Abstract This study offers a novel and quick enrich-ment technology that can be used as a preliminary meth-od to obtain a hydrogen-producing species from the bio-logical sludge produced by wastewater treatment. Theinfluences of acidbase enrichment (by sludge pH ad-justment) on the anaerobic hydrogen-producing micro-organisms were investigated using serum bottle assays.The enrichment pH values were controlled at 3, 4, 5, 7,10, 11 and 12 with 1 N hydrochloric acid and 1 N sodi-um hydroxide. For each enrichment pH, the cultivationpH values were controlled at 5, 6 and 7. Based on the ex-perimental results, hydrogen accumulation from sludgewith acid or base enrichment is higher than that of thecontrol. The hydrogen-production potential of the sludgewith acid or base enrichment is 200 and 333 times en-hanced, compared with the control, when the enrichmentpH is 10 and 3, respectively. The enhancement is due to a shortening of the micro-organisms lag-time which occurs at a proper cultivation-pH level.

Introduction

Anaerobic digestion of organic waste produces methanethat can be used as an energy source. However, methaneand its combustion product, carbon dioxide, are bothgreenhouse gases. This leads to focusing more efforts onthe development of clean energy sources. Hydrogen is apromising energy alternative and can be produced in theacidogenesis process during anaerobic degradation of or-ganics (Christopher 1996; Sparling et al. 1997).

Clostridium is an important anaerobic hydrogen-pro-ducing micro-organism (Kataoka et al. 1997; Reimann etal. 1996). Clostridia are Gram-positive, spore-forming,rod-shaped bacteria. Endospores might be very resistant

to heat or harmful chemicals including acid and base andcannot be destroyed easily (Brock et al. 1994). In the ac-idogenic phase of anaerobically digesting organic wastessuch as sewage sludge, hydrogen gas is produced. In ananaerobic digester, hydrogen-utilizing methanogenicbacteria are present and will consume the hydrogen pro-duced. At pH values lower than 6.3 or higher than 7.8,the methanogenesis rate decreases or stops (Van Haandeland Lettinga 1994). Consequently, using a low or highpH environment to prevent hydrogen reduction and toobtain dominant microbes for hydrogen production fromsludge seems to be a feasible method.

This study aimed to investigate the efficiencies of acidbase enrichment (by adjusting sludge pH) and tofind a suitable incubation pH level (by adjusting sub-strate pH) in the cultivation of anaerobic hydrogen-pro-ducing micro-organisms.

Materials and methods

Seed sludge

The sludge was obtained from the sludge-drying bed at the Li-Ming municipal sewage treatment plant (Taichung, Taiwan). Thecollected sludge was screened with a No. 8 mesh (diam. 2.35 mm).The pH, volatile suspended solids (VSS, to express the biomassconcentrations) and total solids (TS) concentrations of the seedsludge were pH 6.81, 33,280 mg l1and 65,130 mg l1, respectively.

Medium composition

The substrate glucose concentration was 20,000 mg chemical oxy-gen demand (COD) l1. The substrate contained sufficient inorga-nics (Endo et al. 1982; mg l1): 5,240 NH4HCO3, 125 K2HPO4,100 MgCl26H2O, 15 MnSO46H2O, 25 FeSO47H2O, 5 CuSO45H2O, 125 CoCl25H2O and 6,720 NaHCO3.

Experimental procedure

Enrichment

pH adjustment was conducted with 1 N hydrochloric acid and 1 Nsodium hydroxide. Six enrichment values were designed at pH 3,

C.-C. Chen () C.-Y. Lin M.-C. LinGraduate Institute of Civil and Hydraulic Engineering, Feng Chia University, P.O. Box 25-123, Taichung, Taiwan 407, Republic of Chinae-mail: p8600082@knight.fcu.edu.twFax: +886-4-24519746

Appl Microbiol Biotechnol (2002) 58:224228DOI 10.1007/s002530100814

O R I G I N A L PA P E R

Chin-Chao Chen Chiu-Yue Lin Min-Cheng Lin

Acidbase enrichment enhances anaerobic hydrogen production process

Received: 10 June 2001 / Received revision: 22 July 2001 / Accepted: 17 August 2001 / Published online: 30 November 2001 Springer-Verlag 2001

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4, 5, 10, 11 and 12. A blank without pH adjustment was also pre-pared. After pH adjustment, the seed sludge, including the blank,was incubated in the dark at 351 C for 24 h.

