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Anaerobic pilot-scale treatment of a tetrachloethylene-rich synthetic effluent with morphological study of granules.


Tetrachloroethylene (PCE) is widely used in industries as solvents, surface coatings, dry cleaning and degreasing agent. U.S. Environmental Protection Agency has designated PCE as one of the priority pollutants listed under National Priority Pollutant (EPA 1977) [1]. PCE is also considered to be major groundwater contaminants [2]. The removals of chlorinated compounds are of prime concern and major area of research because of their known toxic and carcinogenic effects on humans [3].

There are number of treatment methods i.e. adsorption, using UV light, etc have been developed by several researchers which have been found to be effective for the treatment of PCE [4] and [5]. However, these methods involve use of chemicals, adsorbents, reactivation of adsorbent, UV Light sources, etc. which make them costlier and impracticable. In addition, the generation of chemical sludge, exhausted adsorbents etc. used in the processes creates further disposal problems.

Being cost effective, the biological degradation of PCE either aerobically or anaerobically could offer a better alternative. Many researchers found that PCE can successfully be degraded by aerobic treatment method [6] and [7]. However, the volatilization (stripping off) of PCE in aerobic treatment process causing direct air pollution is a major constraint and concern associated with the process [8]. In this context, anaerobic process is gaining more attention among the researchers and scientists for the treatment of PCE [9] and [10].

Wastewater treatment efficiency of a bio-reactor lies in the biomass retention and its proper granulation inside the reactor. The development of granular sludge is the key factor for the successful operation of the upflow anaerobic sludge blanket (UASB) reactor [11], [12], and [13]. With the development of good quality of granular sludge maximum pollutant removal efficiency even at higher organic loading rate can be achieved. The poor granulation, low sludge retention time and in some cases washout of sludge in currently used UASB reactor have been the reported by many researchers [14] and [15]. Hence the present study was undertaken to assess the biodegradation of PCE in anaerobic hybrid reactor (AHR) combining the sludge blanket in the lower part and an inert filter media in the upper part which provided the dual advantages of both suspended and attached growth of microorganisms in a single system and at the same time the sludge washout rate was reduced and the sludge retention time was increased. In this context, the studies on start-up, acclimatization and granulation of biomass have mainly been emphasized in this paper.

Materials and methodology

Experimental set-up

In order to achieve the objectives, a lab scale model of anaerobic hybrid reactor was designed and fabricated as per the guidelines provided by Lettinga et al.. (1991), [16]. Figure 1 shows the schematic view of the experimental set-up. The reactor was made of 6 mm thick transparent acrylic sheet with inner diameter ([empty set]) 100 mm and height 1500 mm. A hopper (150 mm height) was provided at the bottom of the reactor. The feed inlet pipe of diameter 2.5 mm was provided at the centre of hopper bottom to ensure the uniform distribution of feed through the entire sludge bed area. The feed (synthetic wastewater) containing PCE was fed to the reactor through inlet pipe ([empty set] = 25 mm) using a peristaltic pump. A filter media (300 mm height) consisting of 25 mm diameter PVC pipe was provided below the outlet. The outlet pipe of 2.5 mm diameter was provided at the top of the reactor with the provision of sampling ports to facilitate sampling. Gas Liquid Solid Separator (GLSS) device made of square pyramid with bottom dimensions, 80 mm x 80 mm and side slopes of 50[degrees] was provided at the top to facilitate the separation of gas and biomass and effluent. Gas outlet pipe of 25 mm diameter was provided at the apex of the reactor for facilitating outlet of biogas produced in the reactor.


Experimental protocol

The anaerobic sludge was brought from the waste water treatment plant treating domestic waste water. The sludge was first screened to remove debris using 150 [micro]m sieves. 5 L of sludge with volatile suspended solids (VSS) concentration of 15 g /l was transferred to the AHR. The start up study was carried out by feeding the synthetic wastewater containing 1000 mg/l of COD at a hydraulic retention time (HRT) of 24 hours. The composition of synthetic wastewater is given in Tables 1 and 2 respectively.

After the start up of the reactors, the acclimatization study was performed in two phases. In the first phase of acclimatization, the influent COD concentration (1000 mg/l) was kept constant and the influent PCE concentration was gradually increased from 5 mg/l to 50 mg/l (5, 10, 15, 20, 30, 40, 50 mg/l) in a step wise manner. In the second phase of the acclimatization, the influent PCE (50 mg/l) concentration was kept constant while the influent COD concentration was gradually increased from 1000 mg/l to 2000 mg/l (1250, 1500, 1750 and 2000 mg/l) in a step wise manner. The reactor was run for 10 days at steady state condition. The HRT of 24 hrs was kept constant throughout the study.

