Abstract

Mixed microbial culture collected from the wastewater treatment plant of Indian Institute of Technology Guwahati (IITG) was further grown in anaerobic condition in presence of sulfate where lactate was added as a carbon source. Sulfate addition was increased stepwise up to 1,000 mg l−1 before phenol was added at increasing concentrations from 10 mg l−1 to 300 mg l−1. Kinetics of sulfate, phenol and chemical oxygen demand reduction were studied and experimental findings were analyzed using various bio-models to estimate the bio-kinetic coefficients. This is the first detailed report on kinetics and bio-kinetic studies of sulfate reduction in presence of phenol. Experimental results showed that there was no inhibition of sulfate reduction and microbial growth up to 100 mg l−1 phenol addition. However, inhibition to different degrees was observed at higher phenol addition. The experimental data of microbial growth and substrate consumption in presence of phenol fitted well to the Edward model (R2 = 0.85, root mean square error = 0.001011) with maximum specific growth rate = 0.052 h−1, substrate inhibition constant = 88.05 mg l−1 and half saturation constant = 58.22 mg l−1. The characteristics of the cultured microbes were determined through a series of analysis and microbial tests.

INTRODUCTION

Phenol and its compounds, owing to toxicity, have been categorized in the list of priority pollutants by the US Environmental Protection Agency (Huang et al. 2014; Yagmur et al. 2017). This toxicity is related to two main processes: (i) unspecified toxicity related to hydrophobicity of the individual compound and (ii) formation of free radicals. The concentration of phenol in surface water generally varies between 0.01 and 2.0 μg l−1. The sources of phenol include oil refineries, chemical plants, explosives, resins, coke manufacture, coal conversion plants, pesticide and textile industries (Wang et al. 2014; Wirth et al. 2015). These compounds have high stability, high toxicity and are carcinogenic in nature even at low concentrations. Hence, phenol-containing wastewater needs careful handling before it is discharged to the receiving water bodies. The bio-reduction using micro-organisms is a viable and well proven method for phenol remediation (Wu et al. 2016; Kiliç & Dönmez 2017). However, wastewaters containing phenol in the range of 5–500 mg l−1 are generally considered suitable for treatment by biological processes (Monteiro et al. 2000). The utilization of phenol as a substrate can be accomplished when the microbial mass can be acclimatized to it for a sufficient time. Although it is toxic, phenol can be utilized as a carbon source by various micro-organisms. For example Pseudomonas putida (Ray & Banerjee 2015; Kurzbaum et al. 2017; Lin et al. 2017), Candida tropicalis (Basak et al. 2013; Phalgune et al. 2013; Mahgoub et al. 2014), Acinetobacter calcoaceticus (Jiang et al. 2013; Lin 2017), and Alcaligenes eutrophus (Ahmad et al. 2017) have been widely used to study the biodegradation of phenol. However since isolation of single strains is both difficult and limited in field applications, it is urged that mixed microbial consortia could be used for the biodegradation of phenol (Ray & Banerjee 2015). In sulfate bio-reduction, sulfate-reducing bacteria (SRB) utilize sulfate as the electron acceptor in the presence of a suitable electron donor, preferably a carbon source. So far various organic compounds such as lactate, acetate, methanol, ethanol, and molasses have been used as electron donors to study the effect on sulfate reduction (Jing et al. 2013; Mattei et al. 2014; Xu et al. 2014). Phenolics along with sulfate appearing in various industrial sources such as coke oven plants, paper mills, pharmaceuticals, and tanneries pose a huge environmental hazard and hence remediation of such pollutants becomes necessary. However, the investigation of sulfate reduction in presence of phenol is limited. Boopathy (1985) studied sulfate reduction using phenol as a sole carbon source and a sulfate-reducing bacterium obtained from swine manure, which could degrade 51.7 mg l−1 (0.55 mM) of phenol. Mort & Dean Ross (1994) found sulfate reduction abilities in bacteria obtained from freshwater river sediments, which consisted of SRB, when phenol was introduced as the carbon source without any prior acclimation. However, the biodegradative phase for phenol reduction by acclimated cultures varied from 25 to 40 days. Lin & Lee (2001) studied sulfate reduction in an anaerobic bio-film process composed of mixed culture of SRB and phenol-utilizing bacteria (PUB). They described that simultaneous removal of phenolic compounds and sulfate follows metabolic pathways in which biodegradation of phenolic compounds to simple organic acids was carried out by PUB, while bio-reduction of sulfate to sulfide was carried out by SRB by utilizing simple organic compounds as the electron donor and sulfate as the terminal electron acceptor. To describe substrate biodegradation, it is necessary to evaluate the kinetics and bio-kinetics. While kinetics deals with the study of substrate degradation through determination of various reaction rate coefficients, bio-kinetics involves determining a relationship between the specific growth rate and the substrate concentration, which affects the biodegradation of any pollutant. Knowledge of such behavior is necessary for improvements in the process control and removal efficiency.