Cultivation

For each enrichment pH value, the enriched seed sludge was culti-vated at pH values of 5, 6 and 7. During cultivation the hydrogenproduction was monitored. These hydrogen-production experi-ments were performed in serum vials with a working volume of100 ml, using a batch test. The vials were initially gassed with ar-gon and then the pH-enriched seed sludge (35 ml) and substrate(35 ml) were added. The vials were placed in a reciprocal water-bath shaker (Reciprocation: 3.0 cm150 strokes min1), with thetemperature controlled at 351 C. The contents were drawn out at6, 12, 18, 24, 36, 48 and 72 h, using a syringe to determine thevolatile fatty acids (VFA) content and concentration. The total gasproduction and its composition were determined to measure thehydrogen production. Each experimental condition was carried outin triplicate.

Analyses

Hydrogen gas and liquid VFA were determined with a Shimadzu(Japan) GC-14A gas chromatograph equipped either with a ther-mal conductivity detector (stainless column at 55 C, injectiontemperature 90 C, Ar as carrier gas, with Porapak Q packing,Shimadzu C-R3A Chromatopac integrator) or a flame ionizationdetector (glass column at 145 C, injection temperature 175 C, N2as carrier gas, with FON packing, Shimadzu C-R6A Chromatopacintegrator). VSS and TS were measured according to the proce-dures in the APHA standard methods (APHA 1995).

Results

Acid enrichment (pH 35)

In the acid enrichment, the enrichment values were pH 3,4 and 5. As an example, Fig 1 shows the time course ofhydrogen production in the acid enrichment (pH 3). Incultivation at pH 6 and 7, a marked hydrogen productionwas observed. However, no hydrogen production wasdetermined for the blank and for cultivation at pH 5.These results indicated that cultivation pH affected hy-drogen production. Table 1 lists the hydrogen evolutionsafter 72 h incubation and the ratio of hydrogen evolutionto that of the blank after acid and base enrichments atvarious levels of cultivation pH. The ratios ranged over0.3333 and acid enrichment produced higher ratios. Forthe experiments with acid or base enrichment, most of

them produced no methane. Only the cultivations withenrichment at pH 5 and pH 12 had methane production(1.2 ml, 13.5 ml, respectively) when cultivated at pH 7.However, these amounts were rather small, comparedwith that of the blank (72 ml). These results revealedthat, with the acid and base enrichments, the methaneevolution was significantly inhibited.

As Table 1 reveals, for acid enrichment (except forcultivation at pH 5), the hydrogen evolution after 72 hdigestion at various levels of enrichment pH was in theorder: pH 3>pH 4>pH 5>blank. This result reveals thatthe hydrogen production of acid enrichment was higherthan that of the blank. Moreover, the effect of cultivationpH on hydrogen evolution was pH 7>pH 6>pH 5. ThepH 7 cultivation enhanced the micro-organisms to pro-duce hydrogen gas from 0.24 ml (blank) to 80, 58.2 and33.9 ml at enrichments pH 3, 4 and 5, respectively. Thisimplies that pH 7 was the best cultivation value of thetested pH range. The hydrogen production potential ofacid enrichment (compared to the blank) was enhancedfrom 0.3 times (at enrichment pH 3, cultivation pH 5) to333 times (at enrichment pH 3, cultivation pH 7).

