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Page 1: Hydrogen utilization rate: A crucial indicator for anaerobic digestion process evaluation and monitoring

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Journal of Bioscience and BioengineeringVOL. 117 No. 4, 519e523, 2014

Hydrogen utilization rate: A crucial indicator for anaerobic digestionprocess evaluation and monitoring

Yin-ping Hou,1,* Dang-cong Peng,1 Xu-dong Xue,2 Hong-ye Wang,1 and Li-ying Pei1

Department of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, PR China1 and Shaanxi Provincial Academy ofEnvironmental Science, No. 49 Chang’an Road, Xi’an, Shaanxi Province 710061, PR China2

Received 10 June 2013; accepted 6 October 2013Available online 9 November 2013

* CorrespondE-mail a

(Y.-p. Hou).

1389-1723/$http://dx.doi

Hydrogenotrophic methanogens had been considered as key species for the anaerobic digestion (AD) of industrialwastewater and municipal sludge. However, how to evaluate the activity of the hydrogenotrophic methanogens was lessstudied. In this study, a volumetric device and a test procedure were developed for measuring the specific hydrogenutilization rate (HUR) of anaerobic sludge. Results showed that HUR values were highly influenced by sludge concen-trations because of limitation on H2 mass transfer. The critical value of sludge concentration in the test bottle should notbe higher than 1 gVSS/L. Under such condition, the kinetics of HUR would not be limited by H2 mass transfer and themaximal value of HUR could be obtained. Field survey confirmed that HUR exhibits a good relationship with specificmethanogenic activity (SMA) and reactor performance. An anaerobic system with a relatively high HUR was found to bebeneficial for maintaining H2 partial pressure in an appropriately low level. Moreover, such system was thermody-namically favourable for the syntrophic degradation of volatile fatty acids. As a crucial parameter of the anaerobicprocess, HUR could be used as a key indicator for evaluating and monitoring AD processes.

� 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Anaerobic digestion; Hydrogenotrophic methanogens; Hydrogen utilization rate; H2 partial pressure; Specific methanogenic activity]

Anaerobic digestion (AD) is widely recognised as a sustainabletechnology for biological wastewater treatment. During this pro-cess, at least three groups of microbes, including acidogenic andacetogenic bacteria as well as methanogenic archaea, participate inorganic compounds conversion into CO2 and CH4 (1). Acetogenesis,in which intermediates such as propionate and butyrate areoxidized to acetate, is a key step in organic methanogenic conver-sion. In order to keep acetogenesis process thermodynamicallyfeasible, low H2 partial pressure (<10 Pa) has to be maintained(2e7). H2 partial pressure has been considered as an indicativeparameter for regulating acetogenesis and for evaluating anaerobicdigester performance (8). However, studies on full-scale anaerobicsystems have found that H2 determination, combined with ther-modynamic calculations, is not sufficient for providing meaningfulinformation on actual AD systems (9,10). The metabolic potentialsof anaerobic sludge cannot be assumed to be the same, even if theH2 partial pressure of different anaerobic systems is maintained onan equivalent level. Therefore, determining other parametersappropriate for evaluating anaerobic degradability of contaminantsfully is essential.

In AD process, methanogenic archaea, which catalyze the ter-minal stage of the process, are generally divided into two maingroups, i.e., acetoclastic methanogenes (convert acetate into CH4)and hydrogenotrophic methanogenes (convert H2/CO2 into CH4),

ing author. Tel.: þ86 13402987449; fax: þ86 029 82201354.ddresses: [email protected], [email protected]

e see front matter � 2013, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2013.10.006

based on their available substrate. Although acetoclastic meth-anogenes have a major role in CH4 production (approximately 70%of CH4 is formed from acetate), hydrogenotrophic methanogenesalso play a key role in the process (11) by maintaining low partialpressure of H2, which is necessary for the growth of intermediatesyntrophic bacteria (3,12,13). Schmidt and Ahring (14) reported thatthe addition of H2-utilizing methanogenes to disintegrating gran-ules increases the degradation rate of both propionate and buty-rate. Accordingly, the enrichment of active hydrogenotrophicmethanogens will enhance the degradation efficiency of interme-diate volatile fatty acids (VFAs) in an anaerobic system; however,few studies have focused on the development of efficient anaerobicprocesses by enriching hydrogenotrophic methanogens (15,16).Therefore, determining the activity of hydrogenotrophic metha-nogens, which can be used to indicate the regulating capacity of H2partial pressure by this type of methanogens in reactors, is veryessential.

