See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/222908659 Effluents with soluble metabolites generated from acidogenic and methanogenic processes as substrate for additional... Article in International Journal of Hydrogen Energy · February 2009 DOI: 10.1016/j.ijhydene.2008.11.060 CITATIONS READS 37 42 5 authors, including: Srikanth Sandipam S Venkata Mohan 55 PUBLICATIONS 1,235 CITATIONS 349 PUBLICATIONS 8,533 CITATIONS IndianOil R&D Centre Indian Institute of Chemical Technology SEE PROFILE SEE PROFILE Mamilla Prathima Devi Dr. Lenin Babu Mannem 18 PUBLICATIONS 416 CITATIONS 13 PUBLICATIONS 298 CITATIONS Indian Institute of Chemical Technology Indian Institute of Chemical Technology SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Microbial Mediated Desalination System for wastewater treatment View project Bio-electrochemical treatment (BET) System for Effective treatment of Complex Wastewater assoiacted with Bioelectricity Generation View project All content following this page was uploaded by Dr. Lenin Babu Mannem on 27 December 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. international journal of hydrogen energy 34 (2009) 1771–1779 Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/he Effluents with soluble metabolites generated from acidogenic and methanogenic processes as substrate for additional hydrogen production through photo-biological process S. Srikanth, S. Venkata Mohan*, M. Prathima Devi, M. Lenin Babu, P.N. Sarma Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnka, Hyderabad 500 007, Andhra pradesh, India article info abstract Article history: The feasibility of utilizing effluents generated from acidogenic [producing biohydrogen Received 16 October 2008 (H2)] and methanogenic [producing methane] processes was studied for additional H2 Received in revised form production by terminally integrating with photo-biological process employing enriched 17 November 2008 mixed culture. Experimental data has depicted enhanced process efficiency with respect to Accepted 18 November 2008 additional H2 production and substrate degradation through photo-biological process. Available online 21 January 2009 However, the efficiency was found to depend on the process used in the first stage along with nature and composition of the substrate. Acidogenic process in the first stage had Keywords: more positive influence on photo-biological H2 production [synthetic wastewater – Mixed culture 14.40 mol/Kg CODR and 15.16 mol/Kg CODR (with vitamins); dairy wastewater – 13.29 mol/ Acidophilic Kg CODR and 13.70 mol/Kg CODR (with vitamins)] over the corresponding methanogenic Dark fermentation process. Effluent generated from acidogenic treatment of dairy wastewater yielded high Wastewater treatment substrate degradation rate (SDR) [1.20 Kg COD/m3 day and 1.34 Kg COD/m3 day (vitamins)] Total volatile fatty acids followed by synthetic wastewater [0.92 Kg COD/m3 day and 1.05 Kg COD/m3 day (vitamins)]. Among the studied experimental variations chemical wastewater evidenced poor H2 production and SDR. Vitamin solution showed positive influence on both H2 production and wastewater treatment irrespective of the experimental variations studied. ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. 1. Introduction Increasing demand for energy and the resulting crisis in association with the green house pollution from the combustion of fossil fuels led for search towards alternative and eco-friendly energy sources. In this direction, a great deal of attention is being focused on the usage of hydrogen (H2) as an alternative energy source which may play a pivotal role in future energy supply. H2 production by biological route is considered to be advantageous compared to traditional methods due to its environmental friendly nature. Biological H2 production processes can be classified as biophotolysis by algae/ cyanobacteria, photodecomposition by photosynthetic bacteria and dark fermentation by anaerobic bacteria [1–6]. Most of these processes occur at ambient temperatures and pressures, are less energy intensive and more environmental friendly and thus opening a new avenue for the utilization of renewable energy sources such as wastewater which are inexhaustible [1,6–11]. A practical and efficient H2 generation process is a growing concern among the research fraternity [9] and various strategies