Base enrichment (pH 1012)Comparing the hydrogen evolution values of each en-richment pH at each cultivation pH (Table 1), it is shownthat the cultivation pH effects on hydrogen production

Table 1 Hydrogen evolution(ml) from acid or base enrich-ment, after 72 h incubation.Data are given as meanSD,n=3, with (ratio of hydrogenevolution to that of the blank)in parentheses

Cultivation pH Enrichment pH

3 4 5 Blank (6.81) 10 11 12Blank (8.45) 0.240.02 5 0.060.01 0 11.61.0 0 4.00.3 0.110.01 0.90.08

(0.3) (48) (17) (0.5) (4)6 62.16.5 55.85.3 35.03.4 12.11.2 47.94.5 45.54.3 28.62.5

(259) (233) (146) (50) (200) (190) (119)7 80.07.8 58.25.4 33.93.1 13.31.1 46.34.3 9.60.8 19.71.7

(333) (243) (141) (55) (193) (40) (82)

Fig. 1 Hydrogen evolution in the acid enrichment experiment (en-richment at pH 3). mL Millilitres

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was in the order: pH 7=pH 6>pH 5. This implies thatpH 7 and pH 6 were the best cultivation levels for en-hancing hydrogen production. The pH 7 cultivation levelenhanced the hydrogen production by micro-organismsfrom 0.24 ml (blank) to 46.3, 9.6 and 19.7 ml at enrich-ment pH 10, 11 and 12, respectively. The hydrogen pro-duction potential for base enrichment was enhanced0.5 times (at enrichment pH 11, cultivation pH 5) to200 times (at enrichment pH 10, cultivation pH 6), com-pared with the blank (Table 1).

VFA concentrations after enrichment

Table 2 lists the acid and base enrichment vial contentcharacteristics. With no enrichment, the intermediate an-aerobic digestion products were acetic (HAc), propionic(HPr), butyric (HBu) and valeric (HVa) acids and theirconcentrations ranged over 7102,690 mg COD l1. HBuand HVa were major component (about 35% for each).In base or acid enrichment, the intermediate productswere HAc, HPr and HBu, with HBu as the major compo-nent (7085%). This indicates that, after enrichmenttreatment, the HBu concentrations increased markedly.Figure 2 shows the HBu concentrations after 72 h diges-tion. The HBu production was observed to be cultivationpH-dependent. Cultivation at pH 6 and pH 7 produced

higher HBu concentrations. These VFAs might resultfrom the decrease in pH within the vials. For cultivation,the pH was adjusted before starting the cultivation. Thefinal pH values after 72 h incubation ranged over4.04.8, 4.64.9 and 5.96.6 for cultivation at pH 5, 6and 7, respectively.

Discussion

Enhancement of hydrogen production after enrichment

Table 1 reveals that, in the acid and base enrichments,the hydrogen evolution was dependent on both enhance-ment pH and cultivation pH. This might be because, dur-ing the enrichments, the hydrogen-utilizing methanogenswere killed or inhibited, but clostridia were not. The bac-terial endospore has a complex, multilayered structureand differs structurally from the vegetative cell. Theseendospores are very resistant to heat, drying, radiation,acids and chemical disinfectants; and they cannot be de-stroyed easily, even by harsh chemicals (Brock et al.1994). The control of pH is fundamental to the mainte-nance of optimal bacterial growth and/or conversion pro-cesses in anaerobic microbial systems (Stronach et al.1986). Some investigators have obtained hydrogen pro-duction with pure cultures of clostridia at pH rangesof 6.07.0 (Kataoka et al. 1997; Reimann et al. 1996;Taguchi et al. 1995). Therefore, at the proper cultivation-pH (pH 67), the clostridia were alive and produced alarge amount of hydrogen. Based on the experimental re-sults, by properly adjusting the cultivation-pH value, theoptimum conditions for acidogenesis and hydrogen pro-duction during the anaerobic degradation of organicsmay be obtained.