A mass balance of PCE was carried out to evaluate the percentage of influent PCE converted to several intermediates like trichloroethylene (TCE), Dichloroethylene (DCE), vinyl chloride (VC) and ethylene.

The granulation studies were conducted in parallel in order to study the changes in the characteristics of the sludge granules treating PCE at different stages.

Analytical chemicals

The chlorinated organic compounds used in the study were PCE (Merck India, 98%), trichloroethylene (TCE) (Merck India, 98%), trans-1, 2-dichloroethylene (trans-DCE) (Aldrich Chemical, Wisconsin, USA, 98%), cis-1, 2-dichloroethylene (cis-DCE) (Aldrich Chemical, Wisconsin, USA, 98%), and vinyl chloride (VC) (Sigma Aldrich, India, 98%). Standard gases were, 5% (v/v) methane (Span gas India) and 108 ppm ethylene (Span gas India).

Analytical methods

pH, alkalinity, COD, VSS, and sludge volume index (SVI) were analysed as per the standard method [17] and the rate of biogas production was measured by water displacement method.

The PCE, TCE, DCE, and VC were analysed by injecting 100ul headspace sample to Gas Chromatograph (GC 2000A) equipped with 8"x1/8" OD, stainless steel column packed with OV-101 on 60/80 Chromosorb WHP. The oven temperature was kept 160 [degrees]C and injector and detector temperatures were kept at 200[degrees]C. The carrier gas nitrogen was passed at a flow rate of 20 ml/min. Hydrogen and zero air was used to fuel the flame. Calibration standards were prepared from stock solutions of 100 mg/l of PCE, TCE, DCE, and VC in methanol. Each sample was analyzed in triplicate and the average readings were considered for estimating the concentration of PCE, TCE, DCE, and VC. The analysis of methane and ethylene was carried out by GC equipped with FID and fitted with N-octane on Porasil-C 80/100 mesh, 2m x 3m stainless steel packed column. Oven temperature was maintained at 40[degrees]C, whereas injector and detector temperatures were kept at 100[degrees]C. Nitrogen was used as carrier gas at a flow rate of 20ml/min.

The average settling velocity of the sludge was determined by a column of 5 cm inner diameter and 1m height filled up with water. A measured volume of sludge (5ml) was added to the column. The amount of sludge settled at the bottom was collected after fixed intervals (0.5, 1, 1.5, 2.5, 5, 7.5, 15 minutes) and suspended solid was determined for each sample, which showed the fraction of the sludge settled in that interval of time [18]. The average settling velocity was calculated as:

Avg. Settling Velocity (m/h) = ([summation]Wt. fraction settled x Settling Velocity)/ Total Wt. of sludge sample (1)

Size distribution of the granules were determined by taking the sludge samples from the bottom, middle and top of the sludge bed and analyzed to find out the size distribution across the sludge bed height. Stokes law was used to calculate the size of the granules. According to stokes law the settling velocity of the particle through a column can be described by the expression given in Eq. (1)

[V.sub.c] = [{g. ([[rho].sub.s]-[rho]) [d.sup.2]}/ 18[micro]] (2)


[V.sub.c] = settling velocity of the particle, m/h

g = acceleration due to gravity = 9.81 m/[s.sup.2]

d = diameter of the particle, mm

[[rho].sub.s] = density of the particle (granules)

[rho] = density of the fluid (water), at 20[degrees]C = 998.2 kg/[m.sup.3]

[micro] = dynamic viscosity of the fluid, (water) at 20[degrees]C = 1.002 x [10.sup.-3] N-S/[m.sup.2]

The percentage of granules in the sludge was determined by the formula given below:

Percentage of granules = ([X.sub.r]/X) x 100 (3)


[X.sub.r] = VSS of the granular fraction of the sludge in mg

X = VSS of the total sludge sample in mg

The SEM analysis of the sludge were carried out. For this, the granular sludge samples were washed with 0.1 M phosphate buffer three times and fixed in phosphate buffered gluteraldehyde (96%) overnight. Then fixed samples were washed with 0.2 M sodium cacodylate buffer five times and subsequently dehydrated. Dehydration was accomplished by passing the fixed samples through a graded series of acetone-distilled water solution including 30, 50, 70, 90, 95 and 100 % acetone. During dehydration one solution was removed carefully with a fine pipette or syringe and the next poured on. Dehydration was done with gradual increase in temperature so that 90-95% strength solution is reached at room temperature. After dehydration the samples were dried by critical point drying method. The dried samples were mounted on a sample stub and were sputter-coated with gold-palladium in a sputter coating unit (Polarson SC 7610) and viewed under a Camera Scanning Electron Microscope (FEI, Quanta-300).

