Kinetics of anaerobic treatment of slaughterhouse wastewaterin batch and upflow anaerobic sludge blanket reactor

Jes�uus Rodr�ııguez-Mart�ıınez *, Ivan Rodr�ııguez-Garza, Estaban Pedraza-Flores,Nagamani Balagurusamy, Gerardo Sosa-Santillan, Yolanda Garza-Garc�ııa

Department of Biotechnology, Chemistry Faculty, Universidad Autonoma de Coahuila, Blvd. V. Carranza and J. C�aardenas V. Saltillo,Coahuila, ZC 25280, Mexico

Received 3 July 2001; received in revised form 4 June 2002; accepted 5 June 2002

Abstract

The kinetics of anaerobic treatment of slaughterhouse wastewater in batch and upflow anaerobic sludge blanket (UASB) reactors

was investigated. Different concentrations of organic matter in slaughterhouse wastewater did not change the first order kinetics of

the reaction. In batch digesters, methane and nitrogen production stopped after 30–40, 20–30 h, respectively, and in UASB reactors

it was terminated after 30–40 days. The constant of velocity was 3.93 and 0.23 h�1 respectively, for methane and nitrogen pro-

duction. The yield coefficient, Yp was 343 and 349 ml CH4 per g of chemical oxygen demand at standard temperature and pressure

conditions for batch reactors and UASB reactor, respectively.

� 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Slaughterhouse wastewater; Anaerobic treatment; Kinetics denitrification; Methanogenesis

1. Introduction

Anaerobic treatment is widely employed for treat-ment of most of the industrial wastewaters containinghigh concentrations of soluble organic matter (Jeris,1983). The advantages of anaerobic treatment are widelyreported by many workers (Fiestas, 1984; Olthof andOleszkiewick, 1982). Conventional anaerobic treatmentof slaughterhouse wastewater has also been reported byseveral workers (Rodr�ııguez-Mart�ıınez et al., 1997; Borjaet al., 1995; Polprasert et al., 1992; Sayed et al., 1984,1987). Borja et al. (1993) investigated the influence ofdifferent supports on the kinetics of anaerobic puri-fication of slaughterhouse wastewater. The kinetics ofvarious processes of anaerobic digestion, viz., denitrifi-cation, sulfate reduction and methanogenesis in batchreactors as well as upflow anaerobic sludge blanket(UASB) reactors fed with slaughterhouse wastewater isnot yet fully investigated.

As nitrate and sulfate are present in considerablequantities in slaughterhouse wastewater, it is importantto assess the influence of these substrates on biometha-nation of this wastewater. It is well known that presenceof sulphate is inhibitory to methanogenesis in marineand fresh water sediments (Balderston and Payne, 1976)and in anaerobic digesters (Lawrence et al., 1966). Thetoxicity of sulfide or free H2S produced by microbialreduction of sulphate and competition for electron do-nors between sulphate-reducing bacteria (SRB) andmethanogens are considered to be two important factorsresponsible for this inhibitory effect (Kristjansson et al.,1982; Kroiss and Wabnegg, 1983). The aim of thispresent work was to carry out a kinetic study on variousprocesses that occur during anaerobic treatment ofslaughterhouse wastewater in batch and UASB reactors.

2. Methods

2.1. Wastewater and inoculum

The wastewater used in this study was collected fromthe municipal slaughterhouse of Saltillo, Coahuila (Mex-ico). The wastewater production was approximately

Bioresource Technology 85 (2002) 235–241

*Corresponding author. Tel.: +52-844-4100-722; fax: +52-844-4155-

752.

E-mail address: jrodrigu@alpha1.sal.uadec.mx (J. Rodr�ııguez-

Mart�ıınez).

