- Email: [email protected]
Acidogenesis driven by hydrogen partial pressure towards bioethanol production through fatty acids reduction
Energy 118 (2017) 425e434
Contents lists available at ScienceDirect
Energy journal homepage: www.elsevier.com/locate/energy
Acidogenesis driven by hydrogen partial pressure towards bioethanol production through fatty acids reduction Omprakash Sarkar, Sai Kishore Butti, S. Venkata Mohan* Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 January 2016 Received in revised form 26 October 2016 Accepted 4 December 2016
H2 partial pressure drives the reduction of carboxylic acid (short chain fatty acids) formed as primary metabolites in acidogenic fermentation to form bioalcohols. Microbial catalysis under the influence of H2 partial pressure was evaluated in comparison with a reactor operated at atmospheric pressure under identical conditions. Carboxylic acid reduction gets regulated selectively by the influence of elevated pressures and redox conditions, resulting in the formation of alcohols. The non-equilibrium of the intra and extracellular H2 ions causes the anaerobic bacteria to alter their pathways as a function of interspecies H2 transfer. Ethanol production was quantified, as acetic acid was the major carboxylic acid synthesised during acidogenesis. H2 pressure influenced the electrochemical activity which was reflected in the distinct variation of the electron transfer rates and the catalytic activity of redox mediators (NADþ/ NADH, flavoproteins and iron-sulphur clusters). The bioprocess depicted in this communication depicted a non-genetic regulation of product formation, understanding the acidogenic metabolism and alternate route for alcohol production. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biohydrogen Redox mediators Acetic acid Alcohol Bioethanol
1. Introduction Microorganisms as biocatalyst are capable of producing a wide spectrum of valuable products under structured conditions which have immense significance in the present times of energy crisis and natural resources depletion [1,2]. Anaerobic bacteria (AB) have the metabolic versatility to ferment a wide range of substrates and produce a spectrum of diverse biobased products viz. alcohols, carboxylic acids, hydrogen (H2), methane (CH4) [3e5]. Anaerobic fermentation can broadly be categorised into acidogenesis, solventogenesis and methanogenesis. Initially, organic carbon gets converted to different short chain carboxylic acids viz. acetic acid (2C), propionic acid (3C), butyric acid (4C), succinic acid (4C), valeric (5C), etc. along with biogas (H2, CO2, etc.) by the function of acidogenic bacteria (Eqs. (1)e(3)). Bioalcohol like ethanol, butanol, isopropanol, etc. get synthesised during solventogenesis or lead to methanogenesis (CH4, CO, CO2, etc.) [6,7]. C6H12O6 þ 2NAD þ H2O /2 CH3COOH þ 2NADH2 þ 2H2 þ 2CO2 þ 4 ATP (DGr ¼ 216 kJ/mol) (1)
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Venkata Mohan). http://dx.doi.org/10.1016/j.energy.2016.12.017 0360-5442/© 2016 Elsevier Ltd. All rights reserved.
C6H12O6 þ 2NADH2 / 2CH3CH2COOH þ 2NAD þ 2H2O þ 2ATP (DGr ¼ 364 kJ/mol) (2) C6H12O6 / CH3CH2CH2COOH þ 2H2 þ 2CO2 þ 3ATP (DGr ¼ 266 kJ/mol)
(3)
This bioconversion process is a cascade of complex biochemical reactions, where operational parameters like substrate concentration, retention time, reactor design, temperature, headspace gas composition, pressure, pH, etc. were found to alter the products specificity and concentration [5,8e11]. Every reaction is governed by a threshold energy and reaction potential for enzymatic catalysis (activation energy and free energy), which could be regulated by varying physical parameters like pressure, temperature etc. [3,12,13] Research on acidogenic fermentation has so far primarily focused on H2 production, providing empirical results pertaining to the effects of pH, sparging methods and dilution rates [14e16]. However, as compared to gaseous products (H2 and CH4), liquid biofuels (ethanol, butanol, etc.) offer comparatively beneficiary utilities and advantages such as high energy density, easy storage and transportability. One mole of ethanol has two times higher heating value than 2 mol of H2 and ethanol has a higher energy density of 23.4 GJ/m3 when compared to compressed H2 at 200 bars of 1.95 GJ/m3 [17]. With the pertinent properties like reduction in
426
O. Sarkar et al. / Energy 118 (2017) 425e434
green house gases, high combustibility and as a precursor molecule for various bio-based product syntheses, ethanol finds its applicability in as fuel in the automobile industry, for power generation in thermal plants, as fuel in fuel cells and platform chemical in cogeneration chemical industry, etc. In this study, an attempt was made to selectively regulate acidogenic synthesis through non-genetic methods. The headspace of the reactor is saturated with H2 and maintained under pressure, which leads to the reduction of short chain/volatile fatty acids (VFA; carboxylic acids) to form alcohols with possible chain elongation of the carboxylic acids. The role of reductive energy in the form of NADH/NADþ ratio and dissolved H2 partial pressure were studied to determine their possible role in the reduction of VFA to alcohols [18e22]. Bioelectrochemical analysis was used to correlate the process regulation monitoring H2 as an external driving reductive force by the activity of redox mediators. 2. Materials and methods 2.1. Biocatalyst and waste feedstock Anaerobic sludge was collected from an anaerobic bioreactor treating complex wastewater and used as parent inoculum. Initially, the parent culture was sieved to separate the grit using a nylon filter and the resulting sludge was used as the inoculum. Parent culture (10% v/v) was enriched in the design synthetic wastewater (DSW) (NH4Cl-0.5 g/l, KH2PO4- 0.25 g/l, K2HPO4-0.25 g/ l, MgCl2-0.3 g/l, CoCl2-25 mg/l, ZnCl2-11.5 mg/L, CuCl2-10.5 mg/L, CaCl2-5 mg/L, MnCl2-15 mg/L, NiSO4-16 mg/L, FeCl3e25 mg/L) for five cycles prior to inoculating the bioreactors (COD, 5 g/l; 48 h; pH 6) and each reactor was operated for triplicates. 2.2. Acidogenic bioreactors Two acidogenic bioreactors viz., the high-pressure reactor (HPR) and control reactor (CRT) were designed and fabricated with borosil glass to have a total/working volume of 1.0/0.7 L (Fig. 1). Both reactors were designed to bear high pressure of 4 bars with double layer jackets of borosil glass (inside) and high-density polyethene (HDPE; outside). Inner borosilicate chamber (14 6 cm) was closed to have anaerobic microenvironment intended for acidogenic reaction. The outer layer of HDPE (16 8 cm) was filled with water to monitor leaks; to keep reaction temperature constant and provide safety during operation under high-pressure conditions. The main vessel has three ports for sample collection, gas inlet/outlet and effluent drain out along with safety valve and pressure releasing knob. The H2 gas and DSW feed nozzles were placed directly into the reactors headspace using elliptical top heads. Specially designed nozzles maintain symmetrical sparging of gas into reactors headspace enabling efficient contact with liquid phase contents. Drain port was provided at the bottom of the reactor to flush out the reactor contents whenever required. The bioreactors were equipped with safety valves (to release pressure above 2 bars). CRT was operated under identical conditions, but without any additional pressure in the headspace (at atmospheric pressure). 2.3. Experimental design The pH of DSW was set to 6.0 using 0.1 N HCl and/or 0.1 N NaOH before feeding the reactors. After the inoculation (10%v/v), pH was re-adjusted to 6.0. Vacuum was created in both the reactors to remove dissolved oxygen and to maintain anaerobic conditions using a vacuum pump. HPR was adjusted to a final headspace pressure of 2 bar using H2 passed through a cylinder. The pressure of the headspace was monitored using a manometer every hour.
During the experiment, the pressure drop was noted in the HPR with time and headspace pressure was re-adjusted to 2 bars by passing H2 gas. Control (CTR) reactor was operated under the same conditions at atmospheric pressure. Both the systems were operated at ambient temperature (30 ± 2 C) for fermentation period of 48 h. Stirring was provided using magnetic stirrer (120 rpm) for even distribution of the reactants. Gas and liquid samples were taken once in every 8 h to perform the analysis. 2.4. Analytical methods The process parameters of the bioreactors were assessed by monitoring chemical oxygen demand (COD - closed refluxing titrimetric method), total volatile fatty acids (VFA) and redox conditions [23]. Buffering capacity (b) was estimated based on the acidbase titrations employing auto-titrator (Mettler Toledo DL50). The sample was divided into two parts of 3 mL each prior to the test. The first part was titrated with 0.1 N HCl till the end point of 1.9 pH and the second part was titrated against 0.1 N NaOH till the end point of 12 pH. The volume of acid/base added against the pH change was plotted. The slope of the tangent to the curve at the sample pH (when no acid/base was added) was derived by fitting a quadratic curve over the data. The buffering capacity (b) can be calculated using the equation, where, C is the concentration of acid or base (mol), Vs is the volume of sample (mL), m is the slope of the tangent on the curve (Eq. (4)).
b¼
C Vs m
(4)
The degree of acidification (DOA) represents (Eq. (5)) the extent of acidification achieved due to the production of carboxylic acids in relation to substrate (as COD) degradation [24,25]. Si represent initial substrate concentration measured in COD as mg/L and Sf is net VFA concentration (final-initial) expressed as theoretical equivalents of COD (in mg/L, HAc, 1.066; HPr, 1.512; HBu1.816).