in this direction were reported [3–6,12–17]. Lightdependent (photo-biological) or light independent (dark) fermentative conversions of organic substrates manifest * Corresponding author. E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.11.060 1772 international journal of hydrogen energy 34 (2009) 1771–1779 diverse metabolic pathways and substrate utilization pattern during H2 production [1,10,18–22]. In the case of dark fermentation, the organic substrate was converted to H2 or CH4 along with organic acids as metabolic (soluble) by-products mainly by acidogenic bacteria [1,5,6,23]. Generation and accumulation of soluble acid metabolites causes sharp drop in the system pH and inhibit the H2 production process. Persistent acidophilic conditions result in suppression of methanogenic process and accounts only for 15% of the available energy conversion from the organic source in the form of H2 [6]. Usage of unutilized carbon associated with soluble metabolites present in the effluents for additional H2 production will sustain the practical applicability of the process. Further, photosynthetic organisms are capable of utilizing organic acids as substrate besides the original substrates to produce additional H2 [1,5,6,12,13,22,23]. Therefore, integration of photo-biological process after dark fermentation could result in additional H2 production and substrate degradation efficiency. This route appears to be an ideal one which might lead to highest possible theoretical yield [6,8,20]. An attempt was therefore made in this communication to study the feasibility of utilizing soluble metabolites along with residual carbon source present in effluents generated from dark fermentation processes (acidogenic and methanogenic) as primary substrates for additional H2 production and substrate degradation through integration of photo-biological process. Fig. 1 – Experimental setup used for photo-biological hydrogen production. effluents generated from first stage mostly constitute of residual carbon associated with total volatile fatty acids (TVFA)/soluble acid metabolites which were used as substrates for subsequent H2 production by photo-biological process as depicted in Table 1. 2.3. 2. Experimental procedure 2.1. Mixed culture Photosynthetic mixed culture acquired from Saroor nagar lake, Hyderabad was used after screening and testing its viability in different wastewaters [6]. The inoculum (pH, 8.36; total volatile fatty acids (TVFA), 754 mg/l) was preserved at 4  C for further use. Prior to use, the inoculum was enriched in a mixture of dairy wastewater and domestic sewage (1:1) along with vitamin solution [2.5 ml/l; thiamine mononitrate, 10 mg/l; riboflavin, 10 mg/l; nicotinic acid, 25 mg/l; niacinamide, 75 mg/l; pyridoxine hydrochloride, 3 mg/l; calcium pantothenate, 50 mg/l; folic acid, 1.5 mg/l; vitamin B12, 15 mcg/l; vitamin C, 150 mg/l; biotin, 260 mcg/l] and incubated in the presence of fluorescent light (4 Klux) at 34  C [6] for 24 h (Fig. 1) 2.2. Substrates Initially, H2 producing acidogenic bioreactor (HBR) and CH4 producing methanogenic bioreactor (MBR) were operated with three substrates [synthetic wastewater ((SW); g/l, glucose-3.0/ 6.0, NH4Cl-0.5, KH2PO4-0.25, K2HPO4-0.25, MgCl2$6H2O-0.3, FeCl3-0.025, NiSO4-0.016, CoCl2-0.025, ZnCl2-0.0115, CuCl20.0105, CaCl2-0.005, MnCl2-0.015) [COD: 3200 mg/l, pH: 6.8, TDS: 1420 mg/l, ORP: 2.6], chemical wastewater (CW) [COD: 9200 mg/l, pH: 7.8, TDS: 18,400 mg/l, ORP: 24.6] and dairy wastewater (DW) [COD: 4800 mg/l, pH: 7.2, TDS: 4200 mg/l, ORP: 10.4]] in the first stage. Feed characteristics varied from simple to complex depending upon the source obtained. The Experimental design In the first stage, HBR was operated under acidophilic condition (pH 6) using selectively enriched mixed consortia and MBR was operated under neutral condition (pH 7) using anaerobic mixed inoculum. Both the processes were operated in biofilm configured systems employing batch mode operation at ambient room temperature with three types of wastewaters for a total cycle period of 48 h (retention time). The performance was evaluated in terms of H2 and CH4 production along with substrate removal efficiency. Effluent from the reactors was collected separately after 48 h of operation. In the second stage, after separating biomass (by settling; 30 min), 150 ml of supernatant (adjusted