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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.
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: piyawadee_bally@yahoo.com **Fermentation for Value Added of Agricultural Products, Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: alissara@kku.ac.th; Correspondence author *** Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: samars@kku.ac.th **Fermentation for Value Added of Agricultural Products, Department of Biotechnology, Khon Kaen University A.Muang, Khon Kaen 40002 THAILAND E-mail: paiboon@kku.ac.th 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 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 ml/h per g (dry weight) Aside from organic acids, R rubrum was able to use dextrose as a substrate with the yield of 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: 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 to create anaerobic condition and autoclaved at 110 ºC for 10 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 and 11 mM Fermentation Ten percent (v/v) inoculum of the seed culture at an optimum concentration of x 105 cells/ml was injected into the hydrogen production media using No 24 x 1” sterile needle and 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 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 The supernatant was recovered and fixed with 200 µl of 0.2N oxalic acid One milliliter of the prepared sample was injected to analyze the organic acid (acetate, propionate and butyrate) concentrations Data analysis Volume of biogas produced was calculated using the mass balance equation (Zheng and Yu, 2005) as follows: VH,i = VH,i-1 + CH,i (VG,i - VG,i-1) + VH (CH,i - CH,i-1) ……… … (1) where VH,i and VH,i-1 are the cumulative hydrogen gas volumes at the current (i) and previous time interval (i-1), respectively; VG,i and VG,i-1 are total biogas volume at the current and previous time interval; CH,i and CH,i-1 are the fraction of hydrogen gas in the headspace at the current and previous time interval; VH is the volume of headspace of serum bottles (20 ml) - 96 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 A modified Gompertz equation (Eq 2) was used to plot the cumulative hydrogen production curves and obtain the hydrogen production potential (P), the hydrogen production rate (R) and lag phase (λ) H = P exp - exp Rm*e (λ - t) + P ………………………… …………(2) In equation 2, H represents the cumulative volume of hydrogen produced (ml), P the hydrogen production potential (ml), Rm the maximum production rate (ml/h), λ the lagphase time (h), t the incubation time (h), and e the exp (1) = 2.718 Parameters (P, Rm and λ) were estimated using the solver function in Microsoft Excel version 5.0 (Microsoft, Inc.) as explained by Khanal et al (2004) The specific hydrogen production potential, Ps (ml/g Chemical Oxygen Demand-COD), was obtained by dividing P by COD of substrate applied, while the specific hydrogen production rate, Rms (ml/l/h), was calculated by dividing Rm by the volume of medium (40 ml) The expression for percent substrate conversion efficiency was calculated using the conversion efficiency equation (Koku, et al., 2002) as follows: % Conversion efficiency = (Actual hydrogen/Theoretical hydrogen) x 100 (3) RESULTS AND DISCUSSION Composition of pineapple waste extract Composition of pineapple waste extract revealed that reducing sugar (glucose) was the main organic substance indicating that pineapple waste extract is a potential substrate for hydrogen production (Table 1) In theory, glucose can be converted to hydrogen by anaerobic bacteria yielding and mol of hydrogen per glucose when butyrate (Eq 4) and acetate (Eq 5) are the by-products, respectively (Hawkes et al., 2002) C6H12O6 C6H12O6 + 2H2O CH3CH2CH2COOH + 2CO2 + 2H2 ………………(4) 2CH3COOH + 2CO2 + 4H2 ……………… (5) Previous researchers demonstrated a successful hydrogen production from sucrose and sugar derivatives Huss et al.