Hydrogen production rate of the enrichments

A hydrogen production rate (HPR) exhibits the hydro-gen production ability of biomass and can be used tocompare the hydrogen production efficiency of the acidand base enrichments. Figure 3 shows the HPR for acidand base enrichments at various levels of cultivation pH.The ranking of HPR after 72 h digestion, at various levels of enrichment pH, was in the order: acid enrich-ment (pH 35) > base enrichment (pH 1012) > blank(pH 6.81). Moreover, the cultivation-pH effect on HPRwas in the order: pH 7>pH 6>pH 5 for acid enrichmentand pH 6>pH 7>pH 5 for base enrichment. The highestHPR values were 1.6 ml g VSS h1 for acid enrichment(pH 3, cultivation pH 7) and 0.730.75 ml g VSS h1 forbase enrichment (pH 1012, cultivation pH 6). The dif-ferent HPR values might suggest that they involve differ-ent sludge micro-organisms after acid or base enrich-ment. These HPR values are comparable to the reportedhydrogen production rates of 0.116.8 ml g VSS h1from cultivated sludge (Lay et al. 1999).

Table 2 Characteristics of acidbase enrichment vial contents (mgchemical oxygen demand l1). Data are given as meanSD, n=3.HAc Acetic acid, HBu normal butyric acid, HPr propionic acid,HVa normal valeric acid

HAc HPr HBu HVa HBu/HAc

Blank (cultivation pH 7)71078 1560159 2690264 2630225 3.78

Acid enrichment (enrichment pH 3, cultivation pH 7)53035 51058 5250375 0 9.88

Base enrichment (enrichment pH 10, cultivation pH 6)68065 75061 4400398 0 6.46

Fig. 2 Butyric acid concentration of acidbase enrichment (blank,pH 6.81). COD chemical oxygen demand, n-HBu normal butyricacid

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Variation of VFA concentrations during enrichment

The formation of hydrogen is accompanied with VFA orsolvent production during an anaerobic digestion pro-cess. Therefore, the distribution of VFA concentrationsand their fractions is a useful indicator for monitoringhydrogen production.

The data in Figs. 2 and 3 show that, generally, theHPR markedly increased at high HBu concentrations.The high HBu concentrations reveal that the reactionwas a butyrate fermentation type. Clostridium speciesare, therefore, considered to be the dominant organismsin the vial, because these organisms are responsible forbutyrate fermentation (Dinopoulou et al. 1988; Yokoi etal. 1997). The HPR (Fig. 3) and the HBu concentrations(Fig.2) show that their levels were generally correlated.This result is consistent with the biochemical pathway ofthe anaerobic degradation of organics.

The roles of acetate and butyrate in the anaerobicdegradation of organics have been described. The avail-able hydrogen from glucose degradation during fermen-tation has been determined, using the butyrate/acetate ra-tio (Nandi and Sengupta 1998; Ueno et al. 1995). C. bu-tyricum is reported to form butyrate and acetate at a con-centration ratio of about 2:1 (White 1995). The buty-rate/acetate concentration ratio has been reported to be0.75 for Butyribacterium methylotrophicum (Annous etal. 1996). In this paper, we also observed a relationshipbetween acetate and butyrate. Table 2 reveals that theHBu/HAc ratio was 3.78 with no pH enrichment, butwas 9.88 and 6.46 in acid and base enrichments, respec-tively. This indicates that, for higher HBu/HAc ratios, ahigher HPR value was obtained.

However, in the metabolic pathways of C. acetobu-tylicum, there was no HPr formation when the micro-organisms anaerobically degraded glucose (Girbal et al.1995; White 1995). The differences between our resultsand former reports result from pure culture versus mixedculture. The micro-organisms in mixed culture had somesymbiotic nature or syntrophic interactions that producedHPr. Another speculation is that, after acid or base en-richment, the surviving or dominant micro-organisms aremicro-organisms other than C. acetobutylicum. More in-

depth investigations on the identification of the micro-organisms, using denaturing-gradient gel electrophoreticanalysis of PCR-amplified genes coding for 16S rRNAgene might solve this problem.