to pH 6 using concentrated orthophosphoric acid and/or 3 N NaOH) along with 20 ml of enriched photosynthetic mixed culture (24 h; Saroor Nagar lake, Hyderabad) was loaded in the flask under anaerobic conditions. After feeding, the flasks were sparged with oxygen free nitrogen gas for a period of 30 s to remove dissolved oxygen and to create an oxygen free microenvironment prior to closing with rubber septum (butyl rubber). Batch experimental protocol was employed to evaluate the experimental objectives using a series of 250 ml conical flasks (with a total working volume of 160 ml) under aseptic anaerobic microenvironment. Twelve experiments were designed and evaluated based on the differential combinations of substrate (Table 2). All the experiments were performed in presence of fluorescent light (4 Klux) in an incubator orbital shaker (100 rpm; 34  C) for 24 h. Experiments were performed in the presence and absence of vitamins (3.2 ml/l) to enumerate its role on process performance. Periodically samples were collected to evaluate the process efficiency along with the quantification of H2 gas produced. 1773 international journal of hydrogen energy 34 (2009) 1771–1779 Table 1 – Characteristics of wastewater used as feed in the first stage of treatment [acidogenic (HBR) and methanogenic (MB)] and resulting process efficiency. Wastewater used as feed Reactor microenvironment SW Acidogenic Methanogenic Acidogenic Methanogenic Acidogenic Methanogenic CW DW TVFA (mg/l)a pH Inlet Outlet Inlet Outlet 6.0 7.0 6.0 7.0 6.0 7.0 6.8 8.1 6.6 8.1 7.2 8.4 360.4 246.2 1440 1260 642.6 726.4 782.2 804.6 422 462.8 574.8 242 SDR (Kg COD/m3 day) (mg/l)a xCOD (%)a H2 yield (mol H2/Kg CODR)a CH4 yield (mol CH4/Kg CODR)a 0.525 1.137 0.263 0.831 0.306 0.875 31.58 68.42 12.76 40.42 16.27 46.51 18.64 – 8.26 – 11.12 – – 21.98 – 16.45 – 18.75 a Based on mean value (n ¼ 2) S.E varied between 0.3 and 3.4%. 2.4. Bio-analytical methods Water displacement method was employed for quantitative estimation of the biogas generated from the process. H2 gas was estimated using a microprocessor based electrochemical gas sensor (ATMI GmBH Inc., Germany). The output signal displayed the percent volume of H2 in the reactor head space, which was converted to mmol. Process performance was also evaluated by estimating the soluble COD (closed refluxing titrimetric method) and TVFA according to the procedures outlined in the standard methods [24]. The separation and quantitative determination of the composition of soluble metabolites [acetic acid (HAc), butyric acid (HBu) and propionic (HPr) along with ethanol (EtOH)] were estimated by high performance liquid chromatography [HPLC; UV–vis detector; C18 reverse phase column – 250  4.6 mm and 5 m particle size; flow rate – 0.5 ml/h; wavelength – 210 nm; mobile phase – 40% of acetonitrile in 1 mN H2SO4 (pH 2.5–3.0); sample injection – 20 ml]. COD removal efficiency (x) was calculated as shown in Eq. (1), where, CSO represents the initial COD concentration (mg/l) in the feed and CS denotes COD concentration (mg/l) in the reactor outlet. x ¼ ½ðCSO  CS Þ=CSO   100 Substrate degradation rate (SDR – Kg COD/m day) was calculated to study the rate and pattern of COD removal where, xt represent substrate removal efficiency (%) at time ‘t’ according to Eq. (2). (2) Organic loading rate (OLR – Kg COD/m3 day) was calculated using Eq. (3), where F represents feed rate (l/day) and V denotes volume of substrate (l ). OLR ¼ f½CSO  F=Vg 3. 3.1. Biohydrogen production Experimental data evidenced the feasibility of photo-biological process in using effluents generated from dark fermentation processes (both acidogenic and methanogenic) as primary substrates for additional H2 production and substrate degradation. Fig. 2 depicts the H2 production profile as a function of the substrates used. H2 production was found to depend mostly on the first stage of treatment adapted and nature of wastewater used as substrate. Details of photo- (1) 3 SDR ¼ ½OLR  xt =100 substrate degradation was observed in the methanogenic process rather than acidogenic process. The higher substrate degradation and biogas yields observed with SW might be attributed to its simple composition which may facilitate easy metabolism. Comparatively, CW and DW were complex in nature and are les bio-degradable. Irrespective of the wastewater used, effluents