(2004) reported that the hydrogen production from sucrose and pulped sugar beet by anaerobic digester sludge from a sewage biosolid mesophilic digester were 1.0 ± 0.1 and 0.90 ± 0.2 mol/mol hexose converted, respectively Fang and Liu (2002) found that the hydrogen production by the seed sludge mixed culture from glucose was 64% with a yield of 2.1 ± 0.1 mol H2/mol glucose In addition, sugar substance such as pure sucrose (Sung et al., 2002), sugar wastewater (Ueno et al., 1996) and diluted molasses (Rune et al., 1995) could be utililized to produce hydrogen - 97 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table Composition of pineapple waste extract Composition Reducing sugar (mg/l) (assumed as glucose) COD (mg/l) Acetate (mM) Propionate (mM) Butyrate (mM) Lactate (mM) Total nitrogen (mM) Values 63,600 34,000 5.28 1.92 5.33 22.45 1.70 In Table 1, it is clearly seen that lactic acid is the most abundant organic acid in pineapple waste extract Other organic acids contained are acetate (5.28 mM), propionate (1.92 mM), and butyrate (5.33 mM) This composition including acetate (Barbosa et al., 2001), malate (Oh et al., 2004), lactate and butyrate (Mao et al., 1986) had been reported to support hydrogen production by photosynthetic bacteria Effect of nitrogen levels and initial acetate concentrations Continuous illumination Upon investigation of the effect of nitrogen levels and initial acetate concentrations on hydrogen production under continuous illumination, results indicated that hydrogen production potentials (P) at low level (3 mM) of nitrogen were significantly lower than at high level (11 mM) of nitrogen (Table 2) This was in contrast with the findings of Kim et al (1980), Oh et al (2004), and Vijaraghavan and Soom (n.d.) who reported that the presence of high nitrogen concentration strongly inhibited hydrogen production by photosynthetic bacteria These microorganisms produce hydrogen by the catalytic activity of nitrogenase, which is inhibited by nitrogen (at hydrogen production only) The results suggested that the hydrogen produced in this study would not have resulted from the activity of nitrogenase but from hydrogenase This was because hydrogenase of photosynthetic bacteria is capable of both hydrogen production and consumption (Koku et al., 2002) It is not inhibited by the presence of nitrogen sources but by carbon monoxide, oxygen and high concentrations of organic compounds (Fissler et al., 1994; Gogotov, 1986) Maness and Weaver (2001) reported that R rubrum contains distinct hydrogenase enzymes viz., (i) uptake hydrogenase which mediates hydrogen uptake during photoautotrophic growth; (ii) formate-linked hydrogenase which is synthesized during growth under low light availability and links formate oxidation to hydrogen evolution; and (iii) carbon monoxide-linked hydrogenase which is synthesized in the presence of CO and couples CO oxidation with hydrogen evolution Therefore, it was concluded that in this study, R rubrum used a formate-linked hydrogenase to produce hydrogen - 98 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table Kinetic parameters for hydrogen production from pineapple waste extract added with various initial acetate concentrations at continuous light illumination Total N conc (mM) Initial HAc* conc (mM) λ** Rm** Rms** P** Ps** H2 yield** (h) (ml/h) (ml/l/h) (ml) ml H2/ g COD ml H2/ g glucose consumed 0.2a 4.3d a,b,c 10 0.4 4.2d d 20 0.8 3.8a b,c,d 11 0.7 4.7c e 11 10 2.2 6.6a d 11 20 0.9 5.3b a,b,c control 5.2 0.5 3.5e *Acetate **Comparison among treatments in different small letters 107.2d 105.0e 94.5f 117.5c 166.2a 132.7b 87.5g 121f 177d 149e 218b 211c 278a 179d 89.0f 130.2d 109.5e 160.2b 155.2c 204.5a 131.4d 44.2g 64.7d 54.5e 79.7b 77.2c 101.7a 52.6f Conversion efficiency** R2 (%) 5.93g 8.67d 7.30e 10.68b 10.35c 13.63a 7.04f 0.97 0.99 0.98 0.99 0.98 0.97 column are significantly different (Duncan, p ≤ 0.05) if mark with At each nitrogen level, hydrogen production was not very much affected by initial concentrations of acetate (Table 2) However, accumulation of hydrogen was greater at high level of nitrogen than at low level (Table 2) This may be due to the fact that the R rubrum cell counts at high level of nitrogen were larger than at low level (Table 