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Published in: Water Research 41:721-730.

BIODEGRADATION OF DISTILLERY SPENT WASH IN ANAEROBIC HYBRID


REACTOR

Gupta, Sunil Kumara, Gupta, S. K.b, Singh, Gurdeepc


a
Sr. Lecturer, Centre of Mining Environment, Indian school of Mines, Dhanbad, 826004, (India) Telefax:
+91-326-2206372 (O), E-mail: skgsunil@gmail.com (Corresponding author)
b
Prof. & Head, Centre for Environmental Science and Engineering, Indian Institute of Technology, Bombay,
Mumbai, 400076, (India) Telefax: +91-22-2207853, E-mail: skgupta@iitb.ac.in
c
Centre of Mining Environment, Indian school of Mines, Dhanbad, 826004, (India) Telefax: +91-326-
2206372 (O), E-mail:s_gurdeep2001@yahoo.com.

ABSTRACT

A lab-scale anaerobic hybrid (combining sludge blanket and filter) reactor was operated in
a continuous mode to study anaerobic biodegradation of distillery-spent wash. The study
demonstrated that at optimum hydraulic retention time (HRT), 5 days and organic loading
rate (OLR), 8.7 kg COD/m3.d, the COD removal efficiency of the reactor was 79%. The
anaerobic reduction of sulphate increases sulphide concentration, which inhibited the
metabolism of methanogens and reduced the performance of the reactors. The kinetics of
biomass growth i.e. yield coefficient (Y, 0.0532) and decay coefficient (Kd, 0.0041 d-1) was
obtained using Lawrence and McCarty model. However, this model failed in determining
the kinetics of substrate utilization. Bhatia et al. model having inbuilt provision of process
inhibition described the kinetics of substrate utilization, i.e. maximum rate of substrate
utilization (R, 1.945 d-1) and inhibition coefficient values (Ki, 0.032 L/mg). Modeling of the
reactor demonstrated that Parkin and Speece, and Bhatia et al. models, both, could be used
to predict the effluent substrate concentration. However, Parkin and Speece model predicts
effluent COD more precisely (within ±2%) than Bhatia et al. model (within ±5-20%) of the
experimental value. Karhadkar et al. model predicted biogas yield within ± 5% of the
experimental value.
(Key words – Anaerobic digestion, spent wash, anaerobic hybrid reactor, sulfide toxicity,
kinetics of biodegradation, mathematical modelling)

1
1 Introduction
Distillery spent wash is perceived as one of the serious pollution problems of the countries

producing alcohol from the fermentation and subsequent distillation of sugar cane

molasses. The distillery spent wash is characterized as one of the caramelized and

recalcitrant wastes containing extremely high COD, BOD, SS, inorganic solids, color and

low in pH (Biradar, 2005, Shin et al., 1992; Saha et al., 2005). Being rich in sugarcane

yield, all the ethanol, in India, is produced by the way of fermentation of molasses and its

subsequent distillation (Inamdar, 1991; Saha et al., 2005). At present, there are 285

distilleries in India that producing 2.7 billion liters of alcohol and generating 40 billion

liters of wastewaters annually (Raghukumar et al., 2004). Various treatment

technologies such as incineration, physico-chemical

treatment, composting, and biological treatment have been

studied by the researchers (Pena et al., 2003; Hayase et al.,

1984, Sheehan and Greenfield 1980, and Gurudatta, 1992, Nandy

et al., 2002; Gupta, 1989; Karhadkar et al., 1990, Kaul and

Badrinath, 1987; Bardiya, 1988 and Goyal et al., 1996).

However, there is no general agreement yet on the most


appropriate method of treatment of spent wash. Since the

inception of anaerobic hybrid reactor (combining the sludge

blanket in the lower part and filter in the upper part), it

has been studied by many researchers (Kimata et al., 1993 and

Guiot and van den Berg, 1985, Ramjeawon et al., 1995, Fang

and Kwong, 1994 Bardiya et al., 1995 and Ozatijrk et al.,

1993) for the treatment of low-medium strength wastewater.