generated from acidogenic process documented higher concentration of TVFA along with carbon (residual) than the methanogenic process irrespective of the wastewater used. (3) Results and discussion During initial stage of process operation considerable variation in biogas (H2 and CH4) production, substrate degradation and TVFA generation was observed with the function of wastewater used as substrate and the process used for substrate metabolism (Table 1). Among the wastewaters studied, SW showed highest biogas yield followed by DW and CW irrespective of the metabolic process used. Higher Table 2 – Design criteria of experiments carried by integrating with photo-biological process. Experiment Feed OLR (Kg no. COD/m3 day) 1 2 3 4 5 6 7 8 9 10 11 12 H-SW H-SW M-SW M-SW H-DW H-DW M-DW M-DW H-CW H-CW M-CW M-CW 2.21 2.21 1.11 1.11 3.59 3.59 2.39 2.39 3.18 3.18 2.12 2.12 Operational conditions Acidogenic Acidogenic; with vitamin Methanogenic Methanogenic; with vitamin Acidogenic Acidogenic; with vitamin Methanogenic Methanogenic; with vitamin Acidogenic Acidogenic; with vitamin Methanogenic Methanogenic; with vitamin All experiments were performed with adopted photosynthetic mixed culture from Saroor Nagar pond in presence of incandescent light source (4 Klux) (temperature 34  C; pH 6.0); OLR, Organic loading rate; SW, Designed synthetic wastewater effluent; DW, Dairy wastewater effluent; CW, Industrial wastewater effluent; H, Effluent from acidogenic bioreactor; M, Effluent from methanogenic bioreactor;. 1774 international journal of hydrogen energy 34 (2009) 1771–1779 [8.93 mmol/day; 9.79 mmol/day (vitamins)] followed by M-SW [8.35 mmol/day; 8.93 mmol/day (vitamins)]. Among the effluents, CW documented lowest H2 production [H-CW – 5.76 mmol/day; 7.49 mmol/day (vitamins); M-CW – 2.30 mmol/ day; 2.59 mmol/day (vitamins)] irrespective of the preliminary treatment processes adapted. However, specific H2 production showed marked deviation with the production observed as depicted below. biological H2 production with respect to experimental variation studied in the descending order of performance is as shown below. H  DW > H  SW > M  DW > M  SW > H  CW > M  CW Higher H2 production was observed with the effluents generated from acidogenic reactor (HBR) irrespective of the nature of wastewater. Among effluents, DW documented higher efficiency of H2 generation followed by SW and CW. Effluents supplemented with vitamins showed marked improvement in H2 production in all the experimental variations studied (Fig. 3a). Outlet of acidogenesis reactor (H-DW) showed maximum H2 production [17.28 mmol/day and 19.87 mmol/day (vitamins)] followed by H-SW [14.40 mmol/ day and 17.28 mmol/day (vitamins)]. Similarly, methanogenic outlet also showed higher H2 production in the case of M-DW a H  SW > H  DW > M  SW > M  DW > H  CW > M  CW Acidogenic reactor outlet treating H-SW showed higher specific H2 yield [14.40 mol/Kg CODR and 15.16 mol/Kg CODR (vitamins)] followed by H-DW [13.29 mol/Kg CODR and 13.70 mol/Kg CODR (vitamins)]. Similarly the methanogenic outlets evidenced higher specific H2 production in the case of M-SW [11.60 mol/Kg CODR; 11.75 mol/Kg CODR (vitamins)] 25 mmol/day mol/Kg COD R H2 Production/H2 Yield 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 Experiment Number b 25 Acidophilic/Anaerobic Photosynthetic H2/CH4 Yield (mol/Kg COD R) 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 Experiment Number Fig. 2 – (a) H2 production and yield during photo-biological operation (b) Biogas (H2 and CH4) yield observed during two stages of the experimental study (acidogenic/methanogenic/photosynthetic). international journal of hydrogen energy 34 (2009) 1771–1779 a 16 14 H2 Yield (mol/kg COD R) 1775 Without Vitamin With Vitamin 12 10 8 6 4 2 0 2.21 1.11 3.59 2.39 3.18 2.12 OLR (Kg COD/m3-day) b 1.6 SDR (Kg COD/m3-day) 1.4 Without Vitamin With Vitamin 1.2 1 0.8 0.6 0.4 0.2 0 2.21 1.11 3.59 2.39 3.18 2.12 OLR (Kg COD/m3-day) Fig. 3 – Variation in (a) Hydrogen yield and (b) substrate degradation rate (SDR) in presence and absence of vitamin supplementation with the function of organic loading rate. followed by M-DW [9.62 mol/Kg CODR; 10.20 mol/Kg CODR (vitamins)]. The CW effluent from both the processes evidenced lower H2 yield among the experimental variations studied [H-CW – 6.70 mol/Kg CODR; 8.23 mol/Kg CODR (vitamins); M-CW – 5.24 mol/Kg CODR; 5.71 mol/Kg CODR (vitamins)]. It was evident from the experimental results that, the initial treatment and characteristics of wastewater (before and after first stage of treatment) appeared to have significant influence on H2 production by photo-biological process. Initial placement of acidogenic process appears to have positive influence on subsequent integration with photo-biological H2 production over the integration of corresponding methanogenic process. This may be attributed to the presence of higher concentrations of TVFA associated with residual carbon after acidogenic process in the effluents. 