3) Glucose, in pineapple waste extract might be consumed to produce hydrogen and resulted to butyrate and acetate production (Figures 1, 2) Figures and illustrated a reduction of glucose concentration and an increase of acetate and butyrate during hydrogen production at each nitrogen level and various initial acetate concentrations There was no decrease in either acetate or butyrate concentration even for long term hydrogen production (Figures and 2) confirming that glucose was utilized to produce hydrogen and consequently resulted to the accumulation of acetate and butyrate Glucose can be consumed as a substrate for hydrogen production by fermentative bacteria yielding and mols of hydrogen per glucose when butyrate and acetate were the by-products, respectively (Hawkes et al., 2002) Fang and Liu, (2002) reported that biogas produced from glucose comprised of 64 ± 2% hydrogen with a yield of 2.1 ± 0.1 mol H2/mol glucose R rubrum was reported to have the capability to use reducing sugar, i.e dextrose, as substrate for hydrogen production yielding mol H2/mol dextrose (Weetall et al., 1981) Hydrogen production rapidly increased within 36 h (Figures 1, 2) and then gradually increased After 60 h, the hydrogen production was stable (Figures and 2) This might be due to the fact that during the acidogenesis period, VFAs (acetate and butyrate) were produced (Figures 1, 2) The VFAs can inhibit hydrogen production depending on their concentrations (Stewart, 1975) A low level of VFA might have no effect on hydrogen production but a high level of VFA could inhibit hydrogen production by photosynthetic bacteria (Zheng and Yu, 2005) High VFA concentrations result to a drop of pH which inhibits the growth of hydrogen producing bacteria (Fan et al., 2005) The pH should be controlled by the addition of alkali to support the hydrogen production during hydrogen fermentation (Hawkes et al., 2002) - 99 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table shows that ammonium sulfate added in the production medium was used to support the growth of R rubrum during the hydrogen production as indicated by the greater number of cells at high level of nitrogen than at low level A drop in the final pH (3.5 to 4.5) indicated that VFA was produced Table Number of R rubrum cells grown in hydrogen production medium added with acetate under continuous illumination Total N conc (mM) 3 11 11 11 control Initial acetate conc (mM) 10 20 10 20 5.2 Final pH 4.2 4.5 3.7 3.7 3.8 3.5 3.8 Maximum number of cells (cells/ml) 2.4x108 2.5x108 2.5x108 3.0x108 3.5x108 3.5x108 1.4x108 Periodic illumination The effect of nitrogen levels and acetate concentrations on hydrogen production under periodic illumination was conducted At each level of total nitrogen, the hydrogen production potential (P) under periodic illumination was found to be significantly higher (Table 4) than continuous illumination (Table 2) In addition, it was clearly seen that hydrogen production yields (88.3 to 122 ml H2/g glucose consumed) under periodic illumination (Table 4, Figure and 4) were significantly higher than hydrogen production yields (44.2 to 101.7 ml H2/g glucose consumed) under continuous illumination (Table 2) These results implied that hydrogen production under periodic illumination was more effective than the continuous illumination and it might be possible to increase hydrogen production yield by R rubrum in a reactor with a sequential batch mode (dark- and light- fermentation) These findings were supported by the work of Oh et al (2004) who found that a hydrogen production by Rhodopseudomonas palustris P4 from combined dark-light fermentation was twice higher than just dark fermentation - 100 - 40 20 200 90 150 60 100 0 2000 1000 30 50 3000 150 4000 120 250 VFAs (mM) 60 4000 mM acetate 300 H2 accumulation (ml) glucose (g/l) 80 150 350 100 Number of cell (10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108 156 204 60 40 20 300 120 250 200 90 150 60 100 0 0 2000 1000 30 50 3000 10 mM acetate VFAs (mM) 80 350 H2 accumulation (ml) glucose (g/l) 100 Number of cells (x10 cells/ml) time (h) 12 18 24 36 48 60 