Degree of Acidification ðDOA%Þ ¼
Sf 100 Si
(5)
2.4.1. Chromatography analysis Alcohol analyses were performed using HPLC (Shimadzu LC20A) employing RI detector (RID20A; Shimadzu) and Rezex Monosaccharide (Phenomenex) column by injecting filtered sample of 20 ml. For elution, a flow rate of 0.5 mL/min was used in an isocratic method with water as the mobile phase and the column temperature was maintained constant at 80 C. VFA composition was analysed using HPLC (Shimadzu LC10A) employing UVeVis detector (210 nm) and C18 reverse phase column (250 4.6 mm diameter; 5 mm particle sizes). Filtered sample (20 ml; 0.22 mm porosity) was injected with mobile phase of 40% acetonitrile in 1 mN H2SO4 (pH, 2.5e3.0) and flow rate of 0.5 mL/h. The headspace gas was sampled through a rubber septum using a gas-tight syringe (Hamilton) and analysed in a gas chromatograph (NUCON 5765) using TCD (1/8” 2 m Heysep Q) column and Argon as a carrier gas. The injector and detector were maintained at 60 C each and the oven was operated at 40 C isothermally. Standards for acids (SUPELCO) and alcohols (SIGMA) were used. 2.5. Bioelectrochemical analysis Bio-electrochemical behavior of biocatalyst during the operation was studied using cyclic voltammetry (CV) techniques (BioLogic-VMP3) by applying a potential ramp to the working electrode
O. Sarkar et al. / Energy 118 (2017) 425e434
427
Fig. 1. Reactor design and configuration delineating the complete operation setup: Spares and components are 1. Barometer 2. Safety valve 3. Temperature and pH probe 4. Needle type valve 5. Sample Ports 6. Pressure releasing knob 7. Thick walled high pressure resist glass reactor 8. Thick walled high pressure resist glass reactor 9. Needled inlet port with Nozzle 10. Water Drain out 11. Effluent drain out, 12. Source gas, Pure H2e 99.9%, (b) Ball and stick model depicting the carboxylic acid to alcohol conversion with their respective gibb's free energy. (c) H2 transfer across the cell membrane and the reaction of NAD with diffused intra-cellular hydrogen.
(anode) over a range of þ1.0 to 1.0 V against an Ag/AgCl (S) reference electrode. Electron transfer rate (Kapp) was derived by recording CV at variable scan rates (0.5e30 mV s1). Laviron theory was used to understand the peak potential variation with varying scan rates and thereby determining the electron transfer rates [26].
Epc ¼ Ec
Epa ¼ Ea
RT anFvc ln anF RTKapp
(6)
RT ð1 aÞnFva ln ð1 aÞnF RTKapp
(7)
The dependence of peak potential on the scan rate (mV s1) was evaluated when DEp 200 mV (DEp ¼ EpcEpa) where Epc and Epa represent cathodic and anodic peak potential, a is transfer coefficient, v represents scan rate, kapp is apparent rate constant which is an indicator for the e transfer rate between the biocatalyst and the electrode, R (8.31 J mol1 K1) and F (96,483 C mol1) refers to constant values, T refers to temperature (298 K) and n represents number of electrons respectively (Eqs. (6) and (7)). 3. Results and discussion 3.1. Carboxylic acids production Carboxylic acids production illustrates the progress of acidogenic/methanogenic process. Compositional variation of fatty acids was analysed through the chromatographic analysis The experimental data documented distinct variations in carboxylic acids production, signifying the apparent change in the metabolic
behavior of experimental variations studied (Fig. 2). The pressure of H2 in the head space of HPR showed marked effect on the acidognesis on the production as well as on the consumption of the fatty acids. Relatively higher VFA synthesis was noticed with HPR (2.4 g/l) compared to CTR (1.9 g/l) with more or less 20% increment in productivity. Both the systems showed a consistent increment in the VFA production with cycle time and approached maximum value at 32nd h of the cycle operation (Fig. 2a). Subsequent increment in the cycle time documented a marked drop in the VFA production in both the systems. The carboxylic acids profile showed presence of acetic (HAc) in higher concentration followed by butyric (HBu) acids and propionic (HPr) in both the systems with time bound concentration variation (Fig. 2b, c and d). Microbial metabolites depict the type of fermentation being carried and its end product formation. The production pattern of HAc and HBu in HPR and CTR more or less resembled till 32nd h of operation with varying concentration and then decreased with extension of HRT. Gradual increment in HAc production from 8th to 24th h was observed in both HPR and CTR with varying concentration and then dropped from 32nd h of operation till the end of cycle operation. Comparatively higher HAc accumulation (1.2 g/l) was observed with HPR compared to CTR (0.8 g/l) depicting ~35% improvement due to the regulated headspace environment (Eq. (8)). Fraction of HBu was lower compared to HAc in both the operation (HPR, 0.75 g/l; CTR, 0.6 g/l) with relative improvement in productivity of 25%. Even though relative less HPr production was observed the variation was distinct with 92% improvement in productivity. 4H2 þ 2CO2 / CH3COOH þ 2H2O (23.4 kJ/mol H2)
(8)
428
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 2. Carboxylic acid Synthesis (mg/l) versus time of operation (hours). (a)Total Carboxylic acid production (mg/l) versus time of operation (h) (b)Acetic Acid (c) propionic acid, (d) Butyric Acid, Production and consumption rate of individual carboxylic acids with respect to time operation (e) Acetic acid, (f) Propionic acid, (g) Butyric acid.
O. Sarkar et al. / Energy 118 (2017) 425e434
3.1.1. Production and consumption of fatty acids The acidogenic process is always dynamic and is influenced by the quantity and composition of carboxylic acids present as well as head gas composition and pressure. In order to understand the dynamics of the process, the production/consumption rates of carboxylic acids were calculated based on the following equations (Eqs. (9) and (10)) [27]. Production rate of carboxylic acids (PRCa) ¼ (VFAmaxVFAint)/ TProd
(9)
Consumption rate of carboxylic acids (CRCa) ¼ (VFAdropVFAmax)/ TProd (10) where, VFAmax represents maximum VFA concentration (g/l/h), VFAint is initial VFA concentration (g/l/h), VFAdrop denotes drop/ consumption in VFA concentration (g/l/h), TProd is production time in hours and TDrop represents concentration dropping/consumption time (hours). The positive and negative values explained the rate of production and consumption of carboxylic acids respectively. Production was calculated from the initial hour to the time of next sampling (8 h) while the consumption was calculated from the point of degradation to end of the cycle for each acid individually. Analysis of the data showed some marked variations with the functional role of headspace gas and metabolite composition. Increasing trend in PRCa was observed with HAc from 8th h to 32nd h operations, which dropped later with increasing fermentation time (Fig. 2e). Highest PRCa was observed with HAc in both systems (HPR, þ55 mg/L/h (24 h); CTR, þ45 mg/L/h (8 h). Similar trends where observed in the case of HBu with the maximum PRCa þ 43 mg/L/h than CTR (þ29 mg/L/h) at 8 h. However, in the case of HPr there was production at 40 h (PRCa, þ 28 mg/L/h) with HPR (Fig. 2f). On the contrary, CTR operated showed gradual production from 8th h and reached maximum at 16th h (þ16.6 mg/L/h). The maximum CRCa was observed at 32nd h with HPR (0.38 mg/L/ h) which is lower than CTR (6.9 mg/L/h at 48 h). The CRCa with HBu was observed to relatively more with HPR (10.8 mg/L/h; 48th h) than CTR (0.9 mg/L/h; 48 h) (Fig. 2f). There was no consumption of HPr in either of the system under study. Post the 32nd h the consumption of carboxylic acids correlated with the alcohol production. Higher HPr synthesis was specially observed with CTR against lower fraction in HPR in the later stages of fermentation time. Higher HPr in HBR is observed compared to CTR as the H2 dependent reaction is limited in the normal headspace gas composition when compared to 100% hydrogen headspace in HPR. Degree of acidification (DOA) calculated in terms of individual carboxylic acid concentrations and mainly influenced by the composition of the carboxylic acids profile in the system [27] (Fig. 3a). Production and consumption pattern of carboxylic acid also influence the DOA. Relatively higher DOA (22.3%; 16th h) was observed with CTR than HPR (21.7%; 8th h) operation might be attributed to the relatively higher fraction of HPr produced. Whereas in HPR, the consumption of HAc and HBu (32nd h) and the production of lower HPr at later stage of cycle operation (40th h) might have resulted in lower DOA. 3.2. Hydroxylates formation from carboxylates Reduction of carboxylic acids resulted in solventogenesis leading to production of alcohols (hydroxylates) (Fig. 3b). This bioconversion is a two-step process where organics are first acidified to carboxylates (HAc, HBu and HPr) and biogas production followed by formation of hydroxylates via alcohol dehydrogenases catalysed reactions [28]. pH conditions and increased reductive gas pressure
429
will have a direct influence on the alcohol formation [29]. Induction of reductive environment in the bioreactor with the increasing concentration of carboxylic acids synthesis enforced the biocatalyst to maintain its cellular proton concentration. As a function of pressure, HPR and CTR showed variation in the concentration of alcohol formation. External supplemented H2 pressure in the headspace of HPR mediated the conversion of acids to alcohols. With HAc being the major carboxylic acid synthesised, ethanol concentration was higher (Eq. (11)). CH3COOH þ H2 / CH3CH2OH
(11)
Alcohol production was initiated after 16th h in HPR (0.01 g/l) and the maximum production was observed between 40th to 48th h (0.09 g/l). Alcohol production in CTR is non-influenced fermentation with the maximum production observed at 40th h (0.04 g/l). With specific pretreatment the productivity can be increased as the essential biocatalyst has substrate competition with non-essential biocatalyst. 3.3. Gaseous phase analysis Gas and aqueous phase interface will be the determining factor for the reduction process. Induced H2 pressure in the headspace altered the product formation and lead to bioethanol formation. HPR depicted the consumption of H2 as an electron donor for acetate reduction in conjunction with the pressure reduces the activation energy. H2 production was detected at different time interval in CTR. On the contrary, HPR didn't showed H2 production inspite of acetate formation. The consumption of H2 in fermentation process prevent H2 formation and increase the acetate yield [30,31]. As H2 was pressurised in HPR, a marked consumption of H2 was noticed with time (Fig. 4a). Consumption pattern of H2 was well synchronised with the reduced products formed in the biosystem. Initially, at 8th h, the H2 consumption was about 11% and increased as fermentation time matured and observed 22% (18 h). Higher H2 consumption in HPR was observed at 24th h of 35% with indicated the higher metabolic activity to convert H2 to reduce carboxylic acids to respective alcohols. Consumption of H2 gas decreased at 32nd h to 9% followed by 4% at 48th h. Headspace gas composition was concurrently estimated for enumerating other gases formation. H2 gas transfer across the cell membrane is through diffusion and through proton pumps coupled with ATP synthesis. The gas consumed creates a reducing environment in terms of redox microenvironment and catalyses the carboxylic acid conversion to form alcohols. H2 existing either in the external environmental (Hevx) and/or in the intercellular H2 (Hinc). Microbial ability to adapt to the gradient results in the formation of reducing equivalents intracellularly in the form of NADH (Eq (12)).