3.2. Substrate degradation Experiments also evidenced the possibility of additional substrate degradation due to the terminal integration of photo-biological process. Residual carbon source remaining in the outlets generated from acidogenic and methanogenic processes was observed to be utilized by subsequent integration with photo-biological process (Fig. 4; Table 1). However, the rate of substrate degradation was found to depend on the characteristics/nature of the wastewater used as substrate and the process used in the first stage. Visible improvement in the substrate degradation efficiency was also observed after supplementing with vitamins (Fig. 3b). COD removal efficiency showed a decreasing trend with increasing complexity of the substrated. SW which contained simple carbon source in the form of glucose yielded high COD removal efficiency [H-SW – 41.67% and 47.50% (vitamins); M-SW – 60.00% and 63.33% (vitamins)] compared to the other two substrates studied. DW effluents showed lower COD removal efficiency compared to SW [H-DW – 33.33% and 37.18% (vitamins); M-DW – 35.69% and 36.92% (vitamins)]. Among all the substrates studied CW effluent evidenced poor COD removal efficiency [H-CW – 24.93% and 26.38% (vitamins); M-CW – 19.13% and 19.74% (vitamins)] among all the substrates studied. Complex nature of the CW might be the reason for the poor substrate degradation along with lower H2 production observed during the experimental study. Among all the experimental variations studied, higher SDR was observed with the effluent generated 1776 international journal of hydrogen energy 34 (2009) 1771–1779 a 4 OLR/SDR (Kg COD/m3-day) OLR SDR 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 b 70 COD removal efficiency (%) Experiment Number 60 Acidophilic/Anaerobic Photosynthetic 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 Experiment Number Fig. 4 – (a) Variation in SDR in comparison with OLR during photo-biological process (b) COD removal pattern observed during two stages of experimental studies (acidogenic/methanogenic/photosynthetic). from the acidogenic process rather than the methanogenic process effluents as shown below. H  DW > H  SW > M  DW > H  CW > M  SW > M  CW Among the effluents generated from the acidogenic process, DW showed higher SDR [H-DW – 1.20 Kg COD/m3 day and 1.34 Kg COD/m3 day (vitamins)] followed by SW [H-SW – 0.92 Kg COD/m3 day and 1.05 Kg COD/m3 day (vitamins)]. Likewise in the methanogenic effluents also DW evidenced higher SDR [M-DW – 0.85 Kg COD/m3 day and 0.88 Kg COD/ m3 day (vitamins)] compared to SW [M-SW- 0.67 Kg COD/ m3 day and 0.79 Kg COD/m3 day (vitamins)]. CW documented lower SDR irrespective of the initial process [H-CW – 0.79 Kg COD/m3 day and 0.84 Kg COD/m3 day (vitamins); M-CW – 0.41 Kg COD/m3 day and 0.42 Kg COD/m3 day (vitamins)]. Higher substrate degradation observed in the acidogenic outlets might be attributed to the presence of higher carbon source (residual) even after the first stage of treatment. While, in the case of methanogenic outlets the carbon source might be exhausted in the first stage of treatment and the remaining carbon source might be more complex in nature which cannot be degraded easily. Higher SDR with DW might be attributed to the presence of higher concentration of TVFA during the first stage of treatment. TVFA concentration was relatively low in the case of SW which might be the causative for lower substrate degradation observed. 