0960-8524/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

PII: S0960-8524 (02 )00141-4

100 m3/day. Of the animals slaughtered, cattles werelargest in numbers (about 90%) and the rest were swine(about 10%). The inoculum for the study was obtainedfrom an UASB reactor that treats slaughterhousewastewater. The pH of the inoculum was 7.3 and thetotal solids (TS), volatile solids (VS) and volatile sus-pended solids (VSS) content of the inoculum was 44� 3,27:5� 2 and 21� 3 g per liter respectively.

2.2. Reactors and the experimental set-up

Anaerobic batch reactors (120 ml volume) with 40 mlworking volume including 5 ml of granular sludge wereused as batch reactors. Various COD (g l�1) concentra-tions; 11.7, 9.3, 6.8 and 6.5 were prepared by diluting theslaughterhouse wastewater (12.82 g l�1) with distilledwater. The COD of the slaughterhouse wastewater wasincreased to 29 g l�1 by adding blood to the wastewater.To one set of digesters having a COD concentration of29 g l�1, sulphate was added at a level of 1.0 g l�1.Thirty-five milliliters of mineral medium without carbonsource was maintained as control. A UASB reactor [14 l;1.70 m (height)�10:5 cm (inner diameter)] with a waterjacket for temperature control was also used in thisstudy. The UASB reactor was built of acrylic material.The inoculum was incubated in diluted slaughterhousewastewater at 37� 2 �C for 30 days. After this incuba-tion period, continuous feeding of the wastewater intothe UASB reactor was initiated and the COD concen-tration was gradually increased to the original level ofslaughterhouse wastewater (12.82 g l�1). The pH was inthe range of 7.1–8.2 during the treatment process. Thecharacteristics of slaughterhouse wastewater used in thepresent study are given in Table 1.

2.3. Analysis

Methane and molecular nitrogen were analyzed bycapillary gas chromatograph (Varian 3400; Varian An-alytical Instruments, TX, USA) equipped with a thermalconductivity detector and molecular sieve column

(30 m� 0:53 ID) at 50 �C. Helium was used as thecarrier gas and the flow rate was 30 ml/min. The volatilefatty acids (VFA) were identified and quantified using acapillary gas chromatograph (Varian 3300) equippedwith flame ionization detector and DB-FFAP column.The column temperature was programmed from 150 to220 �C. Helium was used as the carrier gas and the flowrate was 30 ml/min. The following parameters viz.,chemical oxygen demand (COD), pH, TS, VS, mineralsolids (MS), total suspended solids (TSS), VSS, mineralsuspended solids (MSS), VFA (in terms of acetic acid),alkalinity, total nitrogen (Kjeldahl), nitrates, phos-phates, oils and greases were analyzed by followingstandard methods (APHA, 1990). Sulfate was deter-mined by following the procedures described by Cotte-nie et al. (1982).

2.4. Statistical analyses

All treatments were conducted in duplicate. Thevalues reported in this work are an average of threesamples for every replication maintained. The data werestatistically analyzed for differences in means at the 5%probability level by general linear models procedure inSAS institute (1996) and by following Steel and Torrie(1980). Gm and KA were calculated by using time seriesprocessor (TSP), International Program, Version 4.0 D,Stanford, California, USA (Borja et al., 1993).

3. Results and discussion

3.1. Variation of methane volume produced with time

The methane produced per hour in the batch reactorwas dependent on the strength of slaughterhousewastewater (Table 2). The methanogenic activity andvelocity of reaction increased with an increase in COD.But methanogenic activity was less in digesters fed withslaughterhouse wastewater containing concentratedblood, although the COD was higher (Table 2). Themolecular nitrogen and methane produced at differentconcentrations of COD in batch digesters are presentedin Figs. 1 and 2 respectively. The rate of methane for-mation in the UASB reactor is shown in Fig. 3. Thefollowing equation (Fiestas et al., 1990) was used tocalculate the kinetics of the present study

G ¼ Gm½1� e�kat� ð1Þwhere G is the volume of methane produced (ml) in timet (days); Gm is the maximum volume of methane accu-mulated at an infinite digestion time and is the productof the initial substrate concentration (S0) and the yieldcoefficient of the product (Yp): Gm ¼ S0Yp; and ka is ap-parent kinetic constant that includes the biomass con-centration (x): ka ¼ kx.