H2 þ NADþ / NADH2 DG ¼ 18 kJ/mol
(12)
As anaerobic culture was used as an inoculum, CH4 formation was observed in the reactor due to prevailing methanogenic activity. The concentration of CH4 along with CO2 increased with fermentation time in HPR indicating the fermentation of glucose (Fig. 4b). CH4 production varied in the range of 4%e12% along with CO2 (7%e31%). In CTR, biogas composition of H2, CH4 and CO2 with varied concentration was observed (Fig. 4c). Higher concentration of H2 was observed at 16th h (13%). H2 concentration increased till 16 h in CTR and then dropped till the end of the cycle. CH4 detected from 8th h (2%) followed increasing till the end of the cycle operation. Evolution of CH4 in CTR due to the methanogenesis correlated well with the synthesis of HPr and consumption of HAc to CH4 (Eq. (13)). On the other hand synthesis of HPr is through
430
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 3. (a) Degree of acidification with respect to time, (b) Alcohol (Bioethanol) production (mg/l) versus time of operation (hours).
Fig. 4. Biogas Composition versus time of operation (hours) in (a) Hydrogen consumption in HPR, (b) gas composition in HPR and (c) gas composition in CTR system.
decarboxylation of succinate, which generates CO2 in the conversion of C4 acids to C3 acids in the succinate pathway [24]. CH3COOH þ 2H2O / CH4 þ CO2
(13)
The forward progress of the reaction is based on the net energy balance on both sides of the equation, the reactants and the products (Gibbs free energy below zero). Exothermic reactions are spontaneous at standard conditions (T ¼ 25 C), glucose to value added products conversion, unlike, compared to glucose fermentation (225.5 kJ) [32]. Therefore, the concentrations and partial pressure of H2 and protons is regulated to study their effect on the product formation with the function of Gibbs free reaction energy (Eq. (14)).
DG ¼ DG þ RT ln(C y/x)
(14)
where C is the H2 diffused relative to its partial pressure (considered to be at saturation- 1 mmol/l), y/x ratio is of NAD/ NADH. To study the feasibility of the reactions with the
increasing pressure the ratio is considered to be 1, resembling equilibrium. The carboxylic acid reduction occurs at 20 kJ/mol as the highest energy required (9.1 kJ/mol, 9.4 kJ/mol and 5.4 kJ/mol for ethanol, propanol and butanol respectively). As the experiment progresses the DG increases making the reaction less feasible until it reaches equilibrium. This possibly suggests that the reduction of the alcohols is not altered by the pressure in the headspace as the standard Gibbs free energy of these reactions is below zero. 3.4. Bio-electrochemical analysis-reductive solventogenesis Cyclic Voltammogram (CV) of both the HPR and CTR are analysed at different time intervals to understand the reductive product formation and the different redox mediator that participate in the carboxylic acid formation and their reduction to alcohols (Fig. 5). Type and size of the reactors, the initial substrate and all the analytical conditions were maintained the same thus any changes observed in the CVs indicates the shift of metabolism under the influence of regulated headspace conditions.
O. Sarkar et al. / Energy 118 (2017) 425e434
431
Fig. 5. Cyclic Voltammetric Analysis of control (a,b,c) and HPR (d,e,f) at different time intervals (8th, 24th and 48th hour), the determination of an electron transfer rate with multiscan cyclic voltammograms. (g) HPR and (h) Control.
432
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 6. (a) Utilisation of glucose as the substrate; glucose concentration (mg/l) versus time of operation (hours). (b) Variation in pH versus time of operation (hours), (c) Buffering Capacity (bmol) versus time of operation (hours).
Voltammograms also give overview on the interaction reactions between the electrode and biocatalyst through alterations in the electrochemical behaviours. The catalytic reduction currents were showing visible variation in HPR (0.006 mA) as compared to CTR and HPR (0.003 mA) but altered metabolism with different mediator peaks with function of time (Fig. 5a, b & c). At the same time intervals peaks at different potentials (biological redox reaction) was observed, which relates to NADþ/NADH (0.56 V vs Ag/AgCl) and cytochrome complex (0.3 V vs Ag/AgCl). Carboxylic acids get reduced in the presence of the carboxylic reductases to form aldehydes and later form alcohols where these reactions occur under the influence of redox electron carriers like NAD, FeS, Cytochromes, FAD, etc. [33] Four moles of reduced ferredoxin facilitates formation of 2 mol of acetate to acetaldehyde, and four more electrons derived from H2 via NADH gets used in the reduction of 2 mol of acetaldehyde to ethanol. Pyruvate oxidation and Acetyl CoA reduction to form acetate is the possible initial reaction occurring in both the systems, as the initial fermentative pathway of glucose breakdown will remain the same. As the reactions progress with time, peaks were observed at 0.1 V vs Ag/ AgCl in both the CTR as well as in the HPR. These peaks can be attributed to membrane-bound flavoproteins along with an ironsulphur cluster of [4Fee4S] associated complex catalysing the proton reduction. There is a reversible peak observed at 0.32 to 0.4 V (vs Ag/AgCl) specifically in the HPR, which could resemble the redox carriers that are functioning with alcohol dehydrogenases that catalyse acetate to acetaldehyde and reducing it to ethanol (Fig. 5d, e&f). The reversible peak showing oxidative and reductive states could be the flavoproteins and iron sulphur mediators which gets reduced and oxidised supplying electrons to acetate [34,35]. This illustrates specific metabolic shift in the regulated biosystem due to H2 partial pressure. The reducing conditions formed in the process due to the developed electron cloud has been determined by the evaluating the electron transfer rates which facilitates to understand the bio-
electro kinetics. The electron transfer rate (Eqs (6) and (7)) derived with HPR (0.282 s1) was relatively higher compared to that of CTR (0.161 s1). With the higher electron density in the HPR, the transfer rate also has been observed to be higher. On the other hand, the HPR showed higher capacitance than the CTR, which is evident from the figure portraying the ion holding capacity of the reactor that is directly proportional to energy available to increase the spontaneity of the reactions [36]. 3.5. Carbon utilisation The glucose assimilation phase was observed until 36th h in both the systems. The H2 partial pressure had no significant effect on the glucose uptake. The degree of utilisation was dependent on the redox conditions maintained in the reactor only for the initial period. The short chain carboxylic acids formed have the capabilities to act as primary substrate over glucose. Under atmospheric pressure, the utilisation percentage was higher (70%) compared to regulated H2 pressure conditions (60%) (Fig. 6a). the maximum utilisation occurred between 12th h and 16th h where the acidogenic activity of the bacteria will be higher. The rate of utilisation became low paced once the carboxylic acids have been synthesised suggesting the possible uptake of them by the microbiome as the substrate over glucose. 3.6. Redox conditions Acidogenic fermentation leads to high intracellular acid concentrations, which require increased proton translocation for the maintenance of pH. Fermentation pH is assumed to serve as a trigger causing bacteria to shift from the production of hydrolyzed products to organic acids and gases to respective solvents. The pH of the bioreactors (HPR and CTR) was not controlled during operation. From the initial pH 6 at 0th h to 48th h, there is a steep drop towards acidification (pH, 4.4) in the HPR. The H2 in the
O. Sarkar et al. / Energy 118 (2017) 425e434
headspace also diffuses to liquid phase and shifts redox condition. In CTR, pH drop was comparatively marginal (6e5.1) (Fig. 6b). This disparity can be attributed to the concentration of carboxylic acids formed. Variation in pH was higher in the HPR operation during initial hours. Drop in pH was marginal till 16th h of operation in both the reactors. The continuous drop in pH in HPR was attributed to production higher production of VFA in the system. pH change was marginal in the control operation, which favoured the methanogenesis resulted in CH4 evolution in the later hour of operation. The results indicated that the relationship between higher propionic acid production and lower H2 evolution were well correlated with themethanogenic activity in the control operation. The increase in pH and the lowering of the carboxylic acid production correlate giving strength to the presence of homoacetogens/acetogens/methanogens to utilise the formed carboxylic acids. Carboxylic acids formation has a prime influence on the reactors buffering capacity. The reactors insitu ability to maintain its pH was measured at different time intervals (Fig. 6c). Variation in concentrations of the carboxylic acids changes systems buffering capacity. The buffering capacity maintained the redox conditions starting at 8th h to 32nd h with the values ranging from 0.024 to 0.028 b mol in the reactors which are having H2 partial pressure in the headspace. CTR showed comparatively lower buffering capacity with the maximum at 24th h of 0.022 ± 0.002 b mol. The trend of buffering capacity showed an increment up to 24th h with the possible reason of carboxylic acid formation and later on showed a downward slope as the formed carboxylic acids gets utilised. Operations at the extend times at 40th h and beyond due to lack of buffering capability moves towards lower pH which also favour alcohol production. Buffering capabilities of the reactor are developed by the formation of short chain carboxylic acids which are weak acids when they are reacting with ions present in reactor they form a buffering agent. Few buffering systems like ammonium bicarbonate (pKa 6.35/9.35), carbonate (pKa 6.35/9.25), formic acid (pKa 9.25) and HAc (pKa 4.75/9.25) [37]. 4. Conclusions Non-genetic routes for shifting the metabolic pathways towards solventogenesis are possible by manipulating the headspace of the reactor with a specific gas at high pressure. This method could be employed for selective biobased product productivity and enhancement of their yields. Selective product synthesis in microorganisms is a function of physiological and operation parameter modifications. Glucose utilisation reactions are spontaneous and there could be no effect of increased pressure, whereas, reduction of carboxylic acids is enhanced by the partial pressure of H2 gas and prevailing redox conditions as a result of hydrogen gas diffusion. Gas composition analysis revealed the consumption of H2, while intracellular redox mediators involved in solventogenesis was reflected in the bioelectrochemical analysis. The negative effect of H2 is not seen, as the electrochemical analysis show the activity of NADH: Fes reactions to be functioning. The alcohol reduction gets mediated by Fe-s cluster proteins and NADH electron transfer mediators which are observed with the electrochemical analysis. Waste valorization offers a positive energy balance in the environment with operational freedom and low-cost productivity rekindling waste biorefinery and green fuel production. These methods offer economic feasibility with options of integration with other acidogenic fermenters for H2, microalgal cultivation for biodiesel and biopolymer production with photobacteria enabling a closed sustainable loop for bio-fuel production.