3.3. Soluble metabolites 3.3.1. Acidogenic/methanogenic processes Composition and distribution of soluble metabolites during H2 generation were often considered as a crucial factor in understanding the metabolic pathway of H2 production process [1,6,9,10]. Therefore, composition of soluble metabolites viz., acetic acid (HAc), butyric acid (HBu) and propionic (HPr) along with ethanol (EtOH) was determined using HPLC during the experimental study (Table 3). Concentration of soluble metabolites and its utilization showed good correlation with H2 production and substrate degradation. Presence of soluble metabolites viz., HAc, HBu, HPr and EtOH was observed 1777 international journal of hydrogen energy 34 (2009) 1771–1779 Table 3 – Variation pattern of soluble metabolites composition during photo-biological H2 production. Inlet composition (%)a,b Expt. No. 1 2 3 4 5 6 7 8 9 10 11 12 Outlet composition (%)b Acetate Butyrate Propionate Ethanol Acetate Butyrate Propionate Ethanol 68.21 71.54 28.66 21.41 14.62 17.77 15.93 10.72 12.10 16.89 18.42 19.21 7.28 1.55 15.78 10.30 16.50 12.08 18.47 16.01 17.92 16.09 17.91 22.84 1.26 1.86 0.94 1.06 21.63 14.89 17.27 33.24 44.64 48.38 41.45 39.31 23.23 25.05 54.61 67.23 47.25 55.26 38.31 40.02 25.34 18.64 22.22 18.64 26.28 31.55 28.78 15.30 3.50 2.08 2.47 2.01 14.02 12.05 18.91 24.64 25.16 29.35 24.67 31.58 15.57 8.89 24.04 5.94 5.36 9.56 9.38 8.68 2.26 2.86 2.94 2.06 47.64 56.88 41.67 58.04 34.54 27.30 36.42 24.51 46.28 36.24 43.61 51.06 33.29 36.15 31.82 34.01 46.08 51.09 35.29 42.16 a Effluent generated from acidogenic/methanogenic process. b Based on mean value (n ¼ 2) S.E varied between 0.2 and 2.9%. in the effluents generated from both the acidogenic and methanogenic processes. The distribution of metabolites varied in accordance with the nature of wastewater and initial process adapted. In the first stage of treatment, H-SW showed a profile with higher HAc concentration (71.54%) followed by the EtOH (25.05%), HBu (7.28%) and HPr (1.86%) which are feasible for effective H2 production. Presence of higher concentrations of HAc indicated feasible environment for acidogenesis. In the case of M-SW, the concentration of EtOH (67.23%) was higher followed by HAc (28.66%), HBu (15.78%) and HPr (0.96%) supporting the possibility of solventogenesis. In the case of DW also the concentration of EtOH was higher (40–55%) followed by HAc, HBu and HPr concentrations almost similar in the range of 14–21% irrespective of the experimental variations studied. Lower concentration of HPr observed in all the experimental variations studied with SW also indicated positive microenvironment towards acidogenic H2 generation. M-DW showed comparatively higher concentration of HPr which is a negative sign with respect to H2 generation. CW evidenced higher concentration of HPr (40–48%) irrespective of the experimental variations which might be attributed to the inhibition of H2 production. While the concentration of other metabolites viz., HAc, HBu and EtOH were more or less similar and varied within a narrow range (14–22%). However, the concentration of EtOH was relatively higher (18–25%). Moreover, higher H2 yields could be observed in association with a mixture of HAc and HBu where as HPr generation will result in consumption or inhibition of H2 production [1,2]. While the production of EtOH strongly supports the metabolic process towards solventogenesis as observed in the case of DW effluents from both the processes along with the methanogenic effluents of SW. Interestingly, in the case of M-CW also, effluents generated from methanogenic process contributed to higher concentration of HAc over the corresponding effluent generated from acidogenic process. However, the concentration of HPr was higher in CW effluents from both the processes compared to SW and DW which supports the least chances of H2 production. Relatively higher concentration of HAc was observed in the effluents generated from acidogenic process compared to the corresponding methanogenic process. 