The aim of the present study is to investigate the effect of phenol on sulfate reduction using mixed microbial consortia. Based on the results obtained, kinetic studies pertaining to sulfate, chemical oxygen demand (COD) and phenol reduction were analyzed. In addition, bio-kinetic coefficients were evaluated using different existing models.

MATERIALS AND METHODS

Seed sludge enrichment

A mixed microbial culture (total solids = 20.506 g l−1, mixed liquor volatile suspended solids, MLVSS = 16.5 g l−1) was obtained from the wastewater treatment plant of the Indian Institute of Technology (IIT) Guwahati. The culture was grown in a 1-litre Erlenmeyer flask by adding lactate and sulfate. The composition of the media with major nutrients in Milli-Q water was (in g l−1): KH2PO4, 0.5; K2HPO4, 0.1; NaHCO3, 0.5–0.8; NH4Cl, 0.1. To this, 1 ml trace nutrient was added to 1 l of media. The composition of trace nutrient media was (g l−1): NaMo4.2H2O, 0.03; CaCl2.2H2O, 0.05; CoCl2.6H2O, 0.02; KCl, 0.05; MgCl2.6H2O, 0.05; NiSO4.6H2O, 0.01; C6H7NaO6, 0.05 and C2H3NaO2S, 0.05. The culture was kept under constant agitation at 180 rpm and temperature of 30 ± 0.2 °C. After due course of time, the culture from the flask was again transferred into a 500 ml aspirator bottle and fed with the same nutrients as described above for a further acclimation phase. This was marked as ‘seed sludge’ in which the MLVSS was found to be 1,224 mg l−1. During this growth period phenol was not added to the culture.