433
Acknowledgements The authors wish to thank the Director CSIR-IICT for the support and encouragement in carrying out this work. This work was supported by Department of biotechnology (DBT-No. BT/PR13642/ BBE/117/80/2015). Nomenclature HPR Hydrogen Pressure Reactor VFA Volatile Fatty Acids CV Cyclic Voltammograms DSW design synthetic wastewater HAc Acetic acid HPr Propionic acid HBu Butyric acid Hevx Extracellular Hydrogen Hinc Intracellular Hydrogen NADH: Fed Nicotinamide Adenine Dehydrogenase: Ferridoxin DOA Degree of Acidification AB Anaerobic bacteria CRT Control reactor HDPE High density polyethylene References [1] Choi YJ, Lee SY. Microbial production of short-chain alkanes. Nature 2013;502: 571e4. [2] Annie Modestra J, Navaneeth B, Venkata Mohan S. Bio-electrocatalytic reduction of CO2: enrichment of homoacetogens and pH optimization towards enhancement of carboxylic acids biosynthesis. J CO2 Util 2015;10:78e87. [3] Shafiei E, Davidsdottir B, Leaver J, Stefansson H, Asgeirsson EI, Keith DR. Analysis of supply-push strategies governing the transition to biofuel vehicles in a market-oriented renewable energy system. Energy 2016;94:409e21. [4] Jin Y, Illukpitiya P. Cost minimization of supplying biomass for ethanol biorefineries. Energy 2016;96:209e14. http://dx.doi.org/10.1016/j.energy.2015. 12.031. [5] Venkata Mohan S, Bhaskar YV, Murali Krishna P, Rao NC, Lalit Babu V, Sarma PN. Biohydrogen production from chemical wastewater as substrate by selectively enriched anaerobic mixed consortia: influence of fermentation pH and substrate composition. Int J Hydrogen Energy 2007;32:2286e95. [6] Kandylis P, Bekatorou A, Pissaridi K, Lappa K, Dima A, Kanellaki M, et al. Acidogenesis of cellulosic hydrolysates for new generation biofuels. Biomass Bioenergy 2016;91:210e6. [7] Venkata Mohan S, Nikhil GN, Chiranjeevi P, Reddy CN, Rohit MV, Kumar AN, Sarkar O. Waste biorefinery models towards sustainable circular bioeconomy: Critical review and future perspectives. Bioresour Technol 2016;215:2e12. [8] Arslan D, Steinbusch KJJ, Diels L, De Wever H, Hamelers HVM, Buisman CJN. Selective carboxylate production by controlling hydrogen, carbon dioxide and substrate concentrations in mixed culture fermentation. Bioresour Technol 2013;136:452e60. [9] Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010;463:559e62. [10] Chen Y, Luo J, Yan Y, Feng L. Enhanced production of short-chain fatty acid by co-fermentation of waste activated sludge and kitchen waste under alkaline conditions and its application to microbial fuel cells. Appl Energy 2013;102: 1197e204. [11] Spirito CM, Richter H, Rabaey K, Stams AJM, Angenent LT. Chain elongation in anaerobic reactor microbiomes to recover resources from waste. Curr Opin Biotechnol 2014;27:115e22. [12] Xiong M, Deng J, Woodruff AP, Zhu M, Zhou J, Park SW, et al. A bio-catalytic approach to aliphatic ketones. Sci Rep 2012;2:311. [13] Marshall CW, Labelle EV, May HD. Production of fuels and chemicals from waste by microbiomes. Curr Opin Biotechnol 2013;24:391e7. [14] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T. Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol 2000;73:59e65. [15] Zheng X-J, Yu H-Q. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. J Environ Manag 2005;74:65e70. [16] Li C, Fang HHP. Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 2007;37:1e39. [17] Steinbusch KJJ. Liquid biofuel production from volatile fatty acids. 2010. [18] Mohanakrishna G, Venkata Mohan S. Multiple process integrations for broad perspective analysis of fermentative H2 production from wastewater treatment: technical and environmental considerations. Appl Energy 2013;107: 244e54.
434
O. Sarkar et al. / Energy 118 (2017) 425e434
[19] Costello DJ, Greenfield PF, Lee PL. Dynamic modeling of a single-stage highrate anaerobic reactordI. Model derivation. Water Res 1991;25:847e58. [20] Ruzicka M. The effect of hydrogen on acidogenic glucose cleavage. Water Res 1996;30:2447e51. [21] Rodríguez J, Kleerebezem R, Lema JM, van Loosdrecht MCM. Modeling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 2006;93:592e606. [22] Mu Y, Yu HQ, Wang G. Evaluation of three methods for enriching H2producing cultures from anaerobic sludge. Enzyme Microb Technol 2007;40:947e53. [23] APHA. Standard methods for the examination of water and wastewater. twentieth ed. Washington DC, USA: American Public Health Association/ American Water Works Association/Water Environment Federation; 1998. [24] Dahiya S, Sarkar O, Swamy YV, Venkata Mohan S. Acidogenic fermentation of food waste for volatile fatty acid production with co-generation of biohydrogen. Bioresour Technol 2015;182:103e13. [25] Amulya K, Venkateswar Reddy M, Venkata Mohan S. Acidogenic spent wash valorization through polyhydroxyalkanoate (PHA) synthesis coupled with fermentative biohydrogen production. Bioresour Technol 2014;158:336e42. [26] Laviron E. Adsorption, autoinhibition and autocatalysis in polarography and in linear potential sweep voltammetry. J Electroanal Chem Interfacial Electrochem 1974;52:355e93. [27] Sarkar O, Kumar AN, Dahiya S, Krishna KV, Yeruva DK, Venkata Mohan S. Regulation of acidogenic metabolism towards enhanced short chain fatty acid biosynthesis from waste: metagenomic profiling. RSC Adv 2016;6:18641e53. [28] Steinbusch KJJ, Hamelers HVM, Buisman CJN. Alcohol production through volatile fatty acids reduction with hydrogen as electron donor by mixed cultures. Water Res 2008;42:4059e66.
ndez EV, Sepúlveda-Escribano A. From biodiesel [29] Serrano-Ruiz JC, Ramos-Ferna and bioethanol to liquid hydrocarbonfuels: new hydrotreating and advanced microbial technologies. Energy Environ Sci 2012;5:5638e52. [30] de Kok S, Meijer J, Van Loosdrecht MCM, Kleerebezem R. Impact of dissolved hydrogen partial pressure on mixed culture fermentations. Appl Microbiol Biotechnol 2013;97:2617e25. [31] Nie Y, Liu H, Du G, Chen J. Acetate yield increased by gas circulation and fedbatch fermentation in a novel syntrophic acetogenesis and homoacetogenesis coupling system. Bioresour Technol 2008;99:2989e95. [32] Amend JP, Shock EL. Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiol Rev 2001;25: 175e243. [33] Krishna KV, Sarkar O, Venkata Mohan S. Bioelectrochemical treatment of paper and pulp wastewater in comparison with anaerobic process: integrating chemical coagulation with simultaneous power production. Bioresour Technol 2014;174:142e51. [34] Srikanth S, Venkata Mohan S, Lalit Babu V, Sarma PN. Int J hydrogen energy 2010;35:10693e700. [35] Gottumukkala LD, Sukumaran RK, Venkata Mohan S, KuttuvanValappil S, Sarkar O, Pandey A. Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium sporogenes BE01: bio-electrochemical analysis of the electron transport mediators involved. Green Chem 2015;17:3047e58. [36] Veer Raghavulu S, Suresh Babu P, Kannaiah Goud R, Venkata Subhash G, Srikanth S, Venkata Mohan S. RSC Adv 2012;2:677e88. [37] Dinamarca MA, Aranda-Olmedo I, Puyet A, Rojo F. Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures. J Bacteriol 2003;185:4772e8.