3.3.2. Photo-biological process Utilization of TVFA was observed in most of the experimental variations studied during photo-biological H2 generation and this enumerated their participation as a substrate in the subsequent metabolic process (Fig. 5a). H2 production was observed in all the experimental variations studied except CW and DW effluents generated from methanogenic process. Acidogenic effluents showed relatively good TVFA utilization with all the three feeds studied and this also corroborated well with the corresponding H2 production rate. Among the wastewaters, SW showed higher utilization of soluble TVFA. Relatively low utilization of TVFA was observed with CW. Distribution of soluble metabolites showed marked variation after photo-biological process (Table 3). Irrespective of first stage of treatment used, the pattern of metabolite consumption was almost the same in the subsequent photo-biological process. Utilization of HAc (15–25%) associated with EtOH (35– 50%) production was observed with both acidogenic (H-SW) and methanogenic (M-SW) outlets after photo-fermentation. Utilization of HAc along with formation of EtOH during H2 production indicated the metabolic flow associated with the solventogenesis. Increase in HBu formation (25–30%) associated with lower HPr concentration (2–2.5%) was observed irrespective of the experimental variation indicating a positive sign for photo-biological process. In the case of DW effluent from both processes, almost complete utilization of HAc (2.5%) was noticed with a decreasing EtOH concentration (30– 36%). The formation of HPr (40–58%) was higher compared to all other soluble metabolites in this case. However, HBu concentration was observed to be higher (15–24%) in the absence of vitamins. On the contrary, with CW outlet from both the processes, increase in EtOH (35–50%) was observed along with the stable concentration of HAc (12–24%). Stable HAc concentration observed might be attributed to the transformation of residual substrate (during initial treatment). Consumption of HPr (25–35%) and HBu (5–10%) was observed suggesting the efficacy of photosynthetic culture. The system pH showed gradual increase with time in all the experimental variations studied which might be attributed to the strong reducing phase observed during substrate 1778 international journal of hydrogen energy 34 (2009) 1771–1779 a 900 Inlet Outlet 800 VFA (mg/l) 700 600 500 400 300 200 100 0 1 2 3 4 5 6 7 8 9 10 11 12 Experiment Number b 10 Inlet Outlet 8 pH 6 4 2 0 1 2 3 4 5 6 7 8 9 10 11 12 Experiment Number c 110 Inlet Outlet 85 ORP (mV) 60 35 10 -15 1 2 3 4 5 6 7 8 9 10 11 12 -40 -65 Experiment Number Fig. 5 – Change in (a) TVFA, (b) pH and (c) ORP from inlet to outlet during photo-biological operation. metabolism (Fig. 5b). pH microenvironment is crucial during photo-biological H2 production because of nitrogenase activity which is crucial and functions well between pH range of 6–7 [6,25]. Methanogenic effluents showed higher up shift in pH towards basic microenvironment rather than the corresponding acidogenic effluents. Generally, basic pH does not support H2 production and leads to solventogenesis. The generation of EtOH during the process also corroborated with the observation. 4. Conclusions Experimental results evidenced the possibility of additional H2 production associated with higher substrate degradation by integrating acidogenic and methanogenic processes with photo-biological process. The residual carbon source along with soluble metabolites present in the effluents generated from acidogenic and methanogenic processes were reutilized as primary substrates through subsequent photo-biological process integration (terminal) leading to overall process efficiency based on H2 production and wastewater treatment. However, H2 production and substrate degradation were found to depend on the process used in the first stage and nature and composition of the substrate along with the presence of vitamins. Acidogenic process of treatment appeared to have more positive influence over the corresponding methanogenic process in photo-biological H2 production. This observation could be attributed to the international journal of hydrogen energy 34 (2009) 1771–1779 presence of excessive concentration of soluble metabolites along with higher concentration of residual carbon source. The presence of vitamins showed a positive influence on both H2 production and substrate degradation. The process described in this study documented the possibility of higher rates of H2 generation and substrate removal. This was made possible by reusing the unutilized substrate from acidogenic and methanogenic processes by integrating with photo-biological process. Acknowledgments The authors wish to thank the Department of Biotechnology (DBT), Government of India, New Delhi for funding the presented research study (Project No. BT/PR/4405/BCE/08/312/ 2003). 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