However, the quantitative information on the process kinetics

and modeling aspects for this reactor capable of treating

high strength industrial wastewater is limited.

2
The process kinetics plays an important role in

development and operation of biological treatment systems. A

sound knowledge of process kinetics and mathematical models

are extremely essential for optimizing performance and

predicting the behavior of reactors in order to exercise

better control on process design and its operation. Numerous

mathematical models have been developed for the purpose of

defining the performance of the anaerobic reactors. Lawrence

and McCarty model (1970) used simplest Monod-type substrate

consumption equation and described that substrate consumption

is growth associated process. Bhatia et al. model (1985b)

demonstrated that methanogenesis is independent of cell

growth. Karhadkar et al. model (1990) has incorporated

process inhibition in determining the kinetics and predicting

the performance of anaerobic digester. Although many steady

state models have been developed to describe the behavior of

anaerobic reactors, the kinetic model incorporating the

equations for the consumption various type substrate is

required. Hence, the present study was undertaken to study

the biodegradation of spent wash in hybrid reactor with the

following specific objectives:

• To study the effect of hydraulic retention time (HRT)


on the performance of the reactor;

• To determine the kinetics of bacterial growth and


substrate utilization;

• To identify the mathematical models for prediction of


effluent substrate.

2 Materials and Methodology

3
2.1 Experimental set-up & design

The experimental set-up of the anaerobic hybrid reactor was designed as per the guidelines

given by Lettinga and Hulshoff (1991) and is shown in Fig. 1. The reactor was made of 6

mm thick transparent acrylic sheet with inner dimensions of 100 mm x 100 mm, height

1500 mm. The reactor was provided with hopper bottom of 150 mm length with top and

bottom widths of 25 mm and 100 mm, respectively. The feed inlet pipe of diameter 2.5 mm

was provided at the hopper bottom and the feeding system was designed to ensure the

uniform distribution of feed to the entire of the sludge bed area. The outlet pipe of diameter

2.5 mm was provided at the top of the reactor with the provision of sampling ports to

facilitate sampling. Gas Liquid Solid Separator (GLSS) device is made of square pyramid

with bottom dimensions, 80 mm X 80 mm. The gas collector of trapezoidal shape is

provided at the top with inclined walls at 500 and provision of gas outlet pipe.

The actual spent wash collected from SSK Distilleries Ltd., Niphad, Nasik and

neutralized with the lime to a pH of 7.0±0.5. The neutralized wastewater was fed to the

reactor at various HRT and organic loading to study the effect of HRT on the performance

of the reactor as shown in the experimental design of the study in (Fig. 2).

2.2 Analytical methods

Alkalinity, pH, COD, BOD5, TSS, TDS, SO4, Cl-, sulphide and VSS were analyzed in

conformity to Standard Methods for the Examination of Water and Wastewater (APHA,

AWWA, and WPCF, 1989).

Biogas generation was measured by using water displacement method for a

collection period of 3 hrs, twice in a day and average value for the particular day has been

estimated. For measuring the biogas, the vent pipes of the reactors are connected with 5

liter aspirator bottle filled with water. As the biogas enters the bottle filled with water, it

displaces same volume of biogas through the stop cock which was collected and measured

4
to find out total amount of biogas produced during the given period. Methane content of the

biogas was measured by injecting 1 ml of biogas through gas chromatograph equipped with

Thermal Conductivity Detector (TCD) using Porapack - Q (80-100), SS column 8' X 1/8"

(Karim, 2001; Karhadakar, 1988). The analysis with Porapack column was carried out at

oven temperature 1100C and detector temperature 2000C. The carrier gas, hydrogen, was

applied at a flow rate of 40-50 ml/min.

Volatile fatty acid in the effluent was measured by injecting 2 µl of filtered and

acidified samples through gas chromatograph equipped with Flame Ionization Detector

(FID) using 10 % FFAP on 60/80 Chromosorb WHP/0.1 % H3PO4 SS column. The analysis

was carried out at an oven temperature of 1500C, injector temperature of 1800C and

detector temperature of 2500C. Hydrogen and zero air were used to fuel the flame while

nitrogen as carrier gas was applied at the flow rate 20 mL/min.