Contents lists available at ScienceDirect
Energy journal homepage: www.elsevier.com/locate/energy
Acidogenesis driven by hydrogen partial pressure towards bioethanol production through fatty acids reduction Omprakash Sarkar, Sai Kishore Butti, S. Venkata Mohan* Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 January 2016 Received in revised form 26 October 2016 Accepted 4 December 2016
H2 partial pressure drives the reduction of carboxylic acid (short chain fatty acids) formed as primary metabolites in acidogenic fermentation to form bioalcohols. Microbial catalysis under the influence of H2 partial pressure was evaluated in comparison with a reactor operated at atmospheric pressure under identical conditions. Carboxylic acid reduction gets regulated selectively by the influence of elevated pressures and redox conditions, resulting in the formation of alcohols. The non-equilibrium of the intra and extracellular H2 ions causes the anaerobic bacteria to alter their pathways as a function of interspecies H2 transfer. Ethanol production was quantified, as acetic acid was the major carboxylic acid synthesised during acidogenesis. H2 pressure influenced the electrochemical activity which was reflected in the distinct variation of the electron transfer rates and the catalytic activity of redox mediators (NADþ/ NADH, flavoproteins and iron-sulphur clusters). The bioprocess depicted in this communication depicted a non-genetic regulation of product formation, understanding the acidogenic metabolism and alternate route for alcohol production. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biohydrogen Redox mediators Acetic acid Alcohol Bioethanol
1. Introduction Microorganisms as biocatalyst are capable of producing a wide spectrum of valuable products under structured conditions which have immense significance in the present times of energy crisis and natural resources depletion [1,2]. Anaerobic bacteria (AB) have the metabolic versatility to ferment a wide range of substrates and produce a spectrum of diverse biobased products viz. alcohols, carboxylic acids, hydrogen (H2), methane (CH4) [3e5]. Anaerobic fermentation can broadly be categorised into acidogenesis, solventogenesis and methanogenesis. Initially, organic carbon gets converted to different short chain carboxylic acids viz. acetic acid (2C), propionic acid (3C), butyric acid (4C), succinic acid (4C), valeric (5C), etc. along with biogas (H2, CO2, etc.) by the function of acidogenic bacteria (Eqs. (1)e(3)). Bioalcohol like ethanol, butanol, isopropanol, etc. get synthesised during solventogenesis or lead to methanogenesis (CH4, CO, CO2, etc.) [6,7]. C6H12O6 þ 2NAD þ H2O /2 CH3COOH þ 2NADH2 þ 2H2 þ 2CO2 þ 4 ATP (DGr ¼ 216 kJ/mol) (1)
* Corresponding author. E-mail addresses: [email protected], [email protected] (S. Venkata Mohan). http://dx.doi.org/10.1016/j.energy.2016.12.017 0360-5442/© 2016 Elsevier Ltd. All rights reserved.
C6H12O6 þ 2NADH2 / 2CH3CH2COOH þ 2NAD þ 2H2O þ 2ATP (DGr ¼ 364 kJ/mol) (2) C6H12O6 / CH3CH2CH2COOH þ 2H2 þ 2CO2 þ 3ATP (DGr ¼ 266 kJ/mol)
(3)
This bioconversion process is a cascade of complex biochemical reactions, where operational parameters like substrate concentration, retention time, reactor design, temperature, headspace gas composition, pressure, pH, etc. were found to alter the products specificity and concentration [5,8e11]. Every reaction is governed by a threshold energy and reaction potential for enzymatic catalysis (activation energy and free energy), which could be regulated by varying physical parameters like pressure, temperature etc. [3,12,13] Research on acidogenic fermentation has so far primarily focused on H2 production, providing empirical results pertaining to the effects of pH, sparging methods and dilution rates [14e16]. However, as compared to gaseous products (H2 and CH4), liquid biofuels (ethanol, butanol, etc.) offer comparatively beneficiary utilities and advantages such as high energy density, easy storage and transportability. One mole of ethanol has two times higher heating value than 2 mol of H2 and ethanol has a higher energy density of 23.4 GJ/m3 when compared to compressed H2 at 200 bars of 1.95 GJ/m3 [17]. With the pertinent properties like reduction in
426
O. Sarkar et al. / Energy 118 (2017) 425e434
green house gases, high combustibility and as a precursor molecule for various bio-based product syntheses, ethanol finds its applicability in as fuel in the automobile industry, for power generation in thermal plants, as fuel in fuel cells and platform chemical in cogeneration chemical industry, etc. In this study, an attempt was made to selectively regulate acidogenic synthesis through non-genetic methods. The headspace of the reactor is saturated with H2 and maintained under pressure, which leads to the reduction of short chain/volatile fatty acids (VFA; carboxylic acids) to form alcohols with possible chain elongation of the carboxylic acids. The role of reductive energy in the form of NADH/NADþ ratio and dissolved H2 partial pressure were studied to determine their possible role in the reduction of VFA to alcohols [18e22]. Bioelectrochemical analysis was used to correlate the process regulation monitoring H2 as an external driving reductive force by the activity of redox mediators. 2. Materials and methods 2.1. Biocatalyst and waste feedstock Anaerobic sludge was collected from an anaerobic bioreactor treating complex wastewater and used as parent inoculum. Initially, the parent culture was sieved to separate the grit using a nylon filter and the resulting sludge was used as the inoculum. Parent culture (10% v/v) was enriched in the design synthetic wastewater (DSW) (NH4Cl-0.5 g/l, KH2PO4- 0.25 g/l, K2HPO4-0.25 g/ l, MgCl2-0.3 g/l, CoCl2-25 mg/l, ZnCl2-11.5 mg/L, CuCl2-10.5 mg/L, CaCl2-5 mg/L, MnCl2-15 mg/L, NiSO4-16 mg/L, FeCl3e25 mg/L) for five cycles prior to inoculating the bioreactors (COD, 5 g/l; 48 h; pH 6) and each reactor was operated for triplicates. 2.2. Acidogenic bioreactors Two acidogenic bioreactors viz., the high-pressure reactor (HPR) and control reactor (CRT) were designed and fabricated with borosil glass to have a total/working volume of 1.0/0.7 L (Fig. 1). Both reactors were designed to bear high pressure of 4 bars with double layer jackets of borosil glass (inside) and high-density polyethene (HDPE; outside). Inner borosilicate chamber (14 6 cm) was closed to have anaerobic microenvironment intended for acidogenic reaction. The outer layer of HDPE (16 8 cm) was filled with water to monitor leaks; to keep reaction temperature constant and provide safety during operation under high-pressure conditions. The main vessel has three ports for sample collection, gas inlet/outlet and effluent drain out along with safety valve and pressure releasing knob. The H2 gas and DSW feed nozzles were placed directly into the reactors headspace using elliptical top heads. Specially designed nozzles maintain symmetrical sparging of gas into reactors headspace enabling efficient contact with liquid phase contents. Drain port was provided at the bottom of the reactor to flush out the reactor contents whenever required. The bioreactors were equipped with safety valves (to release pressure above 2 bars). CRT was operated under identical conditions, but without any additional pressure in the headspace (at atmospheric pressure). 2.3. Experimental design The pH of DSW was set to 6.0 using 0.1 N HCl and/or 0.1 N NaOH before feeding the reactors. After the inoculation (10%v/v), pH was re-adjusted to 6.0. Vacuum was created in both the reactors to remove dissolved oxygen and to maintain anaerobic conditions using a vacuum pump. HPR was adjusted to a final headspace pressure of 2 bar using H2 passed through a cylinder. The pressure of the headspace was monitored using a manometer every hour.
During the experiment, the pressure drop was noted in the HPR with time and headspace pressure was re-adjusted to 2 bars by passing H2 gas. Control (CTR) reactor was operated under the same conditions at atmospheric pressure. Both the systems were operated at ambient temperature (30 ± 2 C) for fermentation period of 48 h. Stirring was provided using magnetic stirrer (120 rpm) for even distribution of the reactants. Gas and liquid samples were taken once in every 8 h to perform the analysis. 2.4. Analytical methods The process parameters of the bioreactors were assessed by monitoring chemical oxygen demand (COD - closed refluxing titrimetric method), total volatile fatty acids (VFA) and redox conditions [23]. Buffering capacity (b) was estimated based on the acidbase titrations employing auto-titrator (Mettler Toledo DL50). The sample was divided into two parts of 3 mL each prior to the test. The first part was titrated with 0.1 N HCl till the end point of 1.9 pH and the second part was titrated against 0.1 N NaOH till the end point of 12 pH. The volume of acid/base added against the pH change was plotted. The slope of the tangent to the curve at the sample pH (when no acid/base was added) was derived by fitting a quadratic curve over the data. The buffering capacity (b) can be calculated using the equation, where, C is the concentration of acid or base (mol), Vs is the volume of sample (mL), m is the slope of the tangent on the curve (Eq. (4)).
b¼
C Vs m
(4)
The degree of acidification (DOA) represents (Eq. (5)) the extent of acidification achieved due to the production of carboxylic acids in relation to substrate (as COD) degradation [24,25]. Si represent initial substrate concentration measured in COD as mg/L and Sf is net VFA concentration (final-initial) expressed as theoretical equivalents of COD (in mg/L, HAc, 1.066; HPr, 1.512; HBu1.816).