84 108 156 204 40 20 300 120 250 200 90 150 60 100 30 50 0 VFAs (mM) 60 H2 accumulation (ml) glucose (g/l) 80 4000 20 mM acetate 3000 2000 1000 350 100 Number of cells (x10 cells/ml) time (h) 12 18 24 36 48 60 84 108 156 204 time (h) Figure Effect of low level of total nitrogen (3 mM) and initial concentrations of acetate on hydrogen production by R rubrum under continuous illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 101 - 60 40 20 300 120 250 200 90 150 3000 60 2000 100 1000 30 50 0 150 0 4000 4000 80 150 VFAs (mM) H2 accumulation (ml) glucose (g/l) mM acetate 350 100 Number of cells (X10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108 156 204 60 40 H2 accumulation (ml) glucose (g/l) 80 20 300 120 250 200 90 150 3000 60 2000 100 1000 30 50 0 150 0 350 VFAs (mM) 10 mM acetate 100 Number of cells (10 cells/ml) Time (h) 4000 12 18 24 36 48 60 84 108 156 204 60 40 20 H2 accumulation (ml) glucose (g/l) 80 300 120 250 200 90 150 350 VFAs (mM) 20 mM acetate 100 3000 60 2000 100 30 0 1000 50 Number of cells (x10 cells/ml) Time (h) 12 18 24 36 48 60 84 108 156 204 Time (h) Figure Effect of high level of total nitrogen (11 mM) and initial concentrations of acetate on hydrogen production R rubrum under continuous illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 102 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table Kinetic parameters for hydrogen production from pineapple waste extract added with various initial acetate concentrations at periodic light illumination Total N conc (mM) Initial HAc* conc (mM) λ** Rm** Rms** P** Ps** H2 yield** (h) (ml/h) (ml/l/h) (ml) (ml H2/ g COD) (ml H2/ g glucose consumed) 3.5d 11.0a 275a 271e 184.7e 98.2e b,c a,b b b b 10 0.7 10.5 262.5 309 227.2 112b b,c b c c c 20 9.5 237.5 302 222 109.2c 0.6 a c 7d a a 11 0.2 7.9 196 337 247.7 122a b,c c e d d 11 10 0.7 7.2 287 211 104d 179.2 a,b c e c c 11 20 0.4 7.2 179.5 303 222.7 109.7c c d f c c control 0.8 137.5 300 220.8 88.3f 5.5 *Acetate **Comparison among treatments in column are significantly different (Duncan, different small letters R2 Conversion efficiency** (%) 13.2c 15b 14.6b 16.3a 13.9b,c 14.7b 11.8d 0.98 0.97 0.97 0.96 0.97 0.97 0.97 p ≤ 0.05) if mark with Table suggests that ammonium sulfate added in the production medium was used to support the growth of R rubrum during the hydrogen production This is indicated by the slightly higher number of cells that resulted in the production medium added with nitrogen than in the control (no nitrogen added) Table Number of R rubrum cells grown in hydrogen production medium added with acetate under periodic illumination Total N conc (mM) 3 11 11 11 control Initial acetate conc (mM) 10 20 10 20 5.2 Final pH 4.1 4.8 3.5 3.7 3.7 3.6 3.6 - 103 - Maximum number of cells (cells/ml) 3.0x108 3.3x108 2.9x108 3.2x108 3.5x108 3.1x108 1.2x108 Journal of Water and Environment Technology, Vol.3, No.1, 2005 mM acetate 60 40 20 120 250 200 90 150 60 100 2000 30 0 1000 50 3000 300 4000 VFAs (mM) H2 accumulation (ml) glucose (g/l) 80 150 Number of cells (x10 cells/ml) 350 100 12 18 24 36 48 60 84 108 156 204 Time (h) 40 20 120 250 200 90 150 60 100 3000 2000 1000 30 0 150 4000 300 50 4000 150 VFAs (mM) 60 H2 accumulation (ml) glucose (g/l) 80 350 Number of cells (10 cells/ml) 10 mM acetate 100 12 18 24 36 48 60 84 108 156 204 Time (h) 40 20 120 250 200 90 150 3000 60 100 30 50 0 2000 1000 300 Number of cells (10 cellsml) 60 H2 accumulation (ml) glucose (g/l) 80 350 VFAs (mM) 20 mM acetate 100 12 18 24 36 48 60 84 108 156 204 Time (h) Figure Effect of low level of total nitrogen (3 mM) and initial concentrations of acetate on hydrogen production R rubrum under periodic illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 104 - mM acetate 60 40 20 200 90 150 120 250 3000 60 2000 100 1000 30 50 0 4000 150 300 H2 accumulation (ml) glucose (g/l) 80 350 VFAs (mM) 100 0 Number of cells (x10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108 156 204 10 mM acetate 60 40 20 120 300 3000 250 200 90 150 60 100 2000 1000 30 50 4000 H2 accumulation (ml) glucose (g/l) 80 150 350 VFAs (mM) 100 0 150 Number of cells (x10 