b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e8

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Anaerobic acidification of sugar-beet processing wastes: Effectof operational parameters

Emrah Alkaya, Goksel N. Demirer*

Department of Environmental Engineering, Middle East Technical University, Inonu Bulvari, 06531 Ankara, Turkey

a r t i c l e i n f o

Article history:

Received 20 January 2010

Received in revised form

2 August 2010

Accepted 2 August 2010

Available online xxx

Keywords:

Anaerobic digestion

Sugar industry

Acidification

Volatile fatty acids

* Corresponding author. Tel.: þ90 312 210 58E-mail address: goksel@metu.edu.tr (G.N

Please cite this article in press as: Alkayoperational parameters, Biomass and Bio

0961-9534/$ e see front matter ª 2010 Elsevdoi:10.1016/j.biombioe.2010.08.002

a b s t r a c t

The objective of this study was to maximize the hydrolysis and acidification of sugar-beet

processing wastewater and beet pulp for volatile fatty acid (VFA) production through

acidogenic anaerobic metabolism. Experiments were conducted to determine the optimum

operational conditions (HRT, waste-mixing ratio and pH) for effective acidification in daily-

fed, continuously mixed anaerobic reactors. For this purpose, reactors were operated at

35 � 1 �C with different combinations of HRT (2e4 days), wastewater-pulp mixing ratios

(1:0e1:1, in terms of COD) and pH ranges (5.7e7.5). Increased OLRs, resulting from pulp

addition, increased the amount of acidification products (VFAs) which led to relatively low

operational pH values (5.7e6.8). In this pH range, methanogenic activity was successfully

inhibited and the lowest methane percentages (5.6e16.3%) were observed in the produced

biogas. The optimum operational conditions were determined to be 2-day HRT and 1:1

waste mixing ratio (in terms of COD) without external alkalinity addition. These opera-

tional conditions led to the highest tVFA concentration (3635 � 209 mg/L as H-Ac) with the

acidification degree of 46.9 � 2.1%.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction systems to treat many kinds of solid, industrial and agro-

Anaerobic digestion is an established technology, used for the

treatment of wide variety of organic wastes throughout the

decades. It is one of the several biological processing strate-

gies which produce bioenergy and/or biochemicals while

treating industrial and agricultural wastes [1]. Anaerobic

biodegradation can be separated into two phases in order to

enhance treatment efficiencies and/or produce bio-products.

Two-phase anaerobic systems have been extensively

studied and numerous advantages of phase separation over

conventional anaerobic digestion have been described/

demonstrated in numerous studies [2e8]. Some of these

advantages include, increased process stability and control,

smaller reactor volumes and high tolerance to toxicity and

shock loads. These advantages enable the two-phase anaerobic

67; fax: þ90 312 210 26 4. Demirer).

a E, Demirer GN, Anaerenergy (2010), doi:10.10

ier Ltd. All rights reserved

industrial wastes such as distillery, landfill leachate, coffee,

cheesewhey, dairy, starch, fruit, vegetable solid, food, pulp and

paper, olive mill, abattoir, dye wastewaters, primary and acti-

vated sludge [9].

Along with its applications as the first step of a phase-

separated anaerobic waste treatment configuration, anaero-

bic acidification can be exploited separately for bio-product

formation. For example, Parawira et al. [10] stated that

anaerobic acidification could be useful for the production of

organic acids (e.g. VFA) which have variety of industrial uses.

VFAs are utilized for the manufacture of various organic

compounds and some plastics [11]. Moreover, these organic

acids play an instrumental role as a carbon source in the

removal of nutrients from wastewaters [12]. Recently, acid-

phase anaerobic digestion received considerable attention

6.

obic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

.

Table 1 e Wastewater characteristics (1 h settled).