Degree of Acidification ðDOA%Þ ¼
Sf 100 Si
(5)
2.4.1. Chromatography analysis Alcohol analyses were performed using HPLC (Shimadzu LC20A) employing RI detector (RID20A; Shimadzu) and Rezex Monosaccharide (Phenomenex) column by injecting filtered sample of 20 ml. For elution, a flow rate of 0.5 mL/min was used in an isocratic method with water as the mobile phase and the column temperature was maintained constant at 80 C. VFA composition was analysed using HPLC (Shimadzu LC10A) employing UVeVis detector (210 nm) and C18 reverse phase column (250 4.6 mm diameter; 5 mm particle sizes). Filtered sample (20 ml; 0.22 mm porosity) was injected with mobile phase of 40% acetonitrile in 1 mN H2SO4 (pH, 2.5e3.0) and flow rate of 0.5 mL/h. The headspace gas was sampled through a rubber septum using a gas-tight syringe (Hamilton) and analysed in a gas chromatograph (NUCON 5765) using TCD (1/8” 2 m Heysep Q) column and Argon as a carrier gas. The injector and detector were maintained at 60 C each and the oven was operated at 40 C isothermally. Standards for acids (SUPELCO) and alcohols (SIGMA) were used. 2.5. Bioelectrochemical analysis Bio-electrochemical behavior of biocatalyst during the operation was studied using cyclic voltammetry (CV) techniques (BioLogic-VMP3) by applying a potential ramp to the working electrode
O. Sarkar et al. / Energy 118 (2017) 425e434
427
Fig. 1. Reactor design and configuration delineating the complete operation setup: Spares and components are 1. Barometer 2. Safety valve 3. Temperature and pH probe 4. Needle type valve 5. Sample Ports 6. Pressure releasing knob 7. Thick walled high pressure resist glass reactor 8. Thick walled high pressure resist glass reactor 9. Needled inlet port with Nozzle 10. Water Drain out 11. Effluent drain out, 12. Source gas, Pure H2e 99.9%, (b) Ball and stick model depicting the carboxylic acid to alcohol conversion with their respective gibb's free energy. (c) H2 transfer across the cell membrane and the reaction of NAD with diffused intra-cellular hydrogen.
(anode) over a range of þ1.0 to 1.0 V against an Ag/AgCl (S) reference electrode. Electron transfer rate (Kapp) was derived by recording CV at variable scan rates (0.5e30 mV s1). Laviron theory was used to understand the peak potential variation with varying scan rates and thereby determining the electron transfer rates [26].
Epc ¼ Ec
Epa ¼ Ea
RT anFvc ln anF RTKapp
(6)
RT ð1 aÞnFva ln ð1 aÞnF RTKapp
(7)
The dependence of peak potential on the scan rate (mV s1) was evaluated when DEp 200 mV (DEp ¼ EpcEpa) where Epc and Epa represent cathodic and anodic peak potential, a is transfer coefficient, v represents scan rate, kapp is apparent rate constant which is an indicator for the e transfer rate between the biocatalyst and the electrode, R (8.31 J mol1 K1) and F (96,483 C mol1) refers to constant values, T refers to temperature (298 K) and n represents number of electrons respectively (Eqs. (6) and (7)). 3. Results and discussion 3.1. Carboxylic acids production Carboxylic acids production illustrates the progress of acidogenic/methanogenic process. Compositional variation of fatty acids was analysed through the chromatographic analysis The experimental data documented distinct variations in carboxylic acids production, signifying the apparent change in the metabolic
behavior of experimental variations studied (Fig. 2). The pressure of H2 in the head space of HPR showed marked effect on the acidognesis on the production as well as on the consumption of the fatty acids. Relatively higher VFA synthesis was noticed with HPR (2.4 g/l) compared to CTR (1.9 g/l) with more or less 20% increment in productivity. Both the systems showed a consistent increment in the VFA production with cycle time and approached maximum value at 32nd h of the cycle operation (Fig. 2a). Subsequent increment in the cycle time documented a marked drop in the VFA production in both the systems. The carboxylic acids profile showed presence of acetic (HAc) in higher concentration followed by butyric (HBu) acids and propionic (HPr) in both the systems with time bound concentration variation (Fig. 2b, c and d). Microbial metabolites depict the type of fermentation being carried and its end product formation. The production pattern of HAc and HBu in HPR and CTR more or less resembled till 32nd h of operation with varying concentration and then decreased with extension of HRT. Gradual increment in HAc production from 8th to 24th h was observed in both HPR and CTR with varying concentration and then dropped from 32nd h of operation till the end of cycle operation. Comparatively higher HAc accumulation (1.2 g/l) was observed with HPR compared to CTR (0.8 g/l) depicting ~35% improvement due to the regulated headspace environment (Eq. (8)). Fraction of HBu was lower compared to HAc in both the operation (HPR, 0.75 g/l; CTR, 0.6 g/l) with relative improvement in productivity of 25%. Even though relative less HPr production was observed the variation was distinct with 92% improvement in productivity. 4H2 þ 2CO2 / CH3COOH þ 2H2O (23.4 kJ/mol H2)
(8)
428
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 2. Carboxylic acid Synthesis (mg/l) versus time of operation (hours). (a)Total Carboxylic acid production (mg/l) versus time of operation (h) (b)Acetic Acid (c) propionic acid, (d) Butyric Acid, Production and consumption rate of individual carboxylic acids with respect to time operation (e) Acetic acid, (f) Propionic acid, (g) Butyric acid.
O. Sarkar et al. / Energy 118 (2017) 425e434
3.1.1. Production and consumption of fatty acids The acidogenic process is always dynamic and is influenced by the quantity and composition of carboxylic acids present as well as head gas composition and pressure. In order to understand the dynamics of the process, the production/consumption rates of carboxylic acids were calculated based on the following equations (Eqs. (9) and (10)) [27]. Production rate of carboxylic acids (PRCa) ¼ (VFAmaxVFAint)/ TProd
(9)
Consumption rate of carboxylic acids (CRCa) ¼ (VFAdropVFAmax)/ TProd (10) where, VFAmax represents maximum VFA concentration (g/l/h), VFAint is initial VFA concentration (g/l/h), VFAdrop denotes drop/ consumption in VFA concentration (g/l/h), TProd is production time in hours and TDrop represents concentration dropping/consumption time (hours). The positive and negative values explained the rate of production and consumption of carboxylic acids respectively. Production was calculated from the initial hour to the time of next sampling (8 h) while the consumption was calculated from the point of degradation to end of the cycle for each acid individually. Analysis of the data showed some marked variations with the functional role of headspace gas and metabolite composition. Increasing trend in PRCa was observed with HAc from 8th h to 32nd h operations, which dropped later with increasing fermentation time (Fig. 2e). Highest PRCa was observed with HAc in both systems (HPR, þ55 mg/L/h (24 h); CTR, þ45 mg/L/h (8 h). Similar trends where observed in the case of HBu with the maximum PRCa þ 43 mg/L/h than CTR (þ29 mg/L/h) at 8 h. However, in the case of HPr there was production at 40 h (PRCa, þ 28 mg/L/h) with HPR (Fig. 2f). On the contrary, CTR operated showed gradual production from 8th h and reached maximum at 16th h (þ16.6 mg/L/h). The maximum CRCa was observed at 32nd h with HPR (0.38 mg/L/ h) which is lower than CTR (6.9 mg/L/h at 48 h). The CRCa with HBu was observed to relatively more with HPR (10.8 mg/L/h; 48th h) than CTR (0.9 mg/L/h; 48 h) (Fig. 2f). There was no consumption of HPr in either of the system under study. Post the 32nd h the consumption of carboxylic acids correlated with the alcohol production. Higher HPr synthesis was specially observed with CTR against lower fraction in HPR in the later stages of fermentation time. Higher HPr in HBR is observed compared to CTR as the H2 dependent reaction is limited in the normal headspace gas composition when compared to 100% hydrogen headspace in HPR. Degree of acidification (DOA) calculated in terms of individual carboxylic acid concentrations and mainly influenced by the composition of the carboxylic acids profile in the system [27] (Fig. 3a). Production and consumption pattern of carboxylic acid also influence the DOA. Relatively higher DOA (22.3%; 16th h) was observed with CTR than HPR (21.7%; 8th h) operation might be attributed to the relatively higher fraction of HPr produced. Whereas in HPR, the consumption of HAc and HBu (32nd h) and the production of lower HPr at later stage of cycle operation (40th h) might have resulted in lower DOA. 3.2. Hydroxylates formation from carboxylates Reduction of carboxylic acids resulted in solventogenesis leading to production of alcohols (hydroxylates) (Fig. 3b). This bioconversion is a two-step process where organics are first acidified to carboxylates (HAc, HBu and HPr) and biogas production followed by formation of hydroxylates via alcohol dehydrogenases catalysed reactions [28]. pH conditions and increased reductive gas pressure
429
will have a direct influence on the alcohol formation [29]. Induction of reductive environment in the bioreactor with the increasing concentration of carboxylic acids synthesis enforced the biocatalyst to maintain its cellular proton concentration. As a function of pressure, HPR and CTR showed variation in the concentration of alcohol formation. External supplemented H2 pressure in the headspace of HPR mediated the conversion of acids to alcohols. With HAc being the major carboxylic acid synthesised, ethanol concentration was higher (Eq. (11)). CH3COOH þ H2 / CH3CH2OH
(11)
Alcohol production was initiated after 16th h in HPR (0.01 g/l) and the maximum production was observed between 40th to 48th h (0.09 g/l). Alcohol production in CTR is non-influenced fermentation with the maximum production observed at 40th h (0.04 g/l). With specific pretreatment the productivity can be increased as the essential biocatalyst has substrate competition with non-essential biocatalyst. 3.3. Gaseous phase analysis Gas and aqueous phase interface will be the determining factor for the reduction process. Induced H2 pressure in the headspace altered the product formation and lead to bioethanol formation. HPR depicted the consumption of H2 as an electron donor for acetate reduction in conjunction with the pressure reduces the activation energy. H2 production was detected at different time interval in CTR. On the contrary, HPR didn't showed H2 production inspite of acetate formation. The consumption of H2 in fermentation process prevent H2 formation and increase the acetate yield [30,31]. As H2 was pressurised in HPR, a marked consumption of H2 was noticed with time (Fig. 4a). Consumption pattern of H2 was well synchronised with the reduced products formed in the biosystem. Initially, at 8th h, the H2 consumption was about 11% and increased as fermentation time matured and observed 22% (18 h). Higher H2 consumption in HPR was observed at 24th h of 35% with indicated the higher metabolic activity to convert H2 to reduce carboxylic acids to respective alcohols. Consumption of H2 gas decreased at 32nd h to 9% followed by 4% at 48th h. Headspace gas composition was concurrently estimated for enumerating other gases formation. H2 gas transfer across the cell membrane is through diffusion and through proton pumps coupled with ATP synthesis. The gas consumed creates a reducing environment in terms of redox microenvironment and catalyses the carboxylic acid conversion to form alcohols. H2 existing either in the external environmental (Hevx) and/or in the intercellular H2 (Hinc). Microbial ability to adapt to the gradient results in the formation of reducing equivalents intracellularly in the form of NADH (Eq (12)).