2.3 Reactor start-up and acclimatization of seed culture

The reactor was inoculated with 232.5 g of anaerobically digested sludge (on VSS basis)

collected from sewage treatment plant. Table 1 shows the characteristics of the

anaerobically digested sludge used in the study. The quantity of seed sludge was estimated

as per the guidelines mentioned by Hickey et al. (1991b). The reactor was started using 1%

spent wash (v/v) as substrate and later the dilution was gradually decreased to acclimatize

the biomass for higher concentration of spent wash. The acclimation periods in case of

hybrid and UASB reactors were 185 days and 200 days, respectively.

2.4 Hydraulic retention time study

To study the effect of HRT, the reactor was operated in continuous mode at different

HRT’s ranging from 4 - 8 days. HRT of the reactors was gradually increased i.e. 4 days, 5

days, 6 days, 7 days and 8 days in order to find out the optimum hydraulic retention time at

5
which the COD removal and CH4 yield is maximum. The influent pH and alkalinity was

kept 7.00 ± 0.2 and 3950 ± 50 mg/l as CaCO3, respectively. The reactor was operated at

pseudo steady state condition for 20-25 days at every HRT and average value with standard

deviation for each parameter was worked out. The pseudo study state in the present study

was considered when the variation in effluent COD falls within ±5% of the average value.

2.5 Estimation of true bicarbonate alkalinity

According to Jenkins et al. (1991), the true bicarbonate alkalinity of the reactor is given as

under:
(1)
TBA = ( ALK 4.3 − 0.833VFA)
Where,

TBA = True bicarbonate alkalinity as CaCO3, mg/L

Alk4.3 = Total alkalinity as CaCO3, mg/L

VFA = Volatile fatty acids, mg/L

2.6 Determination of kinetics of biomass growth and substrate utilization

The linearised forms of the Lawrence and McCarty`s Model (1970) as given by Eqs. 2 and

3 were employed for the determining the kinetics of bacterial growth and substrate

utilization, respectively.
(2)
1 = γ m [(S0 − S ) θX ] − K d
θc

( Xθ S0 − S ) = [{(K s k ) *1 S } + 1 k ]
(3)

The linearised form of Bhatia et al Model (1985b) considering the process inhibition due to

toxicity was also employed in the study and is given as Eq. 4:

Sθ ( S 0 − S ) = 1 R + ( K i R ) * I s (4)
Where,

6
θc = Sludge retention time, days

Υm = Yield coefficient, mg biomass/mg substrate

So = Influent substrate concentration, mg/L

S = Effluent substrate concentration after timeθ, mg/L

θ = HRT, days

X = Biomass concentration, mg/L

Kd = Decay coefficient, d-1

Ks = Monond half velocity constant, mg/L

k = Maximum substrate utilization rate, d-1

Ki = Inhibition coefficient, L/mg

Is = Effluent sulfide concentration, mg/L

2.7 Model verification for prediction of effluent substrate

The models Bhatia et al. model (1985b) and Parkin and Speece (Non-competitive) models

(1982) as employed for the prediction of effluent substrate concentration are given by Eqs.

4 and 5, respectively.

S = S0 θ {(1 θ ) + R (1 + K i * I s )}
(5)

S = S0 − kXθ (1 + Tx K i ) (6)
Where,

θ = HRT, days

So = Influent substrate concentration, mg/L

S = Effluent substrate concentration, mg/L

R = Maximum rate of substrate utilization, d-1

7
Ki = Inhibition coefficient, L/mg

Is = Inhibitor (sulphide) concentration, mg/L

k = Maximum rate of substrate utilization, d-1

Tx = Inhibitor (hydrogen sulphide) concentration, mg/L

2.8 Model verification for prediction of biogas yield

Karhadkar et al model (1990) as given by Eq. 6 was employed for prediction of biogas

yield from the reactor. Accordingly, the biogas yield can be expressed by Eq. 7 as follows:

Qgas = GQ(S0 − S ) (7)


Where,

Q = Flow rate of influent, (L3T-1)

G = Conversion factor (M-1L3)

So = Influent COD concentration (ML-3)

S = Effluent COD concentration (ML-3)

Qgas = Biogas yield (L3T-1)

The value of G in the above model was determined experimentally.