Numerous criteria can be used for evaluatingmicrobial activitiesof different microbial groups in AD process; however, few studieshave focused on determining the activity of hydrogenotrophicmethanogens (13,17). Gijzen et al. (18) proposed a hydro-genotrophic methanogenic activity test that uses formic acid assubstrate. However, the consumption rate of formic acid is unableto reflect H2 utilisation potential of hydrogenotrophicmethanogensdirectly. In addition, the test procedure was not elucidated andfactors that influence the hydrogen utilization rate (HUR) test werenot discussed. Leu et al. (19) developed a kinetic model based onthe double Monod relationship for describing H2/CO2 utilizationrate by the methane-producing archaea FJ10. Although model

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Page 2: Hydrogen utilization rate: A crucial indicator for anaerobic digestion process evaluation and monitoring

TABLE 1. Average operational data of the anaerobic systems in the study period evaluated in the survey.

Treatmentprocess

pH Workingtemperature

(�C)

Flow rate(m3/d)

Reactorvolume(m3)

MLVSS(g/L)

CODinf

(mg/L)CODeff

(mg/L)HRT(h)

Nv (gCOD/(L$ d))

SRT(d)

Lab-scale CSTR (with glucoseas the sole carbon source)

CSTR 7.2 35�1 0.0045 0.0045 12.92 4000 110 24 4.0 20.0

Industrial wastewatertreatment facilitiesin Xi’an

GuoWei starchfactory

UASB1 7.0 35�1 750 1300 20.0 8000 1000 e 1200 41.5 4.62 27.0

Hans brewery UASB2 7.1 2000 820 17.30 1800 500 e 700 10 e 12 4.39 36.0Wan Long paper mill UASB3 7.3 1400 300 13.07 1000 300 15 3.33 /Xi’an Coca-ColaBeverages Co.

UASB4 7.1 400 240 15.97 1200 350 10 e 15 5.0 /

520 HOU ET AL. J. BIOSCI. BIOENG.,

prediction agrees well with the experimental value, determinationand optimization of parameters, as well as the subsequent modelsolution and validation are relatively difficult and time-consumingto conduct. Coates et al. (20) developed an assay method bydetecting manometric change of headspace pressure for measuringhydrogenotrophic methanogenic activity of anaerobic sludge.Although this manometric test is easy to conduct, it is not suffi-ciently accurate for reflecting actual activity of hydrogenotrophicmethanogens because balance between the dissolution and evac-uation of CO2 cannot be controlled. Bicarbonate in the inoculumsmay contribute to surplus of CO2 during the experiment. Moreover,the residual organics in inoculums will also produce excess CO2,thus resulting in changes in the liquidegas equilibrium of CO2 inthe test vials (13,21). Therefore, a simple and more reliable testapproach for hydrogenotrophic methanogenic activity should bedeveloped.

In the present study, a volumetric test device and a test proce-dure were developed for measuring HUR of anaerobic sludge. Theinfluences of HUR on specific methanogenic activity (SMA) andreactor performance were also discussed.

FIG. 1. Schematic diagram of the HUR test device: 1, serum bottle; 2, magnetic mixer;3, gas diffuser; 4, rotor; 5, sealing plug; 6, peristaltic pump; 7, balance gas bag (N2);8, valve; 9, micromanometer. The volumetric experimental device was designed formonitoring HUR of anaerobic system.