Results and discussion

Performance of Reactor during start-up study

The performance of the reactor in terms of percentage COD reduction and biogas production during start-up study is shown in Figure 2. It can be seen from the figure that in the beginning (first day) the COD reduction was 52.68% which increased gradually with time and found to be 98.4 [+ or -] 0.5% during the pseudo steady state condition. The reactor took 52 days to arrive at the pseudo steady state conditions. Similar trend in biogas production profile was also observed. The biogas production also increased gradually and found to be 7.47 [+ or -] 0.04 l/d during pseudo steady state condition with methane content 67 [+ or -] 1.5%. The study demonstrated that start-up of anaerobic hybrid reactors can be achieved in 52 days. Similar start-up periods were reported by many other scientists in the literature. [19] and [15]. However, many researchers [13], [20] and [21] have reported long start-up periods of 2 to 3 months to 1 year (or even more) for the anaerobic reactors. This may be attributed to varying nature the compound and other operating conditions stipulated in different studies.


Performance of reactor during acclimatization study

Acclimatization of seed sludge helps in genetic changes in microorganism and induction of biodegradation capability in microorganisms [22]. For the acclimatization of biomass the target compound (PCE) was added in the influent at an initial concentration of 5 mg/l which gradually increased up to 50 mg/l in a stepwise manner. Figure 3 shows the performance of the reactor during the first phase of acclimatization. It can be seen from the figure that in the beginning (influent PCE concentration of 5 mg/l) the PCE reduction percentage was marginal which increased to 99.83 [+ or -] 0.14 % at the during the pseudo steady state condition after 11 days of operation. It was also observed that during each step increase in the influent PCE concentration, the percentage PCE reduction suddenly dropped down and then increases gradually till the pseudo steady state was achieved. The reactor took 181 days to get acclimatised for influent PCE concentration of 50 mg/l at which the percentage PCE reduction was 99.92 [+ or -] 0.01%. Chu and Jewell (1994), reported that influent PCE concentration of 8.2-26 mg/l were reduced to less than 0.2 mg/l (>98% removal) using anaerobic attached film expanded bed reactor [23]. The Similar trend in percentage COD reduction was also found. The percentage COD reduction increased gradually and reached to 98.1 [+ or -] 0.51% after the first phase of acclimatization. It was observed that the biogas production was at the rate of 7.6 [+ or -] 0.04 l/day at steady state condition with 65-66% of methane content in the biogas after this phase of acclimation.


After the completion of first phase of acclimatization, the influent COD concentration was increased from 1000 mg/l to 2000 mg/l in stepwise manner keeping influent PCE concentration constant at 50 mg/l for the second phase of acclimatisation of biomass to influent COD concentration of 2000 mg/l. The performance of the reactor in terms of percentage PCE and COD reduction during the second phase of acclimatization is shown in Figure 4. It was observed that during each step increase in the influent COD concentration, the percentage PCE reduction reduced slightly and then increased gradually till the pseudo steady state was achieved. The percentage PCE reduction (99.85 [+ or -] 0.005%) after this phase of acclimatization was found to be slightly lower than the first phase of acclimatization (99.92 [+ or -] 0.01%). The probable reason of the slight decrease in percentage PCE removal may be due to increase in organic loading rate which might cause competitive inhibition in biodegradation of PCE. The reactor took 72 days for this phase of acclimatization. The COD reduction in this phase was found to be 96.1 [+ or -] 0.72%. The biogas production was found to be 14.63 [+ or -] 0.16, with methane content of 67% at steady state condition after this phase of acclimation. The increment in biogas production in second phase of acclimation is due to the biodegradation of high concentration of organic matter (COD concentration around 2000 mg/l). The findings of the biogas production in this study are quite similar to the findings of Verstrate (1983), who stated that anaerobically 1 kg COD gives rise to 0.5 [m.sup.3] of biogas [24].


Mass balance for PCE biodegradation

The dehalogination by-products of PCE such as TCE, cis-DCE, VC and ethylene were detected in the effluent and biogas at different concentrations throughout the study. Many researchers reported the dechlorination of PCE occurred through TCE, cis-DCE, VC and ethylene [25], [26], [27], and [2]. Ethylene is a non chlorinated environmentally acceptable product.