3 Results and Discussion

3.1 Characterization of spent wash

The characteristics of the spent wash are given in Table 2. It can be seen from the table that

the spent wash is highly acidic in nature with a pH value ranging from 3.3 to 3.9. Apart

from the high COD (90,000-1,30,000 mg/L) and BOD (45,000-60,000 mg/L), spent wash

also contains high dissolve (70,000-78,000 mg/L) and suspended solids (9,000-10,000

mg/L). The SO4- and Cl- concentration in spent wash was found in the range of 6000-6500

8
mg/L and 5500–6000 mg/L, respectively. Analysis of the data demonstrated that the

characteristics of the spent wash are in agreement with the reported values by other

researchers (Preeti et al., 2006 and Karhadkar et al., 1990). A little variation in the

characteristics of spent wash as cited in the literature can be attributed to variation in

sources, storage conditions of molasses, and efficiency of the process adopted for the

extraction of ethanol during fermentation.

3.2 Hydraulic retention time study

The process performance data of HRT study i.e. COD removal; biogas generation and

sulphate reduction are shown in Figures 3, 4 and 5 respectively. It can be seen from Figures

3 and 4 that percent COD removal and methane yield per kg of COD removed, increased

with increase in HRT from 4 to 5 days and later decreased with increase in HRT beyond 5

days. The maximum COD removal, (79± 0.05%) and methane yield (0.344± 0.005%) was

found to be at HRT, 5 days and OLR, 8.70 kg COD/m3.day. Decrease in the performance of

reactor at higher HRT can be attributed to the reduction of sulphate into sulphide, which

might have inhibited the metabolism of methanogenic bacteria and reduced the COD

removal and methane yield. This can be seen from the sulphate reduction profile of the

reactor (Fig. 5). The sulphate reduction continuously increased with increase in HRT,

which subsequently increased the sulphide concentration inside the reactor and decreased

the COD reduction and methane yield at higher HRT. Many researchers (Bal, et al., 2001,

Parkin et al., 1983 and Lawrence et al., 1964) have reported sulphide toxicity to the

Methanogens particularly Acetate Utilizing and H2 Utilizing, who become inactive and

reduce the conversion of intermediates into final end products leading to accumulation of

VFA followed by decrease in methane yield. Bal et al. (2001) have reported that sulphide

concentration more than 100 mg/L is toxic for the acetate and H2 utilizing methanogens.

9
Parkin et al. (1983) have reported that sulphide toxicity to the bacterial population could be

observed at sulphide concentration of 50 mg/L. Lawrence et al.(1964) reported severe

process inhibition at sulphide concentration higher than 200 mg/L.

A relationship between volatile fatty acids, true bicarbonate alkalinity and total

alkalinity was worked out and is shown in Fig. 6. True bicarbonate alkalinity of the effluent

was estimated using the Eq. 1 (Jenkins et al., 1991) and compared with the experimental

value observed during the HRT study. It can be seen from the Figure 6 that the total

alkalinity of the effluent increased with increase in HRT. However, the bicarbonate

alkalinity of the effluent remained almost constant (3910 ± 74.16 mg/l) through out the

HRT study. The increase in total alkalinity with increase in HRT can be attributed to

increase in VFA concentration which contributed to VFA alkalinity in the effluent. Further,

it can also be seen that the observed bicarbonate alkalinity are almost same with the TBA as

estimated by the Jenkins et al. equation with standard deviation of ±18.08 mg/l (Fig. 6).

This indicates that Jenkins et al. formula can be used to find out true bicarbonate alkalinity

in the present case.

The VFA/alkalinity ratio varies from 0.02 to 0.14 at all HRT’s. Moreover, the

effluent alkalinity is higher than the influent alkalinity. This indicates that enough buffering

capacity was present in the reactor due to which decrease in pH was not observed even after

increase in the concentration of VFA in the reactor. The pH of the effluent at all HRT was

found in the range of 7.8 to 8. This indicates that the efficiency of the reactors decreased

due to sulphide inhibition rather than VFA inhibition. The increase in VFA concentration in

the reactor merely represents the incomplete conversion of VFA into final end product

(CH4) due to sulphide toxicity to the methanogens.