Parameters Values (mg/L)

tCOD 6621 � 113.2

sCOD 6165 � 517.1

TS 6062 � 53.0

TSS 665 � 21.2

VSS 335 � 7.1

pH 6.82

Alkalinity 1760 (as CaCO3)

TKN 10

PTotal 2.7

tVFA 1115 � 20 (as H-Ac)

H-Ac 394 � 5

H-Pr 610 � 12

H-Bu 46 � 1

Notations

COD chemical oxygen demand

H-Ac acetic acid

H-Bu butyric acid

H-Pr propionic acid

HRT hydraulic retention time

OLR organic loading rate

ORP oxidation-reduction potential

TS total solids

TSS total suspended solids

TVS total volatile solids

TKN total Kjeldahl nitrogen

VSS volatile suspended solids

VFA volatile fatty acids

Subscripts

t, s total, soluble

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e82

and relevant studies covered a wide range of operational

parameters including; pH [13,14] waste type [15], HRT [2,6,12],

temperature [12,16], mixing of wastes [3]and OLR [17].

Today’s excessive resource use and devastating change in

global environment led to the increased pressure on polluting

industries. Sugar-beet processing industry which falls into

this category, devotes increasing attention to environmental

problems associated with high energy consumption and

production of large amounts of wastes. Recently, sugar plants

take measures to reduce energy consumption, recycle mate-

rials and energy and optimize the operation of sugar process

in order to achieve waste minimization and sustainable

production [18].

As a viable option for waste management in sugar-beet

processing facilities, anaerobic digestion was broadly evalu-

ated. Most of the studies focused on the anaerobic treatment

and biogasification of wastewater and beet-pulp which are

two major waste streams of beet processing plants. Waste-

water from the beet-sugar mill contains high concentrations

of hydrocarbons which makes it suitable for anaerobic bio-

logical degradation [19]. Consequently, there have been

several studies on anaerobic digestion of beet processing

wastewater [19e22]. Contrary to wastewater, beet-pulp is

generally considered as a by-product and utilized for further

use instead of a waste to be treated and disposed. It is mostly

used as animal feed in cattle-raising industry [23]. For this

purpose, beet-pulp may be used directly or could be dried

alone or in combination with molasses [18]. Koppar et al. [24]

advocated that the cost of the beet-pulp drying is increasing

due to the increase in fuel prices and more profitable appli-

cations need to be developed. In some studies, anaerobic

digestion is proven to be a more feasible alternative for the

utilization of beet-pulp [24e26]. Moreover, studies which

cover two-phase anaerobic digestion of beet-pulp were con-

ducted and merits of phase separation were indicated for

biogasification and methane production [23,27,28].

Most of the studies regarding the anaerobic digestion of

sugar-beet processing wastes targeted to the effective bio-

gasification and waste stabilization of each individual waste.

However, acid-phase anaerobic co-digestion of these wastes

together can be used as a practical and economically feasible

waste management option together with the effective

recovery methods of the produced valuable organic acids.

Therefore, the objective of the study was set as maximizing

the hydrolysis and acidification of sugar-beet processing

wastewater and beet-pulp simultaneously in an acidogenic

anaerobic reactor for VFA production. The effect of HRT,

waste-mixing ratio and pH on acidification was investigated

to determine optimum operational conditions at mesophilic

Please cite this article in press as: Alkaya E, Demirer GN, Anaeoperational parameters, Biomass and Bioenergy (2010), doi:10.10

temperatures. The performance of the process was mainly

evaluated in terms of VFA accumulation and the degree of

acidification.

2. Materials and methods

2.1. Waste characteristics

Sugar industry wastes (wastewater and pressed beet-pulp)

were obtained from a private beet-sugar factory located near

Amasya, Turkey during the beet-campaign period. Charac-

terizations of the wastes were carried out and the results were

tabulated in Tables 1 and 2. After characterization, wastes

were kept frozen at �20 �C in order to prevent biological

activity prior to the use in the experimental studies.

Before the characterization and the use in the study,

wastewater was settled for 1-h to remove the inorganic sus-

pended materials which are very common for sugar-beet

processing wastewater. The time period of 1-h was chosen to

represent the typical hydraulic retention time of primary

sedimentation before secondary treatment systems.

In order to achieve physical homogeneity, beet-pulp was

processed as follows: First the frozen beet-pulp was thawed at

roomtemperatureand furtherdriedat105 �Cfor24h.Then, the

dried pulp particleswere grindedby thehelp of a pestle. So, the

homogenized powdered pulp was used for reactor feeding.

2.2. Inoculum

The mixed anaerobic cultures that were used as the inoculum

(seed) source were obtained from the anaerobic sludge

robic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

Table 2 e Pressed beet-pulp characteristics.

