 % Time (h) ESI Figure- S4: Degradation efficiency at different glucose concentrations

11 Glucose conumption (g/l) Power ( W) phase l phase lll UR (mg/l.h) Growth at 600nm phase ll Time (h) ESI Figure- S5:Thermodynamic responses of S. lentus cultivated in glucose 5 g/l limited mineral salt medium: (Heat production rate(-), oxygen uptake rate ( ), bacterial growth ( ), glucose consumption( )

12 ESI Figure-S6 Vero cells treated with Choline Lactate sample with 50% cytotoxicity level seen under microscope

13 Control 24hr 72hr ESI Figure- S7: HPLC Chromatogram showing the progress of dye degradation (a) pure dye (b) 24 th hr (c) 72 nd hr