cells/ml) Time (h) 4000 12 18 24 36 48 60 84 108 156 204 300 60 40 20 H2 accumulation (ml) glucose (g/l) 80 120 250 200 90 150 3000 60 2000 100 30 50 0 1000 350 VFAs (mM) 20 mM acetate 100 Number of cells (x 10 cells/ml) Time (h) 12 18 24 36 48 60 84 108 156 204 Time (h) Figure Effect of high level of total nitrogen (11 mM) and initial concentrations of acetate on hydrogen production R rubrum under periodic illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 105 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Effect of nitrogen levels and initial propionate concentrations Continuous illumination The effect of nitrogen levels and initial propionate concentrations on hydrogen production from pineapple waste extract by R rubrum under continuous-illumination was examined Table shows that the hydrogen production at low level of nitrogen (3mM of total nitrogen) was higher than at high level of nitrogen (11 mM of total nitrogen) Control (no N added) had a lower number of cells than the production media containing nitrogen (Table 7) However, at each level of nitrogen, greatest hydrogen production resulted at initial propionate concentration of 10 mM (Table 6) suggesting that the optimum initial concentration of propionate used by R rubrum to produce hydrogen under continuous illumination was 10 mM The effect of initial concentrations of propionate at low level (3mM) and high level (11mM) of total nitrogen under continuous illumination fermentation on hydrogen production are shown in Figures and 6, respectively Results revealed that propionate added into pineapple waste extract was not used to produce hydrogen by R rubrum due to the fact that at the end of incubation (at 204 hrs), propionate concentrations did not change but glucose concentration decreased Consequently, hydrogen accumulated while acetate and butyrate concentrations increased (Figure 2) In this present study, it was confirmed that R rubrum used glucose in extract to produce hydrogen that consequently resulted to the accumulation of acetate and butyrate Our findings contradict with the work of Phansomboon (1998) who reported that in continuous hydrogen production from propionate, acetate and lactate under anaerobic-light conditions by Rhodopseudomonas sphaeroides 3701, the highest yield of hydrogen was obtained when propionate (2,416 ml H2/g dry weight) was used as a carbon source The author noted that propionate was consumed by Rhodopseudomonas sphaeroides 3701 to produce hydrogen at a slower rate than acetate and lactate Sybesma (1970) and Gest et al (1962) found that propionate could be used as electron donor for hydrogen production by photosynthetic bacteria Lag time of hydrogen production when propionate (Table 6) was added into the media was longer than when acetate was added (Tables 2, 4) This was in agreement with Phansomboon (1998) who found that propionate was consumed by Rhodopseudomonas sphaeroides 3701 to produce hydrogen at a slower rate than acetate and lactate - 106 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table Kinetic parameters for hydrogen production from pineapple waste extract added with various initial propionate concentrations at continuous light illumination Total N conc (mM) Initial HPr* conc (mM) λ** Rm** Rms** P** Ps** H2 yield** (h) (ml/h) (ml/l/h) (ml) (ml H2/ g COD) (ml H2/ g glucose consumed) 3.9a,b,c 5a 2.6c 4.2a,b 3.2b,c 4.2a,b 3.5b,c 97.5c 125.2a 66.2f 105e 80e 105e 87.5d 2.5d 10 0.8c 20 0.1a 11 7.3f 11 10 0.1a 11 20 3.8e control 1.2 0.5b * Propionate **Comparison among different small letters 181c 225a 125d 111e 194b 125d 179c 133c 165.5a 92d 81.5e 142.2b 92d 131.4c 66.2c 82.5a 45.7e 40.7f 71b 45.7e 52.6d Conversion efficiency** R2 (%) 8.9c 11a 6.1e 5.5f 9.5b 6.1e 7d 0.98 0.97 0.98 0.99 0.97 0.99 0.97 treatments in column are significantly different (Duncan, p ≤ 0.05) if mark with Table Number of R rubrum cells grown in hydrogen production medium added with propionate under continuous illumination Total N conc (mM) Initial propionate conc (mM) Final pH 3 11 11 11 control 10 20 10 20 1.2 3.5 3.6 3.7 3.5 3.5 3.6 3.8 - 107 - Maximum number