MATERIALS AND METHODS

Sludge samples Sludge samples were respectively from five differentanaerobic reactors. A summary of the compiled average operational data for thestudy period is shown in Table 1. To enable the residual matrix to achieve completeconsumption, portions of all sludge samples were elutriated with oxygen-free waterand reacclimatized for 8 he12 h at 35�C prior to HUR and SMA determination.

The experimental HUR test device A new volumetric experimental device,shown in Fig. 1, was designed for monitoring HUR of anaerobic systems. The reactorwas made of glass and had a volume of 1.0 L. A higher height-to-diameter ratio (H/D:3) was selected to maintain highly efficient H2 transmission. Liquid agitation wasachieved by employing a magnetic stirrer and an H2 supplement that uses a gaslift system by recirculating headspace gas through a peristaltic pump. To makepressure within the reactor in equilibrium to atmospheric pressure, amicromanometer was used to monitor gas pressure in the vial headspace andinert gas N2 served as the balance gas. The reactor sealing test was conductedbefore HUR testing.

HUR test procedure Using the device shown in Fig. 1, the detailedexperimental procedures are as follows. Firstly, an appropriate sludge sample witha buffer solution was deposited into the reactor, and liquor pH was maintained atapproximately 7.0. The buffer solution contained 0.2 g NH4Cl, 0.08 g KH2PO4 and2.0 g NaHCO3 per litre of oxygen-free water. The reactor was sealed with gas-tightrubber septa. Secondly, an appropriate volume of pure H2 gas was introduced intothe reactor to replace the supernatant buffer solution. The peristaltic pump wasopened to cycle H2 gas continuously in the reactor. Meanwhile, the magneticstirrer was turned on to launch the test. To balance gas pressure in the reactorheadspace with atmospheric pressure, a U gauge was used to monitor pressurewithin the reactor and inert gas N2 served as a balance gas. Since the test wasinitiated, headspace gas was sampled regularly (once per 0.5 h) and analyzed bygas chromatography (GC). At the end of the HUR test, the amount of biomasspresent in the reactors was quantified in terms of volatile suspended solids (VSS)by ashing the sludge pellet obtained via gravimetric method (22).

HUR calculation The decrease in H2 concentration obtained by GC could beconverted into HUR with a unit of mL-H2/(gVSS$h) using Eq. 1 and a unit of gCOD/(gVSS$d) using Eq. 2, as follows:

HUR’ ¼ dcH2

dt� V � 1

X(1)

HUR ¼ 0:633� 24� HUR’ ¼ 0:633� 24� dcH2

dt� V

X(2)

where dcH2=dt is the H2 consumption rate in the reactor (h�1), V is the headspace

volume (mL), X is the total biomass in the reactor (gVSS) and 0.633 is the conversioncoefficient of hydrogen to oxygen at 35�C.

Gas analyses H2 and CH4 were measured using an Agilent gas chromato-graph (Agilent 6890N GC, Agilent Technologies, CA, USA), equipped with a TDX-01packed column (2 m � 0.3 mm) and a thermal conductivity detector (TCD). Theinert gas argon was selected as the carrier gas at a flow rate of 49.9 mL/min. Thecolumn, injection port and detector temperatures were 100�C, 120�C and 160�C,respectively. The headspace gas in the reactor was sampled using a 500 mL pressure-lock syringe (Unimetrics, CA, USA), followed by direct injection into the columnthrough a septum. The gas volume percentage (Ci) was got from the data-processingsoftware of the GC. H2 partial pressure was calculated according to the followingequation:

pH2¼ 101325� CH2

(3)

SMA test To examine the specific maximum anaerobic uptake rate of diversesubstrates for generating CH4, the SMA test was conducted in 250 mL serum bottlesat 35 � 1�C under anaerobic conditions.