PCE fed to the reactor can theoretically be removed by adsorption in the sludge, biological degradation or stripping into the gas phase. Since the sludge was in the saturated condition the removal of PCE by adsorption is neglected. Hence biological degradation and stripping was considered for the dehalogination of PCE. The probable reason of stripping into gas is due to volatile nature of these compounds as well as due to high rate of biogas production from the sludge bed of the reactor. Using influent PCE concentration as a parameter, a mass balance considering the relative distribution of PCE, TCE, cis-DCE, VC and ethylene in liquid and gas phase was carried out. The stripped fraction in the produced gas was converted to the corresponding hypothetical liquid phase concentration using Henry's law [28]. It was observed that out of total influent PCE fed to the reactor approximately 1.58% , 1.15%, 8.4%, 19% and 62.67% of PCE, TCE, cis-DCE, VC and ethylene respectively remained in the effluent and biogas (Fig.5). Remaining 7.2% was un accountable. In previous studies, mass balance for PCE demonstrated an almost 95-98% conversion of PCE and intermediates in an anaerobic fixed bed column [26] and 98.9% conversion of PCE and intermediates in an expanded-bed granular activated carbon anaerobic reactor [29].

Characterization of granular sludge

In order to study the granulation of the flocculent biomass, the anaerobic sludge were characterized for various parameters i.e., average settling velocity, SVI, VSS, morphology (shape & size) and SEM analysis.

Average settling velocity and SVI of granular sludge

The average settling velocity was increased from 27.55-35.5 m/h after the completion of startup period which further increased to 47.15 m/h after first phase of acclimatization and finally reached to 63.35 m/h after completion of the second phase of acclimatization (Figure 6). This may be attributed to the conversion of flocculate sludge into compact granular sludge. The decrease in SVI of the sludge during the start up (from 30.55-27 ml/g SS), first phase (from 27 to 21.1 ml/g SS) and second phase (from 21.1-13.54 ml/g SS) of acclimatization indicates the gradual compaction of flocculent sludge into more compact granular sludge which in turn leads to increase in settling velocity and decrease in SVI of granules (Figure 6). The value of SVI found in this study is comparable to the value of SVI (10-20 ml/g SS) reported for the good quality of granules [30] and [31].


VSS profile of granular sludge

The VSS profile of sludge increased gradually from 15 g/l to 24.9 g/l after the entire phase of acclimation (Fig.7). This also indicates the build-up of concentrated sludge inside the reactor. Gupta and Gupta (2005) reported an increase in VSS concentration of sludge from 14000 mg/l to 32000 mg/l in hybrid reactor and 30000 mg/l in UASB reactor treating distillery spent wash [31]. The marginal increase in VSS profile in the present study is due to the inhibitory effect of PCE on growth of the biomass. Yang and McCarty (2000) reported that high concentrations of PCE, cis-DCE, and ethene were inhibitory to methanogens, and high concentrations of PCE were inhibitory to homoacetogens [32].


Size distribution of the granules across the sludge bed

The morphological study of the sludge revealed that in the beginning almost 100 % of the sludge was < 0.5 mm diameter in size which gradually increased during different phases of reactor operation i.e. start up, first phase and second phase of acclimatization {Figure 8 (a, b, c, and d)}. It can be seen from the figure that in the beginning the entire sludge was flocculent of size < 0.5 mm which increases to > 2 mm at the end of the second phase of acclimatization. The size range of the granules found in this study is comparable with the size of the granules 0.92 mm to 2.1 mm reported by other researchers [33], [15], and [34]. Dubourgier et al.. (1987) who suggested that the granulation mechanism starts by the covering of filamentous Methanothrix by colonies of cocci or rods (acidogenic bacteria), forming microflocs of 10-50 [micro]m. Next, the Methanothrix filaments, due to its particular morphology and surface properties, might establish bridges between several microflocs forming larger granules (>200 [micro]m). Further development of acidogenic and syntrophic bacteria favours the growth of the granules [35].

The size distribution of the granules through the sludge bed demonstrated that granules of larger diameter (> 2mm) were mainly concentrated at the lower active zones while smaller sized granules (< 0.5-2 mm) were at the top and middle zones of the sludge bed.

SamSoon et al.. (1987) and Moosbrugger et al.. (1993) reported that the presence of larger size granules at the lower part indicates that granules have been essentially formed at the bottom of the sludge bed and the biomass mainly existing up to lower part of the sludge bed. When these granules move towards top, the loosely packed fraction of granules get sheared off and thus making the size smaller [36] and [37]. The above observations supports the hypothesis given by Wentzel et al.. (1994) that the granulation takes place mainly in the lower active zone and breakup in the upper zones [38]. Sponza (2001) observed an initial granules development after 1.5 months of start-up, which grew at an accelerated pace for 7 months and then became fully grown at a maximum diameter of 2.5 mm treating PCE in an UASB reactor [39].