Analytical methods

Samples drawn from the culture vials were kept inside centrifuge tubes in triplicate for determining various parameters. Dissolved sulfide was determined as per the procedure described by Cord-Ruwisch (1985). Biomass cell concentration was quantified by measuring optical density at 600 nm using a UV/visible spectrophotometer (Model: lambda 45, Perkin Elmer, USA) with distilled water as the reference. The pH of the samples was measured using a digital pH meter (Thermo Scientific, Orion 3 Star, USA). Samples meant for residual sulfate, COD and phenol determination were drawn in separate centrifuge tubes in triplicate. All these samples were centrifuged at 8,000 rpm for 5 minutes to remove the cell debris for further analysis. For sulfate determination, 0.2 ml of zinc acetate and 0.1 ml of NaOH were added to fix the sulfide in the mixture (Somasundaram et al. 2009), while samples meant for COD determination were acidified with 2–3 drops of H2SO4 and purged with N2 gas instead of stirring, to release the sulfides present as gaseous sulfide (Sabumon 2008). To reduce the interference of sulfur compounds during phenol estimation, samples were acidified to pH 4.0 by adding and stirring briefly. Sulfate was measured by turbidimetric method, COD was measured by closed reflux titrimetric method and phenol was measured using direct photometric method (APHA 2005). To identify the nature of bacteria in the culture, samples were diluted and stained stepwise with crystal violet, iodine solution, 95% ethanol and safranine on a glass plate. The slides were left for air drying for 15 minutes after which they were tested under an upright fluorescence microscope (model: CX 41, Olympus, Japan). The bacterial culture for field emission scanning electron microscopy (FESEM) was fixed with 2.5% glutaraldehyde and then washed with distilled water thrice. The washed sample was then sequentially dehydrated using acetone with concentrations increasing from 30% to 90% in 15% increments, with 10 min exposure time. Finally, the sample was dehydrated with 100% acetone. After dehydration, the dried sample was gold sputtered and then examined under FESEM Σ (Sigma), Carl Zeiss, Germany. For identification of the bacterial colony, the microbial culture was sent to geneOmbio Technologies Pvt. Ltd, Mumbai, India, for terminal restriction fragment length polymorphism (TRFLP) analysis. Using the primers specific to 16S rDNA from the bacterial genome, PCR (polymerase chain reaction) amplification was carried out. The genemapper analysis provided an electropherogram with varied peaks for different bacterial species. The online library tool was used to identify the organisms characterized by the peaks.

Sulfate bio-reduction in batch cultures

Table 1 gives a description of the batch studies carried out in this experimental work. About 10% (v/v) of the mixture from the aspirator bottle containing acclimatized sludge was transferred into eight 1-l aspirator bottles to get around 400 ± 50 mg l−1 of biomass concentration (as MLVSS). Phenol of varied concentrations starting from 10 mg l−1 to 300 mg l−1 were added into these bottles, while the one with 0 mg l−1 phenol served as the reference. The contents in aspirator bottles were purged with nitrogen gas to maintain anaerobic conditions. The initial pH was adjusted to 7.0 ± 0.2. The contents from each bottle were then transferred into 60 culture vials (in triplicate) of 15 ml capacity under constant stirring and preventing any prolonged exposure to air. The culture vials thus served as self-sacrificing small batch reactors and were kept in a temperature-controlled horizontal shaker at 180 rpm and 30 ± 0.2 °C. For determination of various parameters as listed in the ‘Analytical methods’ section, any three tubes were drawn from the shaker at an interval of 24 hours and various parameters were investigated.

Table 1

Batch studies in presence of phenol

Batch test Initial parameters (mg l−1)
 
COD/sulfate
 
Lactate COD Sulfate Phenol Lactate COD/sulfate Phenol COD/sulfate Total COD/sulfate 
BT-1 1,500 1,000 1.5 1.5 
BT-2 1,500 1,000 10 1.5 0.02 1.52 
BT-3 1,500 1,000 35 1.5 0.08 1.58 
BT-4 1,500 1,000 50 1.5 0.12 1.62 
BT-5 1,500 1,000 100 1.5 0.24 1.74 
BT-6 1,500 1,000 150 1.5 0.36 1.86 
BT-7 1,500 1,000 225 1.5 0.53 2.03 
BT-8 1,500 1,000 300 1.5 0.71 2.21 
Batch test Initial parameters (mg l−1)
 
COD/sulfate
 
Lactate COD Sulfate Phenol Lactate COD/sulfate Phenol COD/sulfate Total COD/sulfate 
BT-1 1,500 1,000 1.5 1.5 
BT-2 1,500 1,000 10 1.5 0.02 1.52 
BT-3 1,500 1,000 35 1.5 0.08 1.58 
BT-4 1,500 1,000 50 1.5 0.12 1.62 
BT-5 1,500 1,000 100 1.5 0.24 1.74 
BT-6 1,500 1,000 150 1.5 0.36 1.86 
BT-7 1,500 1,000 225 1.5 0.53 2.03 
BT-8 1,500 1,000 300 1.5 0.71 2.21 