Parameter Value (mg/L)

Moisture (%) 85 � 0.1

TS (%) 15 � 0.1

TVS (% TS) 94 � 0.01

COD (g/g dry weight) 1.22 � 0.15

TKN (% TS) 7.28

PTotal (% TS) 1.0 � 0.28

Table 3 e Operational parameters of the reactors.

Reactor Feed HRT(days)

Waste mixingratio (WW:P)

OLR(g COD/L-day)

R1 Stock #1 2 1:0 2.7

R2 Stock #2 2 1:0.5 4

R3 Stock #3 2 1:1 5.4

R4 Stock #1 4 1:0 1.35

R5 Stock #2 4 1:0.5 2

R6 Stock #3 4 1:1 2.7

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e8 3

digesters of the municipal wastewater treatment plant of

Ankara. Prior to the use as inoculum, it was concentrated by

gravity settling for 24 h. By this way, the VSS concentration

was increased up to 41,212 � 2324 mg/L and then used in the

experiments.

2.3. Basal medium

In order to supply adequate micronutrients for an optimum

microbial growth, reactors were daily fed by basal medium

(BM) which contains the following constituents (concentra-

tions are given in parentheses as mg/L): NH4Cl (1200),

MgSO4 7H2O (400), KCl (400), Na2S 9H2O (300), CaCl2 2H2O (50),

(NH4)2 HPO4 (80), FeCl2 4H2O (40), CoCl2 6H2O (10), KI (10),

MnCl2 4H2O (0.5), CuCl2 2H2O (0.5), ZnCl2 (0.5), AlCl3 6H2O (0.5),

NaMoO4 2H2O (0.5), H3BO3 (0.5), NiCl2 6H2O (0.5), NaWO4 2H2O

(0.5), Cysteine (10) [29].

2.4. Analytical methods

Solids, tCOD (for beet-pulp), TKN, PTotal and alkalinity deter-

minations were carried out as described in Standard Methods

[30] pH values were measured with a pH meter (HI 8314,

Hanna Instruments) and a pH probe (HI 1230, Hanna Instru-

ments). tCOD (for wastewater) and sCOD (0.45 mm pore-sized

filters were used) determinations were performed by using

a spectrophotometer (SN 05827, PC Multidirect) and vials

specifically prepared for the determination range of

0e1500 mg/L. ORP values of the reactors were measured with

a pH meter (PH 510, Eutech) equipped with an ORP electrode

(Recorder S-500 C, Sensorex).

Daily biogas productions were measured with a water

replacement device and the compositions were periodically

determined with a gas chromatograph (Thermo Electron Co.)

equipped with a thermal conductivity detector (TCD).

Produced biogases were separated as hydrogen (H2), carbon

dioxide (CO2), oxygen (O2), methane (CH4) and nitrogen (N2) by

using serially connected columns (CP-Moliseve 5A and CP-

Porabond Q) at a fixed oven temperature of 45 �C. Helium was

used as carrier gas at 100 kPa constant pressure. The inlet and

detector temperatures were set to 50 �C and 80 �C respectively.

Prior to the gas chromatography injections, series of

pretreatments were conducted for VFA measurements: First,

samples were filtered through 0.22 mmpore-sized filters. Then

the samples were diluted with deionized water to assure the

VFA concentration of the sample to be in the range of pure

VFA calibration of gas chromatograph. After filtering and

dilution, the samples were acidified with 98% formic acid to

a pH less than 2.5, in order to convert the fatty acids to their

undissociated forms (i.e. acid forms).

Please cite this article in press as: Alkaya E, Demirer GN, Anaeroperational parameters, Biomass and Bioenergy (2010), doi:10.10

The gas chromatograph (Thermo Electron Co.), used for

biogas composition determinations, was also used for the

periodical VFA measurements. However the column, the

detector and the operational conditions were different: Nukol

column (Model 25,326, 15 m � 0.53 mm) was used to separate

VFAs (acetic, propionic, butyric, iso-butyric, valeric, iso-vale-

ric, caproic, iso-caproic and heptanoic acids). Flame ionization

detector (FID) was used for this purposewhichwas adjusted to

280 �C as operating temperature. Helium was used as carrier

gas with a constant flow rate of 6 mL/min and the inlet

temperature was kept at 250 �C. Oven temperature was

initially set to 100 �C with 2 min holding time and then

increased to 200 �C with 8 �C/min ramping.