Table 1

Characteristics of the slaughterhouse wastewater

Characteristics Influent to

UASB reactor

Effluent from

UASB reactor

COD (g/l) 12.82 1.43

pH 7.5 8.19

Sulphate (g/l) 0.97 0.24

Phosphate (g/l) 0.41 0.25

VSS (g/l) 26.5 3.55

TSS (g/l) 58.20 5.66

VFA (acetic acid) (g/l) 0.88 0.325

Alkalinity (CaCO3) (g/l) 0.53 0.58

Grease and oils (g/l) 0.25 0.12

Nitrogen (Kjeldahl) (g/l) 0.531 0.150

Nitrates (mg/l) 0.96 0.21

236 J. Rodr�ııguez-Mart�ıınez et al. / Bioresource Technology 85 (2002) 235–241

Eq. (1) is in agreement with the finding empiricallyestablished by Roediger (Edeline, 1980). Almost con-stant biomass concentration (x) in the digesters (Table2) assisted in comparison and interpretation of the re-sults. Using the same parameters as indicated in Eq. (1)for the methane and nitrogen production, it is demon-strated that the reactions are of first order kinetic model(Figs. 4 and 5) (Winkler, 1986; Edeline, 1980; McCartyand Mosey, 1991). The mean values of ka (with limit ofconfidence of 95%) for the methane and nitrogen for-mation was 3:93� 0:05 and 0:23� 0:01 per day andhour respectively, for batch reactors. At a higher con-

centration of COD (12.3 g COD/l), Eq. (1) did not ex-hibit a pattern characteristic of a linear model. Thisindicated that the formation velocity of methane can beinfluenced by different concentrations of COD (Figs. 6and 7). Conversely molecular nitrogen appeared firstamong all the electron acceptors present in the systembecoming detectable usually during the first 10 h ofincubation (Figs. 2 and 8). This is in accordance withthe earlier reports of Westermann and Ahring (1987)and Lens et al. (1998) that nitrates if present arefirst used as electron acceptors during the anaerobicdigestion. Afterwards, H2S formation was observed

Fig. 1. Kinetics of methane formation from slaughterhouse wastewater at different concentrations of COD; n ¼ 5.

Fig. 2. Kinetics of molecular nitrogen formation from slaughterhouse wastewater at different concentrations of COD; n ¼ 5.

Table 2

Denitrification and methanogenic activity in batch reactors fed with slaughterhouse wastewater

COD (g/l) Denitrification Methanogenesis Granular sludge,

VSS (g/l)Activity

ðg=g of VSS=dayÞ � 103Velocity (V)

ðg=l=dayÞ � 10�2

Activity CH4

ðg=g of VSS=dayÞ � 102Velocity (V)

(g/l/day)

29 1:17� 0:020a 2:51� 0:019a 1:37� 0:14a 0:295� 0:055a 21:4� 1:497a

29þ 1 g sulphate 0:986� 0:024a 2:18� 0:038a 0:44� 0:05b 0:0986� 0:011b 22:2� 2:3a

12.3 6:78� 0:78b 14:93� 1:101b 2:78� 0:031c 0:613� 0:041c 22� 2:16a

11.7 5:89� 0:58c 11:90� 1:171c 2:48� 0:021c 0:51� 0:128c 20:2� 2:7a

9.3 4:72� 0:47d 10:1� 1:28d 2:28� 0:05c 0:49� 0:02c 21:4� 2:4a

6.8 3:66� 0:38e 7:92� 1:28e 2:08� 0:024a 0:45� 0:028a 21:6� 2:4a

6.5 2:54� 0:35f 5:44� 0:88f 2:05� 0:024a 0:44� 0:028a 21:4� 2:4a

Different superscripts indicate significant differences in values of means (P < 0:05, Scheffe’s test).

