Utilization of Lactic Acid Rich Medium and Cassava Starch Wastewater for Biological Hydrogen Production
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International Journal of GEOMATE, Aug., 2020, Vol.19, Issue 72, pp. 173 - 179 ISSN: 2186-2982 (P), 2186-2990 (O), Japan, DOI: https://doi.org/10.21660/2020.72.5675 Special Issue on Science, Engineering and Environment UTILIZATION OF LACTIC ACID RICH MEDIUM AND CASSAVA STARCH WASTEWATER FOR BIOLOGICAL HYDROGEN PRODUCTION Piyawadee Saraphirom1, Nonthaphong Phonphuak2, Napapach Chainamom1, Piyarat Namsena1, and *Mullika Teerakun3 1Faculty of Science and Technology, Rajabhat Maha Sarakham University, Thailand; 2Faculty of Engineering, Rajabhat Mahasarakham University, Thailand; 3Faculty of Agricultural Technology, Kalasin University, Thailand *Corresponding Author, Received: 06 July 2019, Revised: 13 Sept. 2019, Accepted: 28 Feb. 2020 ABSTRACT: H2 production from lactic acid rich medium and H2 production from cassava starch wastewater by isolated bacteria were investigated. The isolation of H2 producing bacteria from heat treated sludge was carried out. The H2 production from lactic acid rich medium which used 1.5 g/L sodium acetate, 1.5 g/L sodium lactate and 1 g/L sucrose as carbon sources. The effects of inoculums size of 10, 20, 30 and 40 % (v/v) and incubation temperature of 35, 45 and 55ºC using cassava starch wastewater as the substrate on H2 production by isolation strains were investigated. Results showed 6 isolated strains i.e. isolation code of MK01to MK06 indicated the H2 production capability when cultured in lactic acid rich medium. However, 3 isolated strains i.e. MK01, MK04, and MK06 were preferred to use as seed inoculums for further experiments depend on a high H2 production capacity obtained. Results indicated that the optimal condition of H2 production from cassava starch wastewater was 30% (v/v) inoculum size of MK06 under 35ºC of incubation temperature. The maximum H2 production potential, H2 yield and H2 production rate of 4.20 L H2/L medium, 1.47 L H2/g substrate consumed, and 14.00 mL H2/L/h was obtained. Identification and characterization of MK06 were investigated using 16S rRNA gene sequence analysis followed by phylogenetic analysis. The 100% similarity of 16S rDNA compare with closely related species was indicated Klebsiella pneumoniae subsp. pneumoniae DSM 30104(T). Keywords: Hydrogen production, Lactic acid rich medium, Cassava starch wastewater 1. INTRODUCTION fermentative H2 production, the presence of lactic acid as a metabolite during H2 production is H2 producing bacteria such Clostridiaceae can frequently regarded as a sign of lower H2 production ferment the carbohydrates in agricultural [5]-[7]. Lactic acid has been rarely studied as wastewater to H2, CO2, and volatile organic acids – substrate for H2 production and despite reports of mainly acetic and butyric acids. However, these CH4 production from lactate [7], no considerable H2 bacteria are not able to further break down the production was reported when lactate was used as organic acids to H2 due to the positive Gibbs free the individual substrate [2]. energy of the reaction [1]. The genus Clostridium Although the previous report on H2 production i.e. Clostridium butyricum, Clostridium diolis, and from mixture of lactic acid and acetic acid by C. beijerinckii have been reported their ability to Clostridium diolis showed the promise on efficient produce hydrogen from a mixture of acetic acid and H2 production process in batch scale but the lactic acid [2]. A mole ratio of depletion of acetic information on the H2 production from lactic acid acid to lactic acid of 1:2 could be obtained from the rich wastewater are still lacking. Therefore, this biological H2 production from a mixture of acetic research aims to isolate H2 producing bacteria and lactic acids by C. diolis JPCC H-3 [2]. Cassava which capable to use lactic acid as substrate for H2 starch wastewater is an industrial residue composed production. The effects of inoculum size and mainly of lactic acid bacteria with excess of the incubation temperature on H2 production genera Lactobacillus, and organic acids [3]. The parameters were investigated. Means of H2 cassava starch wastewater is excluded, and it is production parameters which indicated the high returned an effluent with high chemical oxygen efficiency of H2 producing strain were selected to demand [4]. However, this residue presents a produce H2 from cassava starch wastewater. The motivating component for prospective use in results from this study provided high efficiency of innovative products. The cassava starch wastewater hydrogen producing strain and the optimum has a low pH and it is an organic acid rich condition could improve hydrogen yield from lactic wastewater [4]. The major organic acids include acid rich wastewater. lactic acid, acetic