The sludge concentration of the serum bottle was approximately 5 gVSS/L. Ac-etate, propionate and butyrate were used as the substrate for anaerobic microbesgenerating CH4, and the initial concentration was prepared in 4000 mg/L. Prior toaddition into the test bottles, the substrate solution was adjusted to approximatelypH 7.0. CH4 productionwasmeasured at a regular time interval (once every 1 h) after

Page 3: Hydrogen utilization rate: A crucial indicator for anaerobic digestion process evaluation and monitoring

160

170

180

190

200

210

0 0.5 1 1.5 2 2.5

Time (h)

H2vo

lum

e (m

L)

0.5 g/L

0.75 g/L

1.0 g/L

1.5 g/L

2.0 g/L

0

5

10

15

20

25

30

35

40

0.0 0.5 1.0 1.5 2.0 2.5

MLVSS (g/L)

HU

R (

mL

-H2 /

(gV

SS·h

))

A

B

FIG. 2. Effects of sludge concentration on HUR value. To investigate the effect ofbiomass concentration on the HUR test, five sludge concentrations, namely, 0.5, 0.75,1.0, 1.5 and 2.0 gVSS/L were sampled from the lab-scale CSTR reactor to conduct aseries of contrast tests (A), and the results showed that the threshold sludge con-centration was 1 g/L for the maximum HUR test (B).

VOL. 117, 2014 SIGNIFICANCE OF HUR FOR EVALUATING AD PROCESS 521

the test was initiated, and the precise biomass was quantified using the gravimetricmethod (22). Finally, SMA was calculated according to the following equation:

mmax,CH4¼ 1

0:395� dVCH4

dt� 1XV

(4)

where mmax,CH4is the maximum SMA [gCOD/(gVSS$ d)], 1/0.395 is the conversion

coefficient of CH4 to oxygen at 35�C, dVCH4dt is the CH4 production rate (L-CH4/d) and XV

is the total biomass present in the serum bottle (gVSS).

RESULTS AND DISCUSSION

Optimization of assay condition H2 is a poorly soluble gas(Henry’s constant of 7.4 � 10�4 mol/(L$ Pa) at 35�C) (23) andpresents a relatively low mass transfer coefficient. Thus, the H2/CO2 utilization rate of hydrogenotrophic methanogens will belimited by the hydrogen available in the aqueous phase (17,24). Inorder to test the maximum activity of hydrogenotrophicmethanogens, H2 gas has to be transferred to the liquid phase atsuch a rate that its dissolved concentrations do not restrain thekinetics of methanogenesis (25). H2 mass transfer under a steadystate can be expressed by the following equation:

dC=dt ¼ KLa�C* � C

�(5)

where dC/dt is the mass transfer rate, KLa is the total mass transfercoefficient, C* is the saturation concentration of dissolved hydrogenand C is the concentration of dissolved hydrogen.

KLa is the main factor for H2 gas mass transfer rate as indicatedin Eq. 5. Pauss et al. (23) stated that the value of KLa is partlyinfluenced by the volumetric gas supply rate. KLa also depends onthe specific surface area between H2 gas and the liquor phase.

To improve H2 mass transfer rate, a high H/D (3.0) of the reactorand the fine-bubble diffuser was selected. A higher H/D can prolonggas retention time in the liquor. Moreover, the fine-bubble diffusercan make H2 dispersed into water in very fine bubbles, therebyincreasing the area of the liquor/gas interface. Frigon and Guiot (5)indicated that the liquid/gas interface can be increased by biogasrecycling. Accordingly, this previous study employed a 2:1 gas-recycling ratio in the system to enhance mass transfer rate.

The gaseliquid interface area and H2 mass transfer rate aresignificantly affected by sludge concentration. To investigate theeffect of biomass concentration on the HUR test referring to theviewpoint of elsewhere (20), five sludge concentrations, namely,0.5, 0.75, 1.0, 1.5 and 2.0 gVSS/L were sampled from the lab-scaleCSTR reactor to conduct a series of contrast tests. Thereby, theoptimum sludge concentration was obtained.