J. Rodr�ııguez-Mart�ıınez et al. / Bioresource Technology 85 (2002) 235–241 237

indicating the sequence of sulphate reduction after thedisappearance of nitrate ions (Fig. 8). Sulphate reduc-tion reduced methane production either as a conse-quence of competition, due to their higher affinity of thehydrogenase system or due to the inhibition of the ac-tivity of methanogenic bacteria to the presence of hy-drogen sulfide (Westermann and Ahring, 1987). Forkinetics of the methanogenic reaction in the UASB re-actors, methane formation under different organic

loading rates (OLR) coincided with the batch reactorshaving COD concentrations of 6–12 g l�1. If the OLR ishigher than this level, the removal efficiency of UASBreactor decreased. The formation of methane as afunction of organic loading rate is within the rank ofbetween 1.7 and 3.0 (Fig. 9). From the relationshipbetween OLR and hydraulic retention time (HRT), itwas observed that with the OLR higher than 4 g COD/lper day, methane formation decreased as in the batch

Fig. 4. Kinetic formation of methane as a function: ln V0 ¼ lnK þ n ln½Gm�; where V0 is initial velocity of methane and Gm is accumulated methane;

n ¼ 5.

Fig. 3. Kinetic of methane formation in UASB rector; OLR: 1.7–3.0 g COD/l/day; n ¼ 5.

Fig. 5. Kinetic formation of N2 as a function: ln V0 ¼ lnK þ n ln½Gm�; where V0 is initial velocity of N2 and Gm is accumulated nitrogen; n ¼ 5.

238 J. Rodr�ııguez-Mart�ıınez et al. / Bioresource Technology 85 (2002) 235–241

reactor. After this stage the linearity of the reactiondisappeared (Fig. 10).

3.2. Yield coefficient

The yield coefficient Yp at standard temperature andpressure (STP) was determined from the volume of

methane produced and the initial and final COD. Ascan be seen from the results, the methane produced inthe batch reactors was proportional up to the concen-tration of 12.3 g/l of COD (Fig. 2). In the case of theUASB reactor, the yield coefficient was constant. Byfitting G and COD uptake value pairs to a straight line,the yield coefficient under STP conditions was found

Fig. 6. Influence of Gm (accumulated methane) on initial formation velocity of methane; n ¼ 5.

Fig. 7. Influence of initial concentration of COD on initial consumption velocity of COD and their relationship to CH4 formation; n ¼ 5.

Fig. 8. Kinetic formation of N2, H2S and CH4 during anaerobic treatment of slaughterhouse wastewater (29 g COD/l) with 1 g of sulphate; n ¼ 5.

J. Rodr�ııguez-Mart�ıınez et al. / Bioresource Technology 85 (2002) 235–241 239

to be 343� 5 and 349� 5 ml CH4 under STP condi-tions per g COD for the batch and UASB reactors,respectively. The results are in conformation with theearlier report of Borja et al. (1993).

4. Conclusion

Based on these studies, it appears that the reactions ofanaerobic treatment of slaughterhouse wastewater in thisstudy agree with the first order kinetic model. The meanvalues of ka (with limit of confidence of 95%) for themethane and nitrogen formation was 3:93� 0:05 and0:23� 0:01 per day and hour, respectively, in batch re-actors. At higher concentrations of COD (12.3 g COD/l),there was no linearity, which showed that the formationvelocity of methane can be influenced by different con-centrations of COD. The yield coefficient of methane atSTP conditions was found to be 343� 5 and 349� 5 mlCH4 for the batch and UASB reactors, respectively.

Acknowledgements

This work was supported by Consejo Nacional deCiencia y Tecnolog�ııa (CONACYT), Mexico.

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