of cells (cells/ml) 2.5x108 3.1x108 3.8x108 3.0x108 3.6x108 4.0x108 1.2x108 60 40 20 mM propionate 300 120 250 200 150 4000 3000 90 60 2000 100 1000 30 50 150 0 150 4000 H2 accumulation (ml) glucose (g/l) 80 350 Number of cells (x10 cells/m) 100 VFAs (mM) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108156 204 90 150 100 40 20 0 150 4000 12 18 24 36 48 60 84 108156 204 Time (h) 300 20 mM propionate H2 accumulation (ml) glucose (g/l) 60 1000 30 50 80 2000 60 100 3000 250 120 200 90 200 3000 2000 150 60 100 30 50 0 Number of cells (x10 cells/ml) 20 H2 accumulation (ml) glucose (g/l) 40 120 VFAs (mM) 250 80 60 10 mM propionate 300 VFAs (mM) 100 Number of cells (x10 cells/ml) Time (h) 1000 12 18 24 36 48 60 84 108156 204 Time (h) Figure Effect of low level of nitrogen (3 mM) and initial concentrations of propionate on hydrogen production R rubrum under continuous illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 108 - mM propionate 60 40 120 200 90 60 100 1000 30 50 3000 2000 150 20 250 4000 150 VFAs (mM) 80 300 H2 accumulation (ml) glucose (g/l) 100 Number of cells (x10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 0 300 150 4000 250 120 12 18 24 36 48 60 84 108 156 204 60 40 20 200 90 150 3000 2000 60 100 1000 30 50 VFAs (mM) glucose (g/l) 80 H2 accumulation (ml) 100 10 mM propionate 0 0 Number of cells (x10 cells/ml) Time (h) 12 18 24 36 48 60 84 108 156 204 Time (h) 20 mM propionate 40 20 250 120 200 90 150 4000 3000 2000 60 100 30 50 0 1000 propionate Number of cells (x10 cell/ml) 60 H2 accumulation (ml) glucose (g/l) 80 150 VFAs (mM) 300 100 12 18 24 36 48 60 84 108 156 204 Time (h) Figure Effect of high level of nitrogen (11 mM) and initial concentrations of propionate on hydrogen production R rubrum under continuous illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 109 - Journal of Water and Environment Technology, Vol.3, No.1, 2005 Periodic illumination Under periodic illumination, the effect of nitrogen levels and initial propionate concentrations on hydrogen production from pineapple waste extract by R rubrum was investigated Results showed that nitrogen levels and initial concentrations of propionate significantly affected hydrogen production under periodic illumination The highest yield of hydrogen was found at high level of nitrogen (11 mM) and initial propionate concentration of 20 mM (Table 8) In addition, it was found out that the pattern of light had strong effect on hydrogen production when cultures were exposed to periodic illumination as shown in Tables and 8; and Figures and Accordingly, hydrogen yields under periodic illumination were 88.3 to 116.2 ml H2/g glucose consumed (Table 8) and higher than under continuous illumination, which were 40.7 to 82.5 ml H2/g glucose consumed (Table 6) These results suggested that an alternate light and dark cycle was more effective in producing hydrogen by R rubrum than a continuous illumination Koku et al., (2003) explained that total hydrogen gas was more produced in the dark-light cycle due to higher concentration of cells Mayer et al, (1978) elaborated that the dark-light cycle had positive effects on nitrogenase activity High number of R rubrum cell counts was obtained at high total nitrogen concentration (11 mM) and at all initial propionate concentrations (Table 9) indicating that nitrogen was used for cells growth It was worth noting that butyrate was produced during hydrogen fermentation process (Figure 1-6) suggesting that R rubrum converted glucose in pineapple waste extract to butyrate However, high concentration of butyrate was found to inhibit hydrogen production and pigment production of Rhodopseudomonas sphaeroides 3701 (Pansoomboon, 1988) Table Kinetic parameters for hydrogen production from pineapple waste extract added with various initial propionate concentrations at periodic light illumination Total N conc (mM) Initial HPr* conc (mM) λ** Rm** Rms** P** Ps** H2 yield** (h) (ml/h) (ml/l/h) (ml) (ml H2/ g COD) (ml H2/ g glucose consumed) 0.5c 7.3c 182.5d 305b 224.2b 110.5b b c c d d 10 0.2 7.4 185.0 279 205.2 101c a d e b,c b,c 20 0.05 6.7 167.5 303 