10
A relationship between specific gas production and organic loading rate was

established (fig. 7), which yielded a good correlation (R2 = 0.994) between organic loading

rate and specific gas production. This indicates biogas production is strongly correlated

with the organic loading rate and is in accordance with the fundamentals of anaerobic

treatment.

3.3 Kinetics of bacterial growth

In order to determine the kinetics of biological growth, the parameter 1/θc versus (So-S)/Xθ

of Eq. 2 were calculated from the process performance data of the reactor and plotted as

shown in Figure 8. The plot of 1/θc versus (So-S)/Xθ yielded a straight line (R2 = 0.985)

whose slope gives biomass yield coefficient (Y = 0.0532) and intercept gives bacterial

decay coefficient (Kd = 0.0041d-1). These values compare well with the reported values of

Y and Kd by many researchers (Karhadkar et al., 1990, Prakash, 1998 and Karim, 2001)

3.4 Kinetics of substrate utilization

The relationship among the parameters 1/S versus Xθ/(So-S) were plotted (Fig. 9) based on

the performance of the reactor at various HRT. However, no correlation could be

established between these parameters showing non-conformity of Lawrence and McCarty’s

model. For a meaningful model, the values of k and Ks should be positive, which do not

seem to be possible in the present case. The reason for non-compliance of this model for

the present system could be as under:

• The model does not take into consideration the process inhibition. However, in

present case, COD reduction and methane formation is strongly related to sulphide

inhibition as is evident from the HRT study.

11
• Secondly, the model assumes growth-associated methanogenesis, which does not

appear to be true in the present case. The yield coefficient as estimated in

accordance with the model is significantly higher than Kd value indicating

substantial growth of microorganism. However, no substantial growth of biomass

was observed in the present case despite substrate conversion to methane. The fact

is well supported from the mass balance of COD reduction and gas production

(Table 3) which shows that out of total substrate consumed, most of the substrate

(90.2%) is utilized in biogas production while only a little substrate (6.72%) is

utilized for sludge growth indicating non-growth-associated methanogenesis in the

present system. Similar observations were made by Karhadkar et al. (1990) and

Bhatia et al. (1985b).

3.5 Identification of suitable model for the present system

The relationship for the substrate utilization as given by Eq. 4, is not bound with the

limitations of Lawrence and McCarty`s model as discussed earlier (Bhatia et al., 1985b). In

addition, the concept of non-growth-associated methanogenesis as assumed in this model

supports the observations made in this study i.e. high rate of substrate conversion to

methane despite of low biomass yield. Because of the above-mentioned reasons, Bhatia et

al. (1985b) model offering the following advantages over Lawrence and McCarty`s model

is considered to represent the substrate utilization in this case.

• It explains the high rate of substrate conversion to methane despite of low biomass

yield on the basis of non-growth-associated methanogenesis.

• Substrate utilization rate has inbuilt provision for incorporating process inhibition

due to toxicity.

12
3.6 Determination of kinetic coefficients of Bhatia et al. model

A relationship was established among the parameters Sθ/(So-S) versus (Is) based on the

process performance data of reactor at various HRT’s (Fig. 10). The plot of these two

variables established a good correlation (R2 = 0.9743) and gives maximum rate of substrate

utilization (R) and inhibition coefficient (Ki) to be 1.945 mg/mg/d and 0.032 l/mg,

respectively. The kinetics of substrate utilization as obtained in the present case is in good

agreement with the values reported by other researchers (Karhadkar, 1988, and Prakash,

1998).

3.7 Model verification for prediction of effluent substrate

Using Bhatia et al. model (Eq. 5) and Parkin and Speece model (Eq. 6) the effluent

substrate concentration (S) was predicted at various HRTs and compared with the

experimental values as obtained during HRT study. Figures 11 and 12 show the plots of

observed and predicted effluent substrate concentrations using Bhatia et al model, and

Parkin and Speece model, respectively. The comparison of observed and predicted values

reveals a good agreement between the two. Bhatia et al. model predicts effluent substrate

within ±5-20% of the experimental value. However, the model suggested by Parkin and

Speece predicted effluent substrate more precisely (within ±2 % of the experimental

values).