SEM analysis of the granules

The SEM study of granules showed the shape of the granule and a heterogeneous bacterial population on the surface of the granules {Figure 9 (a and b)}. Cocci shaped {Figure 9 (c)} showing the typical morphology of Methanosarcina types of bacterial species as given by Zhao et al., (1985) [40] and long filamentous rods {Figure 9 (d)} showing the typical morphology of Methanothrix types of bacterial species as given by Zinder et al., (1984), [41] and, were present over the surface of the granules. The SEM study of the granulated sludge treating PCE in UASB reactor by Prakash and Gupta, (2000) showed the presence of Methanothrix and Methanosarcina types of bacterial species on the surface of the granules [42]. Fathepure et al., (1987) reported that Methanosacina sp and Methanothrix sp. culture found to be very effective for the treatment of PCE [43].



The anaerobic hybrid reactor was found to be very effective in treating PCE. After the first phase of acclimation, the percentage COD and PCE removal was found to be 98.1 [+ or -] 0.51% and 99.92 [+ or -] 0.01% respectively and after second phase of acclimation, the percentage COD and PCE removal was found to be 96.1 [+ or -] 0.72 and 99.85 [+ or -] 0.005% respectively at steady state condition. The biogas produced during the biodegradation process is very rich in methane which can be used as an economic bio-fuel.

Granules of size (<0.5 mm - >2 mm) with settling velocity of 27.5-63.35 m/h and SVI of 30.55-13.54 ml/g SS with VSS content of 24.9 g/l were developed in the AHR. SEM study of the granules showed the probability of the presence of Methanosarcina and Methanothrix type of species on the surface of granules.

The effluent dehalogination intermediates like TCE, DCE and VC were found to be very less but still these are a matter of prime concern. This AHR in combination with aerobic treatment or physicochemical treatment will be proved to be very effective in PCE removal with negligible accumulation of these dehaloginated intermediates.


The authors are thankful to Professor T. Kumar, Director, Indian School of Mines, (ISM) for providing necessary facilities in carrying out this research. ISMU fellowship to one of the authors (Shibam Mitra) is gratefully acknowledged.


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* Shibam Mitra (1), S.K. Gupta (2) and Gurdeep Singh (3)

(1) Senior Research Scholar, (2) Assistant Professor, (3) Professor and Head

(1, 2, 3) Centre of Mining Environment, Dept. of Environmental Science and Engineering, Indian School of Mines University, Dhanbad-826004, Jharkhand, India

* Corresponding Author E-mail:

E-mail: (2), (3)
Table 1: Composition of synthetic wastewater.

Sl.No Compounds Concentration (mg/l)

1 Sodium acetate 789-1578
2 Methanol 222-444
3 Acetone 151-302
4 PCE 5-50
5 [K.sub.2]HP[O.sub.4] 8.4-16.8
6 K[H.sub.2]P[O.sub.4] 15.2-30.4
7 [(N[H.sub.4]).sub.2] S[O.sub.4] 20-40
8 N[H.sub.4]Cl 100-200
9 NaHC[O.sub.3] 1000-2000
10 Ca[Cl.sub.2] x 2[H.sub.2]O 294

Table 2: Composition of trace metal solution.

Sl.No Compounds Concentration (mg/l)

1 MgS[O.sub.4] x 7[H.sub.2]O 5000
2 Fe[Cl.sup.2] x 4[H.sub.2]O 6000
3 Co[Cl.sub.2] x 4[H.sub.2]O 880
4 [H.sub.3]B[O.sub.3] 100
5 ZnS[O.sub.4] x 7[H.sub.2]O 100
6 CuS[O.sub.4] x 5[H.sub.2]O 1000
7 NiS[O.sub.4] x 8[H.sub.2]O 1000
8 Mn[Cl.sub.2] x 4[H.sub.2]O 5000
9 [(N[H.sub.4]).sub.6] [Mo.sub.7] 640
 [O.sub.24] x 4[H.sub.2]O

Figure 5: Mass balance for PCE at 24 hrs of HRT.

PCE 1.58%
TCE 1.15%
DCE 8.4%
VC 19%
Ethylane 62.67%
Unaccounted 7.2%

Note: Table made form pie chart.
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Author:Mitra, Shibam; Gupta, S.K.; Singh, Gurdeep
Publication:International Journal of Applied Environmental Sciences
Date:Nov 1, 2010
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