Journal of Water and Environment Technology, Vol.3, No.1, 2005 INFLUENCE OF NITROGEN, ACETATE AND PROPIONATE ON HYDROGEN PRODUCTION FROM PINEAPPLE WASTE EXTRACT BY Rhodospirillum rubrum Piyawadee Ruknongsaeng*, Alissara Reungsang**, Samars Moonamart*** and Paiboon Danvirutai** *Graduate College, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: [email protected] **Fermentation for Value Added of Agricultural Products, Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: [email protected]; Correspondence author *** Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: [email protected] **Fermentation for Value Added of Agricultural Products, Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: [email protected] ABSTRACT This research examined the influence of nitrogen, acetate and propionate on hydrogen production from pineapple waste extract by photosynthetic bacteria strain Rhodospirillum rubrum in batch culture. The fermentation conditions used in this study were continuous illumination fermentation (24 hours of light) and periodic illumination fermentation (alternate 12 hours of light and dark). Two levels of total nitrogen (3 mM-low level and 11 mM-high level) with various initial concentrations of acetate or propionate (5, 10 and 20 mM) were added into the production medium. Results indicated that levels of total nitrogen did not affect the production of hydrogen. Neither acetate nor propionate was used as carbon source by R. rubrum but glucose contained in pineapple waste extract was used. Periodic illuminated fermentation was more effective in producing hydrogen than continuous illuminated fermentation. The maximum hydrogen production potential (337 ml), specific hydrogen production rate (11 ml/l/h), specific hydrogen production potential (247.75 ml H2/g COD) and hydrogen yield (122 ml H2/g glucose consumed) occurred upon addition of high level of total nitrogen (11 mM) with 5 mM initial concentrations of acetate under periodic illumination and a working volume of 40 ml. Results indicated that pineapple waste extract could be effectively used as substrate for hydrogen production by R. rubrum without any carbon and nitrogen sources. - 93 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 KEYWORDS: acetate, propionate, hydrogen production, Rhodospirillum rubrum, pineapple waste extract INTRODUCTION Hydrogen is a clean fuel and an environmentally safe energy source. After hydrogen combustion, only water is formed and is exhausted to the atmosphere without causing any air pollution (Emtiazi et al., 2001). Hydrogen can be produced chemically (e.g. gasification of coal), electrochemically (e.g. electrolysis of water) or by the use of microorganisms (Takabatake et al., 2004). The two main systems of microbial hydrogen production are photochemical and fermentative systems. Photochemical system consists of photosynthetic microorganisms such as algae and photosynthetic bacteria (Ike et al., 1997; Melis and Happe, 2001). Fermentative system, on the other hand, is carried out by facultative anaerobes and obligate anaerobes (Joyner and Winter, 1977; Nandi and Segupta, 1998). Among these microorganisms, photosynthetic bacteria had been widely studied as a candidate for hydrogen production because of their ability to convert light energy to hydrogen through photosynthesis (Takabatake et al., 2004). The purple photosynthetic bacterium Rhodospirillum rubrum has the ability to anaerobically produce hydrogen from different kinds of carbon sources such as ethanol, acetate, fructose and most intermediates of the tricarboxylic acid cycle (Pfenning and Trüper, 1974). R. rubrum uses light (photon) to produce hydrogen and maintains energy for growth and metabolism from organic acids (Najafpour et al., 2004). Effective substrates for hydrogen production by R. rubrum include malate (Arik, 1996), fumalate (Sasikala et al., 1995), oxaloacetate (Sasikala et al., 1993) pyruvate (Gorrell and Uffen, 1977), acetate (Mao et al., 1986) and succinate (Klasson et al., 1993). Zürrer and Bachofen (1979) studied hydrogen production by R. rubrum in batch culture using pure lactate or lactic acid-containing waste and the results showed that hydrogen was produced at an average of 6 ml/h per g (dry weight). Aside from organic acids, R. rubrum was able to use dextrose as a substrate with the yield of 6 mol H2/mol dextrose (Weetall et al., 1981). Production of hydrogen by R. rubrum was affected by the addition of nitrogen source. Weetall et al. (1981) reported that an atem inhibited the activity of nitrogenase, the enzyme which mainly catalyzes hydrogen production (Jones and Monty, 1979; Gest et al., 1950). One of the factors determining the feasibility of photohydrogen production process is light illumination pattern. Oh et al. (2004) reported that the hydrogen production by Rhodopseudomonas palustris P4 in dark fermentation was twice lower than combined dark-light fermentation. Various kinds of hydrogen production from renewable organic acids such as acidogenic wastewater (Takabatake et al., 2004), lactic acid fermentation plant wastewater (Sasikala et al., 1991), tofu wastewater (Zhu et al., 1999), sugar industry wastewater (Lee et al., 2002), and dairy industry wastewater (Türkarslan et al., 1998) had been used to produce hydrogen by photosynthetic bacteria. However, information on hydrogen production from pineapple waste extract is limited. - 94 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Pineapple waste consists of the residual peels and cores from pineapple fruit processing industries. Pineapple waste extract contains sugars, organic acids and other substances that can be utilized as substrate in hydrogen production by R. rubrum. In this study, we investigated the possibility to produce hydrogen from pineapple waste extract by R. rubrum under different light illumination patterns. The main objective of this study was to examine the influence of nitrogen, acetate and propionate on hydrogen production from pineapple waste extract by R. rubrum. MATERIALS AND METHODS Microorganisms Rhodospirillum rubrum ATCC 11170 was purchased from the DSMZ–Deutsche Sammlung van Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. The seed culture was prepared by cultivating R. rubrum at 30 oC in a 50-mL sealed stopper serum bottle using the modified ATCC medium: 112 Van Niel’s yeast (pH 7.0). The medium contained the following components per liter: 1 g of K2HPO4, 0.5 of MgSO4, 10 g of yeast extract, 15 mM L-malic acid (C-source) and 10 mM L-glutamic acid (N-source). Squeezed juice of pineapple waste Pineapple waste consisting of residual peels and cores were obtained from the fruit shops on Khon Kaen University campus. Pineapple wastes were squeezed by a presser to extract the juice, then filtered through a thin cloth and kept in the freezer at - 17 ºC until usage. Frozen extract was thawed in a refrigerator at 4oC and centrifuged at 7,000 rpm for 10 minutes to separate the solid matter prior to usage as medium for hydrogen production. Hydrogen production medium Forty milliliters of pineapple waste extract (pH 6.5-7.0) was transferred to a 50-ml serum bottle, capped with a rubber stopper and wrapped 2-3 times with parafilm to prevent gas from leaking. A bottle was flushed with argon gas for 1 min to create anaerobic condition and autoclaved at 110 ºC for 10 min to avoid browning reaction. After sterilization, sodium acetate or sodium propionate was added into pineapple waste extract to obtain final acetate or propionate concentration of 5, 10 and 20 mM. For each concentration of acetate or propionate, ammonium sulfate ((NH4)2SO4) was added to obtain total nitrogen concentrations of 3 and 11 mM. Fermentation Ten percent (v/v) inoculum of the seed culture at an optimum concentration of 2 x 105 cells/ml was injected into the hydrogen production media using No. 24 x 1” sterile needle and 5 ml sterile plastic syringe. Three replicates of serum bottles were incubated at 30 ºC under continuous illumination (24 hours of light) or periodic illumination (12 hours of dark condition alternated with 12 hours of light condition). - 95 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Light intensity was 6,000 lux from cool white fluorescent lamps and measured by a lux meter (Phillips, Japan). Analytical methods The cell concentration of the culture media was determined by cell optical density at 520 nm using Shimadzu UV-1601 spectrophotometer with the modified ATCC medium: 112 Van Niel,s yeast (pH 7.0) as blank. The number of cells at each sample was determined by plate count technique. Glucose concentration in pineapple waste extract was determined by Somogyi-Nelson method using Shimadzu UV-1601 spectrophotometer at 620 nm optical density. The COD of pineapple waste extract was determined by a closed reflux titrimetric method (APHA AWWA and WPCE, 1995). During the experiments, the gas evolved was measured volumetrically by water displacement in a burette and the volume was calculated using the mass balance equation (Zheng and Yu, 2005). Gas samples were taken from the headspace of each serum bottles by a gas-tight syringe. The biogas composition was analyzed by a gas chromatograph (Shimadzu GC-17A) equipped with a thermal conductivity detector (TCD) and 2 m stainless column packed with 5A molecular sieve (Morimoto et al., 2004). The temperature of injector, column and detector were kept at 100, 40 and 100 ºC, respectively. Argon was used as carrier gas at a flow rate of 10 ml/min. Concentrations of acetate, propionate and butyrate in samples were also analyzed by gas chromatography (Shimadzu GC-14A) equipped with flame ionization detector (FID) and integrator (CR-4A). A 2m x 2mm stainless steel, 80/120 mesh 4% carbowax 20 M (Supelco, USA) was used. The oven temperature was maintained at 180 ºC. The injector and detector temperatures were 200 and 250 ºC, respectively. The carrier gas was nitrogen; with the flow rate set at 20 ml/min. Prior to analysis, 1.5 ml of pineapple waste extract was centrifuged at 8,000 rpm for 10 min.