H2 þ NADþ / NADH2 DG ¼ 18 kJ/mol
(12)
As anaerobic culture was used as an inoculum, CH4 formation was observed in the reactor due to prevailing methanogenic activity. The concentration of CH4 along with CO2 increased with fermentation time in HPR indicating the fermentation of glucose (Fig. 4b). CH4 production varied in the range of 4%e12% along with CO2 (7%e31%). In CTR, biogas composition of H2, CH4 and CO2 with varied concentration was observed (Fig. 4c). Higher concentration of H2 was observed at 16th h (13%). H2 concentration increased till 16 h in CTR and then dropped till the end of the cycle. CH4 detected from 8th h (2%) followed increasing till the end of the cycle operation. Evolution of CH4 in CTR due to the methanogenesis correlated well with the synthesis of HPr and consumption of HAc to CH4 (Eq. (13)). On the other hand synthesis of HPr is through
430
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 3. (a) Degree of acidification with respect to time, (b) Alcohol (Bioethanol) production (mg/l) versus time of operation (hours).
Fig. 4. Biogas Composition versus time of operation (hours) in (a) Hydrogen consumption in HPR, (b) gas composition in HPR and (c) gas composition in CTR system.
decarboxylation of succinate, which generates CO2 in the conversion of C4 acids to C3 acids in the succinate pathway [24]. CH3COOH þ 2H2O / CH4 þ CO2
(13)
The forward progress of the reaction is based on the net energy balance on both sides of the equation, the reactants and the products (Gibbs free energy below zero). Exothermic reactions are spontaneous at standard conditions (T ¼ 25 C), glucose to value added products conversion, unlike, compared to glucose fermentation (225.5 kJ) [32]. Therefore, the concentrations and partial pressure of H2 and protons is regulated to study their effect on the product formation with the function of Gibbs free reaction energy (Eq. (14)).
DG ¼ DG þ RT ln(C y/x)
(14)
where C is the H2 diffused relative to its partial pressure (considered to be at saturation- 1 mmol/l), y/x ratio is of NAD/ NADH. To study the feasibility of the reactions with the
increasing pressure the ratio is considered to be 1, resembling equilibrium. The carboxylic acid reduction occurs at 20 kJ/mol as the highest energy required (9.1 kJ/mol, 9.4 kJ/mol and 5.4 kJ/mol for ethanol, propanol and butanol respectively). As the experiment progresses the DG increases making the reaction less feasible until it reaches equilibrium. This possibly suggests that the reduction of the alcohols is not altered by the pressure in the headspace as the standard Gibbs free energy of these reactions is below zero. 3.4. Bio-electrochemical analysis-reductive solventogenesis Cyclic Voltammogram (CV) of both the HPR and CTR are analysed at different time intervals to understand the reductive product formation and the different redox mediator that participate in the carboxylic acid formation and their reduction to alcohols (Fig. 5). Type and size of the reactors, the initial substrate and all the analytical conditions were maintained the same thus any changes observed in the CVs indicates the shift of metabolism under the influence of regulated headspace conditions.
O. Sarkar et al. / Energy 118 (2017) 425e434
431
Fig. 5. Cyclic Voltammetric Analysis of control (a,b,c) and HPR (d,e,f) at different time intervals (8th, 24th and 48th hour), the determination of an electron transfer rate with multiscan cyclic voltammograms. (g) HPR and (h) Control.
432
O. Sarkar et al. / Energy 118 (2017) 425e434
Fig. 6. (a) Utilisation of glucose as the substrate; glucose concentration (mg/l) versus time of operation (hours). (b) Variation in pH versus time of operation (hours), (c) Buffering Capacity (bmol) versus time of operation (hours).
Voltammograms also give overview on the interaction reactions between the electrode and biocatalyst through alterations in the electrochemical behaviours. The catalytic reduction currents were showing visible variation in HPR (0.006 mA) as compared to CTR and HPR (0.003 mA) but altered metabolism with different mediator peaks with function of time (Fig. 5a, b & c). At the same time intervals peaks at different potentials (biological redox reaction) was observed, which relates to NADþ/NADH (0.56 V vs Ag/AgCl) and cytochrome complex (0.3 V vs Ag/AgCl). Carboxylic acids get reduced in the presence of the carboxylic reductases to form aldehydes and later form alcohols where these reactions occur under the influence of redox electron carriers like NAD, FeS, Cytochromes, FAD, etc. [33] Four moles of reduced ferredoxin facilitates formation of 2 mol of acetate to acetaldehyde, and four more electrons derived from H2 via NADH gets used in the reduction of 2 mol of acetaldehyde to ethanol. Pyruvate oxidation and Acetyl CoA reduction to form acetate is the possible initial reaction occurring in both the systems, as the initial fermentative pathway of glucose breakdown will remain the same. As the reactions progress with time, peaks were observed at 0.1 V vs Ag/ AgCl in both the CTR as well as in the HPR. These peaks can be attributed to membrane-bound flavoproteins along with an ironsulphur cluster of [4Fee4S] associated complex catalysing the proton reduction. There is a reversible peak observed at 0.32 to 0.4 V (vs Ag/AgCl) specifically in the HPR, which could resemble the redox carriers that are functioning with alcohol dehydrogenases that catalyse acetate to acetaldehyde and reducing it to ethanol (Fig. 5d, e&f). The reversible peak showing oxidative and reductive states could be the flavoproteins and iron sulphur mediators which gets reduced and oxidised supplying electrons to acetate [34,35]. This illustrates specific metabolic shift in the regulated biosystem due to H2 partial pressure. The reducing conditions formed in the process due to the developed electron cloud has been determined by the evaluating the electron transfer rates which facilitates to understand the bio-
electro kinetics. The electron transfer rate (Eqs (6) and (7)) derived with HPR (0.282 s1) was relatively higher compared to that of CTR (0.161 s1). With the higher electron density in the HPR, the transfer rate also has been observed to be higher. On the other hand, the HPR showed higher capacitance than the CTR, which is evident from the figure portraying the ion holding capacity of the reactor that is directly proportional to energy available to increase the spontaneity of the reactions [36]. 3.5. Carbon utilisation The glucose assimilation phase was observed until 36th h in both the systems. The H2 partial pressure had no significant effect on the glucose uptake. The degree of utilisation was dependent on the redox conditions maintained in the reactor only for the initial period. The short chain carboxylic acids formed have the capabilities to act as primary substrate over glucose. Under atmospheric pressure, the utilisation percentage was higher (70%) compared to regulated H2 pressure conditions (60%) (Fig. 6a). the maximum utilisation occurred between 12th h and 16th h where the acidogenic activity of the bacteria will be higher. The rate of utilisation became low paced once the carboxylic acids have been synthesised suggesting the possible uptake of them by the microbiome as the substrate over glucose. 3.6. Redox conditions Acidogenic fermentation leads to high intracellular acid concentrations, which require increased proton translocation for the maintenance of pH. Fermentation pH is assumed to serve as a trigger causing bacteria to shift from the production of hydrolyzed products to organic acids and gases to respective solvents. The pH of the bioreactors (HPR and CTR) was not controlled during operation. From the initial pH 6 at 0th h to 48th h, there is a steep drop towards acidification (pH, 4.4) in the HPR. The H2 in the
O. Sarkar et al. / Energy 118 (2017) 425e434
headspace also diffuses to liquid phase and shifts redox condition. In CTR, pH drop was comparatively marginal (6e5.1) (Fig. 6b). This disparity can be attributed to the concentration of carboxylic acids formed. Variation in pH was higher in the HPR operation during initial hours. Drop in pH was marginal till 16th h of operation in both the reactors. The continuous drop in pH in HPR was attributed to production higher production of VFA in the system. pH change was marginal in the control operation, which favoured the methanogenesis resulted in CH4 evolution in the later hour of operation. The results indicated that the relationship between higher propionic acid production and lower H2 evolution were well correlated with themethanogenic activity in the control operation. The increase in pH and the lowering of the carboxylic acid production correlate giving strength to the presence of homoacetogens/acetogens/methanogens to utilise the formed carboxylic acids. Carboxylic acids formation has a prime influence on the reactors buffering capacity. The reactors insitu ability to maintain its pH was measured at different time intervals (Fig. 6c). Variation in concentrations of the carboxylic acids changes systems buffering capacity. The buffering capacity maintained the redox conditions starting at 8th h to 32nd h with the values ranging from 0.024 to 0.028 b mol in the reactors which are having H2 partial pressure in the headspace. CTR showed comparatively lower buffering capacity with the maximum at 24th h of 0.022 ± 0.002 b mol. The trend of buffering capacity showed an increment up to 24th h with the possible reason of carboxylic acid formation and later on showed a downward slope as the formed carboxylic acids gets utilised. Operations at the extend times at 40th h and beyond due to lack of buffering capability moves towards lower pH which also favour alcohol production. Buffering capabilities of the reactor are developed by the formation of short chain carboxylic acids which are weak acids when they are reacting with ions present in reactor they form a buffering agent. Few buffering systems like ammonium bicarbonate (pKa 6.35/9.35), carbonate (pKa 6.35/9.25), formic acid (pKa 9.25) and HAc (pKa 4.75/9.25) [37]. 4. Conclusions Non-genetic routes for shifting the metabolic pathways towards solventogenesis are possible by manipulating the headspace of the reactor with a specific gas at high pressure. This method could be employed for selective biobased product productivity and enhancement of their yields. Selective product synthesis in microorganisms is a function of physiological and operation parameter modifications. Glucose utilisation reactions are spontaneous and there could be no effect of increased pressure, whereas, reduction of carboxylic acids is enhanced by the partial pressure of H2 gas and prevailing redox conditions as a result of hydrogen gas diffusion. Gas composition analysis revealed the consumption of H2, while intracellular redox mediators involved in solventogenesis was reflected in the bioelectrochemical analysis. The negative effect of H2 is not seen, as the electrochemical analysis show the activity of NADH: Fes reactions to be functioning. The alcohol reduction gets mediated by Fe-s cluster proteins and NADH electron transfer mediators which are observed with the electrochemical analysis. Waste valorization offers a positive energy balance in the environment with operational freedom and low-cost productivity rekindling waste biorefinery and green fuel production. These methods offer economic feasibility with options of integration with other acidogenic fermenters for H2, microalgal cultivation for biodiesel and biopolymer production with photobacteria enabling a closed sustainable loop for bio-fuel production.