The decrease in H2 gas volume over time under different sludgeconcentrations is illustrated in Fig. 2A. Considerable H2 gas wasconsumed and high H2 gas reductions were observed with highersludge concentrations. Nevertheless, the HUR value that corre-sponds to high sludge concentration was not the maximum rate.The H2 mass transfer was restricted under high sludge concentra-tion, thereby resulting in an underestimation of the methanogenicactivity on H2. HUR remained unchanged until sludge concentra-tion was less than 1 g/L (Fig. 2B). The experiments were repeatedand the same results were obtained. Therefore, 1 g/L sludge con-centration was identified as the threshold of the maximum HURtest. This finding indicates that if the sludge concentration isgreater than the threshold point, H2 gas transfer will be limited andthe determined HUR value will be underestimated. H2 uptake bybiomass will not be restricted by H2 mass transfer limitation andthe maximum HUR can be reached only when the sludge concen-tration is less than the threshold point.

The KLa value of 4.11 � 2.62 h�1 proposed by Frigon and Guiot(5) was used to calculate the theoretical value of HUR.When sludgeconcentrations were 2.0, 1.5, 1.0, 0.75 and 0.5 g/L, the calculated

values of HUR were 34.52 � 22.0, 46.02 � 29.33, 69.03 � 44.0,92.17 � 58.67 and 138.06 � 88.0 mL-H2/(gVSS$h), respectively. Itcan be seen that the calculated values of HUR were greater than theexperimental values. Hence, the experimental results were reliableand the experimental procedure for the HUR test was technicallyfeasible.

The test vials (600mL) containing 400mL sludge/buffer solutionand the residual headspace was filled with anaerobic H2 gas. All thetested vials followed the same speed of liquid agitation and biogasrecycling ratio. In summary, the experimental results demonstratedthat HUR value highly depended on the sludge concentrations inthe test vessel because of H2 mass transfer limitation. The HURvalue increased with decreasing sludge concentration over the testrange, and the maximum HUR was obtained when the sludgeconcentration decreased to 1.0 gVSS/L. Therefore, the critical sludgeconcentration in the test bottle was identified to 1 g/L. Themaximum HUR could be achieved only when the sludge concen-tration was equal to or less than 1.0 gVSS/L. The test results for thesludge concentrations of 0.5 gVSS/L and 0.75 gVSS/L furtherconfirmed the efficiency of the aforementioned optimal sludgeconcentration.

Relationship between H2 partial pressure andHUR Hydrogenotrophic methanogenic species play a key rolein overall AD process. These species maintain an insignificantly lowH2 partial pressure (<10 Pa) that is thermodynamically favorable

Page 4: Hydrogen utilization rate: A crucial indicator for anaerobic digestion process evaluation and monitoring

0

20

40

60

80

100

CSTR UASB1 UASB2 UASB3 UASB4

H2

part

ial p

ress

ure

(Pa)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

HU

R (

gCO

D/(

gVS

S·d

))

H2 partial pressure HUR

FIG. 3. Relationship between HUR and H2 partial pressure in different AD process. H2

partial pressure will be maintained at a low level if an anaerobic reactor possesses arelatively high sludge HUR; But if the H2 partial pressure always maintained at anextremely low level, then the metabolism and growth of the hydrogenotrophicmethanogens will be inhibited because of the low substrate concentration, thusresulting in a relative low activity of this species.

0

20

40

60

80

100

CSTR UASB1 UASB2 UASB3 UASB4

H2

part

ial p

ress

ure

(Pa)

0.0

0.2

0.4

0.6

0.8

1.0

SMA

-pro

pion

ate

(gC

OD

/(gV

SS·d

)

H2 partial pressure SMA-propionate

A

B

0

20

40

60

80

100

CSTR UASB1 UASB2 UASB3 UASB40.0

0.2

0.4

0.6

0.8

SMA

-but

yrat

e (g

CO

D/(

gVS

S·d

))

H2 partial pressure SMA-butyrate

H2

part

ial p

ress

ure

(Pa)

FIG. 4. Relationships between H2 partial pressure and the SMA based on propionate andbutyrate as the substrates. A clear correlation was found between H2 partial pressureand the degradation rates of propionate and butyrate. The decrease in H2 partialpressure can accelerate the degradation rates of propionate (A) and butyrate (B).

y = 1.2069x + 0.5395

R2 = 0.9232

0.0

1.0

2.0

3.0

4.0

5.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0

HUR (gCOD/(gVSS·d)

TSM

A (g

CO

D/(

gVSS

·d)

FIG. 5. Positive correlation between HUR and TSMA. A linear increase in TSMA wasobserved as HUR test value increased from 0.97 gCOD/(gVSS$ d) to 2.46 gCOD/(gVSS$d). This result suggests that a higher HUR of sludge indicates more benefits for pro-pionate and butyrate degradation; thereby the methanogenic activity of anaerobicmicroorganism will be stimulated.