222.7 109.7b f a a f f 11 10.4 260.5 159.5 78.5f 217 c c d e e 11 10 0.5 7.3 182.5 252 185.2 91.2d e b b a a 11 20 8.1 202.7 322 236.7 116.2a d e f c c control 1.2 0.8 5.5 137.5 300 220.8 88.3e * Propionate **Comparison among treatments in column are significantly different (Duncan, p different small letters - 110 - Conversion efficiency* * R2 (%) 14.8b 13.5c 14.7b 10.4f 12.2d 15.6a 11.8e 0.95 0.96 0.96 0.98 0.97 0.96 0.97 ≤ 0.05) if mark with Journal of Water and Environment Technology, Vol.3, No.1, 2005 Table Number of R rubrum cells grown in hydrogen production medium added with propionate under periodic illumination Total N conc (mM) Initial propionate conc (mM) Final pH 3 11 11 11 control 10 20 10 20 1.2 4.1 3.6 3.5 4.1 3.9 3.7 3.6 Maximum number of cells (cells/ml) 2.1x108 1.5x108 1.2x108 1.4x109 2.4x109 2.1x109 1.25108 CONCLUSIONS The conclusions drawn from the study are as follows: (1) At each level of nitrogen, neither acetate nor propionate was used as carbon source to produce hydrogen by R rubrum from pineapple waste extract The results suggested that glucose in extract was used as carbon source instead (2) Light pattern affected the production of hydrogen from pineapple waste extract by R rubrum in which periodic illumination was more effective in producing hydrogen than continuous illumination (3) Pineapple waste extract could be effectively used as substrate for hydrogen production by R rubrum without any carbon and nitrogen sources ACKNOWLEDGEMENT Financial support from the Department of Alternative Energy Conservation Promotion Fund and the National Research Council is very much appreciated - 111 - 40 20 120 250 200 90 150 4000 VFAs (mM) 60 300 H2 accumulation (ml) glucose (g/l) 80 150 350 3000 60 2000 100 1000 30 50 150 4000 5 mM propionate 100 Number of cells (x10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108 156 204 40 20 120 250 200 90 150 60 100 0 3000 2000 1000 30 50 0 VFAs (mM) 60 300 H2 accumulation (ml) glucose (g/l) 80 350 10 mM propionate 100 Number of cells (x10 cells/ml) Time (h) 12 18 24 36 48 60 84 108 156 204 40 20 300 120 250 200 90 150 VFAs (mM) 60 H2 accumulation (ml) glucose (g/l) 80 4000 150 350 3000 60 2000 100 30 0 12 18 24 36 48 60 84 108 156 204 1000 50 20 mM propionate 100 Number of cells (x10 cells/ml) Time (h) Time (h) Figure Effect of low level of nitrogen (3 mM) and initial concentrations of propionate on hydrogen production R rubrum under periodic illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 112 - 60 40 20 150 300 120 250 200 90 150 3000 60 2000 100 1000 30 50 0 4000 H2 accumulation (ml) glucose (g/l) 80 350 VFAs (mM) mM propionate 100 Number of cells (x10 cells/ml) Journal of Water and Environment Technology, Vol.3, No.1, 2005 12 18 24 36 48 60 84 108 156 204 10 mM propionate 60 40 20 120 250 200 90 150 4000 150 300 H2 accumulation (ml) glucose (g/l) 80 350 VFAs (mM) 100 3000 60 100 50 2000 1000 30 0 150 Number of cells (x10 cells/ml) Time (h) 4000 12 18 24 36 48 60 84 108 156 204 60 40 20 120 250 200 90 150 300 H2 accumulation (ml) glucose (g/l) 80 350 VFAs (mM) 20 mM propionate 100 3000 60 2000 100 30 50 0 12 18 24 36 48 60 84 108 156 204 Number of cells (x10 cells/ml) Time (h) 1000 Time (h) Figure Effect of high level of nitrogen (11 mM) and initial concentrations of propionate on hydrogen production R rubrum under periodic illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and number of cells of R rubrum ( ) - 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117 - ... initial propionate concentrations on hydrogen production from pineapple waste extract by R rubrum was investigated Results showed that nitrogen levels and initial concentrations of propionate significantly... mM) and initial concentrations of acetate on hydrogen production by R rubrum under continuous illumination Symbols: Acetate ( ), propionate ( ), butyrate ( ), H2 accumulation ( ), glucose ( ) and. .. concentrations on hydrogen production from pineapple waste extract by R rubrum under continuous-illumination was examined Table shows that the hydrogen production at low level of nitrogen (3mM of total