One of the main interests of this study was to compare the

proportions of the substrates converted to VFAs. For this

purpose, “acidification degree” was calculated for each of the

reactor in order to express the acidification efficiency (Fig. 3).

So, the organic content of initial substrate and VFAs were

based on COD concentrations. By this way, it was possible to

present the related conversion efficiencies for all operated

reactors as:

Degree of acidification ð%Þ ¼ � � Sf=Si

�� 100 (1)

Where; Si: Initial substrate concentration,measured in COD as

mg/L, *Sf: Produced net VFAs (finaleinitial), expressed as

theoretical equivalents of COD concentrations as mg/L. *The

CODequivalents of eachVFA:Acetic acid, 1.066; Propionic acid,

1.512; Butyric acid, 1.816; Valeric, 2.036; Caproic acid, 2.204. [7].

When calculating acidification degrees obtained in the

reactors, initial tVFA concentration (1115� 20mg/L asH-Ac) of

the wastewater was taken into consideration. In other words,

reactors having wastewater as the feed had already VFAs

mainly in the form of acetic, butyric and propionic acids. In

order to determine actual conversion of COD into VFA, initial

tVFA concentration was subtracted from the final tVFA values

giving net tVFA production.

2.5. Experimental set-up and procedures

Six reactors were operated with different combinations of the

operational parameters (HRT, waste mixing ratio and pH)

(Table 3). Reactors were run as duplicates to test statistical

reliability and the presentation of the data (tables and figures)

involves the averaged values of duplicate reactors. 250 mL

reactors with effective volume of 150 mL were operated by

daily fed-batch feeding strategy as continuously mixed

acidogenic reactors. Reactors were kept continuously mixing

obic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e84

at 175 rpm by using a mechanical shaker in a temperature

controlled room (35 � 1 �C) for 41 days.

In order to prevent uncontrolled pH drops and to investi-

gate the effect of higher operational pH (6.9e7.5) on the acid-

ification, first 20 days of operation was carried out by adding

external alkalinity in the form of NaHCO3. In this period,

reactors were supplied with NaHCO3 to set daily initial

concentrations to 6000mg/L by daily additions along with BM.

Then, the reactors were operated 21 more days without

adding any external alkalinity to observe the natural pH drop,

as a result of acidification, and to investigate the effect of

lower operational pH (5.7e7.4) on acidification.

For the purpose of reactor feeding, three different stock

solutions were prepared (Table 4). In order to investigate the

effect of waste mixing ratio on acidification, prepared stock

solutions involved same concentrations of BM but different

mixing ratio of wastewater (WW) and pulp (P) in terms of

tCOD.

As the control parameters of reactor operations, pH, ORP,

biogas production, biogas composition, VFA and sCOD

concentrations weremeasured. Among these parameters, pH,

ORP and biogas production were daily, VFA, sCOD, and biogas

compositions were periodically measured.

Fig. 1 e Temporal changes of reactor conditions: (a) pH;

(b) chemical oxygen demand (soluble); (c) oxidatione

reduction potential; (d) daily gas production.

3. Results and discussions

3.1. pH profiles of the reactors

pH drops were observed with varying rates and extents for all

reactors (Fig. 1a). Organic acid productionswere initiated from

the onset of the reactor operations which resulted in these

expected pH drops (Fig. 2). For the same substrate (stock

solution), 2 days of HRT led to lower pH values than 4 days.

The noticeable differences between the operational pHs were

mainly due to the differences of the methanogenic activities

in the reactors. Since, growth rates of methanogenic micro-

organisms are lower than the other members of anaerobic

consortia, they require more time to survive in anaerobic

systems [31]. So, in this study, 4 days of HRT served as a better

growing condition for methanogens than 2 days. As a result,

methanogenic microorganisms led to VFA consumption and

corresponding buffering effect that prevented further pH

drops at 4-day HRT (R4, R5, R6). This situation was the reason

of higher pH values, observed in these reactors. Indeed, VFA

concentration profiles (Fig. 2) and biogas compositions

(Table 5) of the reactors supported this speculation.

After external alkalinity addition was ceased, pH values

were further decreased and stabilized at lower levels

Table 4 e Compositions of stock solutions.