3.8 Model for prediction of biogas yield

13
As per the Karhadakar et al. model (Eq. 7)), the biogas production at various HRT’s were

predicted and compared with the experimental values (Fig. 13). The value of conversion

factor (G, 0.52 m3/kg COD) was determined experimentally. The comparison of observed

and predicted values reveals a good agreement between the two. The model predicted

biogas yield within ± 5 % of the observed value.

4. Conclusions

The following conclusions can be drawn from the present study:

1. Anaerobic hybrid reactor can successfully be employed for the treatment of

distillery-spent wash. The optimum COD removal efficiency of the hybrid reactor

found to be 79% corresponding to optimum HRT and organic loading rate of 5 days

and 8.7 kg COD/ m3.d.

2. Kinetics of bacterial growth (Y, 0.0532 and Kd, 0.0041 d-1) can be determined using

Lawrence and McCarty model. However, the model fails in determination of

kinetics of substrate utilization.

3. Bhatia et al. model having the inbuilt provision of process inhibition can be used for

determining the kinetics of substrate utilization (R, 1.945 d-1 and Ki 0.032 L/mg).

4. Parkin and Speece model can be more precisely used for modeling the performance

of anaerobic hybrid reactor for the anaerobic treatment of spent wash treatment than

Bhatia et al. model. The model predicts effluent substrate within ± 2 % of the

experimental value.

14
5. Kerhadakar et al. model can be used to predict the biogas yield of the anaerobic

hybrid reactor for the treatment of spent wash. The model predicts gas yield within

±5 % of the experimental value.

15
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Nomenclature

θc = Sludge retention time, days


Υm = Yield coefficient, mg biomass/mg substrate
So = Influent substrate concentration, mg/L
S = effluent substrate concentration after timeθ, mg/L
θ = HRT, days
X = Biomass concentration, mg/L
Kd = Decay coefficient, d-1
Ks = Monod half velocity constant, mg/L
k = Maximum Substrate Utilization Rate, d-1
Ki = Inhibition coefficient, L/mg
Is = Effluent sulfide concentration, mg/L
R = Maximum rate of substrate utilization (T-1)
Tx= Inhibitor (hydrogen sulphide) concentration (ML-3)
Q = Discharge/flow rate of influent (L3T-1)
G = Conversion factor (M-1L3)
TBA4.3 = True bicarbonate alkalinity as CaCO3, mg/L
Alk4.3 = Total alkalinity as CaCO3, mg/L
VFA = Volatile fatty acids, mg/L

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Table Legend

Table 1: Characteristics of Anaerobically digested sludge


Table 2: Characteristics of spent wash collected from S.S.K. Distilleries (Ltd.), Niphad,
Nasik, Maharashtra.
Table 3: Mass balance of COD and biogas yield at optimum HRT, 5 days.

Figure Legend

Fig. 1: Anaerobic Hybrid Reactor.


Fig. 2: Experimental design of HRT study.
Fig. 3: COD removal profile at various HRT’s.
Fig. 4: Biogas generation profile at various HRT’s
Fig. 5: Sulfate removal profile at various HRTs
Fig. 6: Relationship of VFA and various forms of alkalinity
Fig. 7: Relationship between specific gas production and organic loading rate
Fig. 8: Estimation of Kd and Y of the biomass growth using Lawrence and McCarty model
Fig 9: Estimation of k and Ks using Bhatia et al. model.
Fig. 10: Estimation of R and Ki of substrate utilization using Parkin and Speece model.
Fig 11: Comparison of experimental and predicted effluent COD of the reactor using Bhatia
et al. model.
Fig 12: Comparison of experimental and predicted effluent substrate by Parkin & Speece
Model
Fig. 13: Comparison of observed and predicted biogas yield using Karhadkar et al. model.

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