acid, butyric acid, and other components include starch residual. In dark 173 International Journal of GEOMATE, Aug., 2020, Vol.19, Issue 72, pp. 173 - 179 capacity was enriched in lactic acid rich medium for 2. MATERIALS AND METHODS 48 h under anaerobic condition before used as seed inoculums. The cassava starch wastewater used as 2.1 Anaerobic Sludge sole substrate for H2 production consisted of 2.78 g/L starch, 4.59 g/L volatile fatty acid i.e. 0.89 g/L Sludge of an up flow anaerobic sludge blanket acetic acid, 1.33 g/L lactic acid, 1.09 g/L propionic (UASB) was pretreated by the dry heating method acid and 1.28 g/L butyric acid. The variation under 105°C for 2 h for suppressing the methanogen. inoculums size of 10, 20, 30 and 40 % (v/v) was After heating, the UASB sludge was dispersed into adjusted then the effect of incubation temperature at an aqueous suspension by vortexing it in PBS buffer 35, 45 and 55 ºC on H2 production from cassava (pH 7.2, 10 mM Na2HPO4, and 0.13% NaCl) for 5 starch wastewater by isolated bacteria was min. The suspension was diluted serially, plated on investigated. The difference inoculums size was NB medium (pH 6.8, 0.3% beef extract, and 0.5% inoculated into cassava starch wastewater in 25 mL peptone) and incubated at 30°C under anaerobic serum flasks with a liquid volume of 70 mL under conditions. After incubating for 24-36 h, colonies adjusted initial pH of 5.5. Serum flasks were were selected randomly and transferred to fresh NB flushed with N2 to remove O2 in the headspace and plates. to create an anaerobic condition. The bottles were incubated at room temperature and operated in an 2.2 H2 Production Confirmation orbital shaker with a rotation speed of 120 rpm to provide better contact among substrates. After 24 h, Each isolated bacterium obtained was used as the total gas volume was measured by releasing the seed inoculums for the H2 production confirmation pressure in the bottles using wetted glass syringe. experiment. The experiment was accompanied in 25 The 3 mL of biogas produced was collected into mL serum flasks with a working volume of 15 mL. another serum bottle then measured the H2 The H2 production medium was contained 10% concentration by gas chromatography analysis. (v/v) inoculum. The H2 production capability of isolated bacteria was performed by cultured in 2.5 Analytical Method and Kinetics Model synthetic medium (M solution) [8]. Serum flasks were flushed with nitrogen gas to eliminate oxygen H2 content was measured using a gas in headspace and to generate the anaerobic chromatograph under the same analytical condition condition then incubated at room temperature. After as [9]. Helium was used as the carrier gas at a flow 24 h, the total gas volume was measured by rate of 25 mL/min. H2 production was calculated releasing the pressure in serum flasks using the from the headspace measurement of gas wetted glass syringe. composition and the total volume of H2 produced, at each time interval, using the mass balance 2.3 H2 Production from Lactic Acid Rich equation [9]. Medium by Isolated Bacteria VH,i= VH,i-1+CH,i(VG,i - VG,i-1)+VH,0 (CH,i - CH,i-1) Isolated colonies indicated the H2 production capacity obtained from 2.2 were cultured in a Where VH,i and VH,i-1 are the cumulative synthetic medium (M solution) [8]. H2 production hydrogen gas volumes at the current (i) and previous ability of all isolated bacteria was conducted in the time interval (i-1), respectively; VG,i and VG,i-1 are same size of serum flasks with a liquid volume of total biogas volume at the current and previous time 70 mL and 50 mL of headspace volume. The H2 interval; CH,i and CH,i-1 are the fraction of hydrogen production capability of isolated bacteria was gas in the headspace at the current and previous accomplished by cultured in lactic acid rich medium time interval; VH is the volume of the headspace of (M solution contained sodium acetate, 1.5 g/L serum bottles. A modified Gompertz equation was sodium lactate and 1 g/L sucrose as carbon sources). used for adequate H2 production profiles [9]. Total The 10% (v/v) inoculum was inoculated into 120 sugar, volatile fatty acid (VFAs) and dry cell weight mL serum flasks then incubated at room ( DW) are measured according to the procedures temperature and activated in an orbital shaker with described in standard methods [10]. a rotation speed of 120 rpm. The biogas volume was continuously measured and collected by gas-tight H=Pexp{-exp[(Rme/P)( λ-t)+1]} syringe for biogas composition analysis. Where H represents the cumulative volume of 2.4 H2 Production from Cassava Starch hydrogen produced (mL), Ps the hydrogen Wastewater by Isolated Bacteria production potential (mL), Rm the maximum production rate (mL/h), λ the lag-phase time (h), t Isolated strains showed the high H2 production the incubation time (h), and is 2.718281828.