Composition Stock #1 Stock #2 Stock #3

Wastewater (mg/L as tCOD) 5300 5300 5300

Pulp (mg/L as tCOD) e 2650 5300

tCOD (mg/L) 5300 7950 10,600

sCOD (mg/L) 4932 � 72 5211 � 421 5318 � 288

pH 8.02 7.99 8.08

Please cite this article in press as: Alkaya E, Demirer GN, Anaeoperational parameters, Biomass and Bioenergy (2010), doi:10.10

(Fig. 1a). The observed pH decreases were steeper (down to

5.7e7.1) at 2-day HRT since the VFA accumulations (Fig. 2)

were too high for the inherent alkalinity of wastewater to

buffer. In this period, steady-state pH values were higher at

4-day HRT (6.6e7.4).

Additionally, pH patterns of the reactors differed from

each other when they are compared in terms of waste mixing

ratios. For the same HRT, increased OLRs, resulting from pulp

addition, increased the amount of acidification products

(VFAs) which lead to low operational pHs (5.7e6.8). It was

reported that high OLRs result in higher VFA accumulations

and related pH decreases [32]. In this study, the reactors R2

and R3 which received pulp as well as wastewater were

operated in the pH range of 5.6e6.2 without external alka-

linity addition at HRT of 2 days. Since the pH range of 4e6.5 is

accepted as optimum for the growth of anaerobic acidogenic

microorganisms [31], proper pH conditions were established

in R2 and R3.

robic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

Fig. 2 e VFA concentration profiles of the reactors: (a)

volatile fatty acids (total); (b) acetic acid; (c) propionic acid;

(d) butyric acid.

ReactorR1 R2 R3 R4 R5 R6

)%(

eergeD

noitac if id icA

0

10

20

30

40

50

60

70

80

External AlkalinityNo External Alkalinity

Fig. 3 e Observed acidification degrees in the reactors.

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e8 5

3.2. Volatile fatty acid productions

Fig. 2 illustrates the inverse relationship between VFA accu-

mulation and operational HRT of the reactors. 2 days of HRT

yielded higher tVFA concentrations (2159e3635 mg/L as H-Ac),

which is the indication of higher acidogenic activity and/or

lower methanogenic activity. Since the acidogenic micro-

organisms growmuch faster than themethanogens, they could

successively survive inHRTsas lowas2days.Thisalso indicates

that at 4-day HRT (R4, R5, R6) methanogens could be retained

long enough to metabolize VFAs for methane production,

causing to lowerVFA concentrations (1814e2640mg/L asH-Ac).

The composition of the produced biogaswhich indicates higher

methane percentages at 4-day HRT (Table 5) was a strong

evidence of this claim.

Lowest VFA concentrations (1814e2244 mg/L as H-Ac) were

recorded in the reactors,whichwere fed only bywastewater (R1

and R4). As expected, VFA concentrations were proportionally

Please cite this article in press as: Alkaya E, Demirer GN, Anaeroperational parameters, Biomass and Bioenergy (2010), doi:10.10

increased with the increase in amount of pulp added and the

highest concentrationswere observed in2-day-HRTreactor (R3)

which was fed by wastewater and pulp in equal amounts in

terms of COD (1:1). These results indicated that pulp was

successfully acidified together with wastewater, which led to

the increase of the VFA concentrations.

After the cessation of alkalinity addition, VFA concentra-

tions were not changed noticeably. This observation indicated

that, the change in narrow operating pH values (Fig. 1a) did not

result in a major change in the acidification trend of sugar

industry wastes, even in R3 which indicated the steepest

decrease from 6.9 to 5.7. Dinopoulou et al. [33] stated that

acidification degree can be determined by calculating the

proportionof the initial substratewhich isconvertedtoVFAsas

end products. In this study, the highest acidification degrees

(60.3e64.2%) were observed in R1, which was only fed by

wastewater and operated at 2 days of HRT (Fig. 3). The increase

in pulp addition caused a decrease in acidification degree

which was accepted as the direct result of increasing OLR.

Similarly, in a complex wastewater acidification study, the

degree of acidification was found to diminish with increasing

OLR [33]. Still, the reactors which were fed by wastewater and

pulpwith HRT of 2 days (R2, R3) denoted substantial degrees of

acidification (44.1e53.5%) when compared with other studies:

15e60% for complexwastewater [33]; 56% for dairywastewater

[34]; 10.3e43.4% forfish meal processing wastewater [35].