Kinetic analysis

The modified Gompertz equation (Eq. 1) has been usedto describe the progress of cumulative hydrogen produc-tion obtained from a batch experiment (Lay et al. 1999;Lee et al. 1999; Onodera et al. 1999). Using the cumula-tive hydrogen production data obtained from batch ex-periments to fit the modified Gompertz equation, the re-sults are listed in Table 3. The correlation coefficient val-ues ranged over0.9771.0. This indicates that the modi-fied Gompertz equation could also be used to estimatethe hydrogen production potential, maximum hydrogenproduction rate and lag-phase time.

(1)H(t) is the cumulative hydrogen production (ml), P is thehydrogen production potential (ml), Rm is the maximumhydrogen production rate (ml h1), e is 2.71828..., isthe lag-phase time (h) and t is time (h).

Table 3 reveals that the maximum hydrogen produc-tion potential (P) was 81.6 ml (at enrichment pH 3, culti-vation pH 7) and 47.6 ml (at enrichment pH 10, cultiva-tion pH 6) at acid and base enrichment, respectively.These calculated values are consistent with our experi-mental data (80 ml, 47.9 ml; Table 1).

For acid enrichment of pH 3, 4 and 5, it was observedthat, when the P value was high, both the Rm and val-ues were low. However, for base enrichment of pH 10,11 and 12, when the P value was high, the Rm value was

Fig. 3 Hydrogen production rate of acidbase enrichment (blank,pH 6.81). g VSS/h Grams of volatile suspended solids per hour

Table 3 Modified Gompertz equation parameters (see text for de-tails). Data insufficient for simulationEnrichment Cultivation P (ml) Rm (ml h1) (h) RpH pH

3 5 6 61.5 5.1 5.8 0.9997 81.6 3.2 1.9 0.992

4 5 6 56.1 5.1 3.9 0.9997 60.3 2.2 0.6 0.977

5 5 11.8 0.7 10.0 0.9996 35.1 2.4 2.3 1.07 34.1 2.7 2.7 1.0

10 5 3.9 0.9 15.6 1.06 47.6 3.7 3.6 0.9987 47.0 2.1 3.4 0.998

11 5 6 45.1 2.8 3.1 0.9917 39.1 2.4 3.1 0.993

12 5 0.8 0.1 12.4 0.9846 28.4 2.4 5.6 0.9957 20.0 1.0 1.3 0.996

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high, but the value was medium. These results mightsuggest that the sludge micro-organisms possess a rangeof responsive capacities for different adverse circum-stances. Thus, they can display a certain characteristic,such as P, Rm and , or even others, i.e. the difference inhydrogen production (acid enrichment > base enrich-ment, as listed in Table 1) and the difference in VFA pro-duction (as Table 2 shows).

For enrichment at pH 3, where the hydrogen produc-tion enhancement was the most obvious, the at cultiva-tion pH 6 was higher than that at pH 7 (5.8 h, 1.9 h, re-spectively). Because of the value, it is known that un-der the condition of cultivation at pH 6, the micro-organ-isms took more time to modify their physiological stateto adapt to a new environment (Onodera et al. 1999).This indicates that a proper cultivation-pH level couldshorten the lag-phase time and be useful in acclimatinganaerobic micro-organisms for producing hydrogen. Forenrichment pH 4 at cultivation pH 7, a small negative value was observed. This might result from the fact that,in this environmental condition, the micro-organisms didnot have an obvious lag-phase time.

Acknowledgements The authors would like to thank the NationalScience Council of the Republic of China for financially support-ing this manuscript under Contract No. NSC 88-2211-E-035-020.Part of this paper was presented at Biotechnology 2000, the World Congress on Biotechnology, 38 Sept. 2000, in Berlin, Germany.

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