522 HOU ET AL. J. BIOSCI. BIOENG.,

for syntrophic bacteria responsible for degrading intermediateVFAs, including propionate and butyrate (2,26). In an anaerobicreactor with a relatively high sludge HUR, the H2 partial pressurewill be maintained at a low level. As illustrated in Fig. 3, the H2partial pressure of the system used in the present study wasreduced as HUR increased sequentially from CSTR to UASB1, andthen to ASB2.

However, the data of UASB2, UASB3 and UASB4 in Fig. 3 alsoshows that the lack of H2 partial pressure results in the decrease ofsludge HUR sequentially. This finding is in contrast to theassumption that an anaerobic digester with a lower H2 partialpressure has greater sludge HUR. A possible explanation for thesecontradictory results is that increasing H2 concentration, as anavailable substrate for hydrogenotrophic methanogens, signifi-cantly favours the metabolism of these species. However, if H2partial pressure is alwaysmaintained at an extremely low level for asteady operating system, then the metabolism and growth of thehydrogenotrophic methanogens will be inhibited in a certain de-gree because of the low substrate concentration, thus resulting in arelatively low activity of the species.

Effects of H2 partial pressure on SMA-propionate and SMA-butyrate By monitoring H2 partial pressure in five anaerobicreactors, a clear correlation was found between H2 partial pressureand the degradation rates of propionate and butyrate (Fig. 4). Thedecrease in H2 partial pressure can accelerate the degradationrates of propionate and butyrate. This experimental result is inaccordance with theoretical thermodynamic considerations,which indicate that because the oxidation of propionate andbutyrate has a positive Gibbs free energy (6G�), the degradationof propionate and butyrate is possible only when degradationproducts, particularly H2, are effectively removed by themethanogens.

A relatively low H2 partial pressure can result in higher degra-dation rates for propionate and butyrate, namely, SMA-propionateand SMA-butyrate. Combining with the results of Fig. 3 that H2partial pressure can be maintained at a low level if the sludge HURis relatively high in an anaerobic reactor, then it can be concludedthat the degradation rates of propionate and butyrate can beimproved by improving HUR of sludge to ensure that the anaerobicdigester will operate efficiently.

Relationship between HUR and total specific methanogenicactivity Total specific methanogenic activity (TSMA) is definedas the sum of the specific methanogenic activities against acetate(SMA-acetate) and H2/CO2 (SMA-H2/CO2). A linear increase in TSMA

was observed as HUR test value increased from 0.97 gCOD/(gVSS$d) to 2.46 gCOD/(gVSS$ d) (Fig. 5). This phenomenon can beexplained by the hypothesis that an increase in HUR will reduceH2 concentration in the system, thereby improving intermediateVFAs degradation rates. An improvement in intermediatedegradation will allow AD process to avoid acid accumulation,thus enhancing system stability.

In conclusion, a higher HUR of sludge indicates more benefits forpropionate and butyrate degradation, thus resulting in improve-ments in the methanogenic activity of anaerobic microorganism.

Page 5: Hydrogen utilization rate: A crucial indicator for anaerobic digestion process evaluation and monitoring

VOL. 117, 2014 SIGNIFICANCE OF HUR FOR EVALUATING AD PROCESS 523

HUR can therefore be used as a key parameter for evaluating andmonitoring the performance of anaerobic processes.

ACKNOWLEDGMENT

The study was financially supported by a grant from the Na-tional Natural Science Foundation of China (Grant no. 50878178).

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