In all of the reactors, the main acidification products were

H-Ac (40.3e49.2%w/w of tVFA), H-Pr (36.3e42.6%w/w of tVFA)

and H-Bu (3.6e7.5% w/w of tVFA) comprising 88.3e96.2% of

tVFAs. The higher molecular weight VFAs (valeric, caproic,

heptanoic etc.) were produced in insignificant amounts. On

the other hand, alcohols (e.g. ethanol, methanol), other major

metabolites of anaerobic acidification, were not detected at

all. It is a well-known fact that substrate characteristics and

operational conditions play a major role on product distribu-

tion in an acidification reactor [13,14,33]. In this study, the

dominance of H-Ac, H-pr and H-Bu can be associated with the

carbohydrate degradation, since both wastewater [19] and

pulp [23] contains high amounts of sugars. This is relevant

with the literature in which the short-chain fatty acids were

obic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

Table 5 e Steady-state biogas compositions of thereactors.

Reactor External alkalinity No external alkalinity

CH4 (%) CO2 (%) CH4 (%) CO2 (%)

R1 25.3 � 1.7 74.7 � 1.7 23.2 � 5.9 76.8 � 5.9

R2 16.3 � 2.1 83.7 � 2.1 16.7 � 3.3 83.3 � 3.3

R3 6.4 � 1.5 93.7 � 1.5 5.6 � 0.5 94.4 � 0.5

R4 53.4 � 0.4 46.6 � 0.4 49.8 � 2.1 50.2 � 2.1

R5 51.6 � 3.3 48.4 � 3.3 45.1 � 2.6 54.9 � 2.6

R6 44.5 � 2.7 55.5 � 2.7 39.5 � 2.2 60.5 � 2.2

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e86

found to be dominant in acidogenic reactors [14,33]. Parawira

et al. [10], stated that higher molecular weight VFAs are

generally found in protein fermentation.

3.3. Soluble chemical oxygen demand concentrations

sCOD concentration distributions of the reactors were depicted

in Fig. 1b. The highest sCOD concentrations (5422e5826 mg/L)

were observed in R3 which was operated at an HRT of 2 days.

4-day HRT led lo lower sCOD concentrations resulting from

the consumption of VFAs which contribute to sCOD. It can be

seen from Fig. 1 that, there is a direct relationship between VFA

and sCOD concentrations. Since, hydrolysis of particulate

organic matter occurs simultaneously during the acidification

of soluble organics, sCOD concentrations were higher

(3490e5826 mg/L) in the reactors which have pulp addition

together with wastewater.

The effect of pH values on sCOD concentrations was

negligible when the alkalinity-added period was compared

with the period without alkalinity addition. This result indi-

cated that the trend of hydrolysis was not significantly altered

with the decreases in operational pH values.

3.4. Oxidation-reduction potentials

ORP values of anaerobic reactors were inspected to detect the

relative amounts of oxidized materials such as nitrate ions

(NO3�) and sulfate ions (SO4

2�), and reduced materials, such as

ammonium ions (NH4þ). At ORP values between �100 mV and

�300 mV, degradation of organic compounds proceeds

primarily as fermentation and acid formation. On the other

hand, ORP values lower than �300 mV indicates considerable

methanogenic activity in anaerobic conditions [36].

Fig. 1c depicts that, ORPs of reactors were diminished to

steady-state values after the operation of 3e5 days. ORP values

of the reactorswerewithin�226mVand�350mV in the period

of alkalinityaddition.After alkalinity additionwasstopped,ORP

values started to increase and attained steady-state between

�162mV and�300 mV. This observation indicates that, the pH

drops resulting from the cessation of alkalinity addition slightly

altered the circumstances in favor of fermentation and mixed-

acidformation, inhibitingthemethanogenicactivities.This idea

was supported by the results of biogas compositions (Table 5)

which indicate that methane percentages were slightly

decreased along with decreasing pH values.