433
Acknowledgements The authors wish to thank the Director CSIR-IICT for the support and encouragement in carrying out this work. This work was supported by Department of biotechnology (DBT-No. BT/PR13642/ BBE/117/80/2015). Nomenclature HPR Hydrogen Pressure Reactor VFA Volatile Fatty Acids CV Cyclic Voltammograms DSW design synthetic wastewater HAc Acetic acid HPr Propionic acid HBu Butyric acid Hevx Extracellular Hydrogen Hinc Intracellular Hydrogen NADH: Fed Nicotinamide Adenine Dehydrogenase: Ferridoxin DOA Degree of Acidification AB Anaerobic bacteria CRT Control reactor HDPE High density polyethylene References [1] Choi YJ, Lee SY. Microbial production of short-chain alkanes. Nature 2013;502: 571e4. [2] Annie Modestra J, Navaneeth B, Venkata Mohan S. Bio-electrocatalytic reduction of CO2: enrichment of homoacetogens and pH optimization towards enhancement of carboxylic acids biosynthesis. J CO2 Util 2015;10:78e87. [3] Shafiei E, Davidsdottir B, Leaver J, Stefansson H, Asgeirsson EI, Keith DR. Analysis of supply-push strategies governing the transition to biofuel vehicles in a market-oriented renewable energy system. Energy 2016;94:409e21. [4] Jin Y, Illukpitiya P. Cost minimization of supplying biomass for ethanol biorefineries. Energy 2016;96:209e14. http://dx.doi.org/10.1016/j.energy.2015. 12.031. [5] Venkata Mohan S, Bhaskar YV, Murali Krishna P, Rao NC, Lalit Babu V, Sarma PN. Biohydrogen production from chemical wastewater as substrate by selectively enriched anaerobic mixed consortia: influence of fermentation pH and substrate composition. Int J Hydrogen Energy 2007;32:2286e95. [6] Kandylis P, Bekatorou A, Pissaridi K, Lappa K, Dima A, Kanellaki M, et al. Acidogenesis of cellulosic hydrolysates for new generation biofuels. Biomass Bioenergy 2016;91:210e6. [7] Venkata Mohan S, Nikhil GN, Chiranjeevi P, Reddy CN, Rohit MV, Kumar AN, Sarkar O. Waste biorefinery models towards sustainable circular bioeconomy: Critical review and future perspectives. Bioresour Technol 2016;215:2e12. [8] Arslan D, Steinbusch KJJ, Diels L, De Wever H, Hamelers HVM, Buisman CJN. Selective carboxylate production by controlling hydrogen, carbon dioxide and substrate concentrations in mixed culture fermentation. Bioresour Technol 2013;136:452e60. [9] Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010;463:559e62. [10] Chen Y, Luo J, Yan Y, Feng L. Enhanced production of short-chain fatty acid by co-fermentation of waste activated sludge and kitchen waste under alkaline conditions and its application to microbial fuel cells. Appl Energy 2013;102: 1197e204. [11] Spirito CM, Richter H, Rabaey K, Stams AJM, Angenent LT. Chain elongation in anaerobic reactor microbiomes to recover resources from waste. Curr Opin Biotechnol 2014;27:115e22. [12] Xiong M, Deng J, Woodruff AP, Zhu M, Zhou J, Park SW, et al. A bio-catalytic approach to aliphatic ketones. Sci Rep 2012;2:311. [13] Marshall CW, Labelle EV, May HD. Production of fuels and chemicals from waste by microbiomes. Curr Opin Biotechnol 2013;24:391e7. [14] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T. Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol 2000;73:59e65. [15] Zheng X-J, Yu H-Q. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic cultures. J Environ Manag 2005;74:65e70. [16] Li C, Fang HHP. Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol 2007;37:1e39. [17] Steinbusch KJJ. Liquid biofuel production from volatile fatty acids. 2010. [18] Mohanakrishna G, Venkata Mohan S. Multiple process integrations for broad perspective analysis of fermentative H2 production from wastewater treatment: technical and environmental considerations. Appl Energy 2013;107: 244e54.
434
O. Sarkar et al. / Energy 118 (2017) 425e434
[19] Costello DJ, Greenfield PF, Lee PL. Dynamic modeling of a single-stage highrate anaerobic reactordI. Model derivation. Water Res 1991;25:847e58. [20] Ruzicka M. The effect of hydrogen on acidogenic glucose cleavage. Water Res 1996;30:2447e51. [21] Rodríguez J, Kleerebezem R, Lema JM, van Loosdrecht MCM. Modeling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 2006;93:592e606. [22] Mu Y, Yu HQ, Wang G. Evaluation of three methods for enriching H2producing cultures from anaerobic sludge. Enzyme Microb Technol 2007;40:947e53. [23] APHA. Standard methods for the examination of water and wastewater. twentieth ed. Washington DC, USA: American Public Health Association/ American Water Works Association/Water Environment Federation; 1998. [24] Dahiya S, Sarkar O, Swamy YV, Venkata Mohan S. Acidogenic fermentation of food waste for volatile fatty acid production with co-generation of biohydrogen. Bioresour Technol 2015;182:103e13. [25] Amulya K, Venkateswar Reddy M, Venkata Mohan S. Acidogenic spent wash valorization through polyhydroxyalkanoate (PHA) synthesis coupled with fermentative biohydrogen production. Bioresour Technol 2014;158:336e42. [26] Laviron E. Adsorption, autoinhibition and autocatalysis in polarography and in linear potential sweep voltammetry. J Electroanal Chem Interfacial Electrochem 1974;52:355e93. [27] Sarkar O, Kumar AN, Dahiya S, Krishna KV, Yeruva DK, Venkata Mohan S. Regulation of acidogenic metabolism towards enhanced short chain fatty acid biosynthesis from waste: metagenomic profiling. RSC Adv 2016;6:18641e53. [28] Steinbusch KJJ, Hamelers HVM, Buisman CJN. Alcohol production through volatile fatty acids reduction with hydrogen as electron donor by mixed cultures. Water Res 2008;42:4059e66.
ndez EV, Sepúlveda-Escribano A. From biodiesel [29] Serrano-Ruiz JC, Ramos-Ferna and bioethanol to liquid hydrocarbonfuels: new hydrotreating and advanced microbial technologies. Energy Environ Sci 2012;5:5638e52. [30] de Kok S, Meijer J, Van Loosdrecht MCM, Kleerebezem R. Impact of dissolved hydrogen partial pressure on mixed culture fermentations. Appl Microbiol Biotechnol 2013;97:2617e25. [31] Nie Y, Liu H, Du G, Chen J. Acetate yield increased by gas circulation and fedbatch fermentation in a novel syntrophic acetogenesis and homoacetogenesis coupling system. Bioresour Technol 2008;99:2989e95. [32] Amend JP, Shock EL. Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiol Rev 2001;25: 175e243. [33] Krishna KV, Sarkar O, Venkata Mohan S. Bioelectrochemical treatment of paper and pulp wastewater in comparison with anaerobic process: integrating chemical coagulation with simultaneous power production. Bioresour Technol 2014;174:142e51. [34] Srikanth S, Venkata Mohan S, Lalit Babu V, Sarma PN. Int J hydrogen energy 2010;35:10693e700. [35] Gottumukkala LD, Sukumaran RK, Venkata Mohan S, KuttuvanValappil S, Sarkar O, Pandey A. Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium sporogenes BE01: bio-electrochemical analysis of the electron transport mediators involved. Green Chem 2015;17:3047e58. [36] Veer Raghavulu S, Suresh Babu P, Kannaiah Goud R, Venkata Subhash G, Srikanth S, Venkata Mohan S. RSC Adv 2012;2:677e88. [37] Dinamarca MA, Aranda-Olmedo I, Puyet A, Rojo F. Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures. J Bacteriol 2003;185:4772e8.