Moreover, 2-day HRT yielded higher ORP values (�183 mV

to �234 mV) than 4-day HRT especially in the period without

alkalinity addition. In 4-day-HRT reactors, the observed ORP

Please cite this article in press as: Alkaya E, Demirer GN, Anaeoperational parameters, Biomass and Bioenergy (2010), doi:10.10

values were between �273 mV and �318 mV which indicated

higher methanogenic activities than 2-day-HRT reactors.

3.5. Biogas productions and compositions

The direct relationship between the operational HRT and the

amount of daily produced biogas can be observed in Fig. 1d.

Reactors, which were operated with an HRT of 4 days (R4, R5,

R6) produced higher amounts of biogas (27e72 mL/day)

compared to those operated with an HRT of 2 days (19e25 mL/

day). When the biogas productions are interpreted in combi-

nation with their composition (Table 5), it can be postulated

that, higher methanogenic activity was responsible for the

higher biogas productions in 4-day-HRT reactors.

Since, the daily biogas productions were primarily affected

bymethaneproductions, conditions, influencingmethanogenic

activity, also influenced the total biogas productions. So, this

explains why the lowest biogas productions (19e21 mL/day)

were observed in R2 and R3, in which pH values were low

enough (5.7e6.2) to inhibitmethaneproductions tosomeextent.

Thus, pulp addition increased the substrate concentrations and

highest biogas productions (31e72mL/day) were recorded at R5

and R6 which were operated with 4 days of HRT.

In acidogenic anaerobic reactors, methanogenic activity

must be restrained to be able to reach high VFA accumula-

tions. Methanogenic activities and related methane produc-

tions could be suppressed to some extent with 2 days of HRT

(R1, R2, R3). As a result, lower methane percentages

(5.6e25.3%) were detected within the produced biogases of

these reactors when comparedwith the percentages appeared

in the 4-day-HRT reactors (39.5e53.4%).

Furthermore, Table 5 indicates the inverse relationship

between substrate concentrations and methane productions.

It can be observed that, increased substrate concentrations

resulting from pulp additions caused significant decreases in

methane productions; especially at 2-day HRT. The main

reason was the increased VFA accumulation in corresponding

reactors, resulting in the lower operational pHs (5.7e6.9). So, it

was evident that, pulp addition, causing to VFA accumulation,

suppressed the methane production.

When the biogas compositions of 4-day-HRT reactors were

inspected, it was claimed that neither HRTs nor the operational

pHvalues (6.6e7.5)wereacceptable for the targeted inhibitionof

the methanogenic activity. In fact, Yu and Fang [14] stated that

the pH values of acidogenic reactors must be kept below 5.5 to

inhibit methanogenic microorganisms after observing high

methane concentrations (31%) even at pH values as low as 6.5.

After cessation of alkalinity addition,methane percentages

of the reactors were slightly decreased following the

decreases in pH values. The lowestmethane percentage (5.6%)

was observed at R3 in the period without alkalinity addition.

This result indicates that, the optimum conditions for inhi-

bition of methanogenic activity were 2 days of HRT and

5.7e6.2 range of pH in this study.

4. Conclusions

From the obtained results, the following conclusions can be

drawn:

robic acidification of sugar-beet processing wastes: Effect of16/j.biombioe.2010.08.002

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e8 7

� Sugar industry wastewater and beet-pulp can simulta-

neously be converted to VFAs in acidogenic anaerobic reac-

tors with considerable acidification degrees (43.8e52.9%).

� In this study, wastewater was used for dilution of beet-pulp

instead of fresh water, which serves for resource conser-

vation. By this way, external alkalinity addition, a common

application, could also be avoided due to the inherent

alkalinity of the wastewater.

� Increased OLRs, resulting from pulp addition, increased the

amount of acidification products (VFAs) which lead to

relatively low operational pH values (5.7e6.8). In this pH

range, methanogenic activity was inhibited and lowest

methane percentages (5.6e16.3%) were observed in biogas

compositions.

� The optimum operational conditions were selected as 2-day

HRT and 1:1 waste mixing ratio (in terms of COD) without

external alkalinity addition. These operational conditions

led to the highest tVFA concentrations (3635 � 209 mg/L as

H-Ac) with an acidification degree of 46.9% at the highest

OLR of 5.4 g COD/L-d.

Acknowledgments

This study was funded by The Scientific and Technological

Research Council of Turkey through Grant Number 104I127.

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