Sulfate bio-reduction and phenol inhibition kinetics

The experimental data for sulfate, COD and phenol reduction were fitted to zero, first and second order reactions. The values of degradation rate obtained from these models gave an idea of the extent of utilization of phenol and lactate for sulfate reduction. For each batch culture with a certain initial phenol concentration, the specific growth rate (μ, h−1) was measured by performing a least squares regression on the semi-logarithmic plot of biomass growth (X, mgl−1) over time (t, h) in the exponential growth phase. Since phenol was a very significant parameter which affected the growth rate and hence sulfates reduction, a plot of μ (h−1) vs phenol concentration (mg l−1) was developed using various models, Haldane, Webb, Yano, Edward, Aiba and Andrews, in MATLAB 7.0.

RESULTS AND DISCUSSION

Time series and kinetics of sulfate bio-reduction at different phenol concentrations

Sulfate degradation profile with time in the batch cultures is shown in Figure 1. It was noticed that the sulfate concentration reduced by more than 80% within 8 days of batch analysis up to 100 mg l−1 phenol addition. The highest (88%) and lowest (56%) sulfate reduction efficiencies were noticed in batch cultures containing an initial phenol concentration of 100 and 300 mg l−1. Such efficiency relies on the extent of utilization of phenol and lactate by the mixed microbial consortia as substrates for sulfate reduction. It was further observed that in batch cultures containing 225 and 300 mg l−1 phenol, sulfate could be used up less efficiently compared to the other concentrations, probably due to these higher doses of phenol imposing an inhibition effect on the sulfate reducers. This observation was also reflected by the fact that even after 192 hours of batch analysis, around 440 ± 16 mg l−1 of sulfate concentration still appeared in the batch culture containing 300 mg l−1 phenol.

Figure 1

Time series plot of sulfate reduction at different initial phenol concentrations.

Figure 1

Time series plot of sulfate reduction at different initial phenol concentrations.

To study the kinetics of sulfate reduction, sulfate concentration over time was fitted to various orders of reaction (e.g. zero, first and second) and reaction rate coefficients were determined. It was noticed that the experimental results fitted best to zero order reaction. Table 2 gives the reaction rate coefficients obtained after kinetic analysis. The reaction rate coefficient was found to be the maximum in the case of batch culture containing an initial phenol concentration of 100 mg l−1 and then declined reaching a minimum of 2.8 mg l−1h−1 in the culture having initial phenol concentration of 300 mg l−1. The sulfate-reducing potential and reaction rate coefficient thus proved that a concentration of 100 mg l−1 could give better sulfate-reducing efficiency compared to the other conditions. These results also indicate that beyond a certain phenol concentration of 100 mg l−1, sulfate reduction efficiencies declined due to inhibition of phenol concentration on the mixed microbial culture for reduction of sulfate. Such observations were also noticed by García-Cruz et al. (2010), where acute inhibition to the sulfate-reducing bio-film in terms of IC50 (half maximal inhibitory concentration) was observed at 143.8 mg l−1 phenol.

Table 2

Kinetics of sulfate reduction with various reaction rate coefficients

Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-1 y = −3.4648x + 935.03 0.9762 3.46 
BT-2 y = −3.5753x + 924.19 0.9519 3.57 
BT-3 y = −3.9572x + 911.63 0.9968 3.95 
BT-4 y = −4.2441x + 992.84 0.9849 4.24 
BT-5 y = −4.4716x + 981.08 0.9933 4.47 
BT-6 y = −3.5449x + 934.33 0.9466 3.54 
BT-7 y = −3.2515x + 986.83 0.9684 3.25 
BT-8 y = −2.7932x + 996.16 0.9735 2.79 
Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-1 y = −3.4648x + 935.03 0.9762 3.46 
BT-2 y = −3.5753x + 924.19 0.9519 3.57 
BT-3 y = −3.9572x + 911.63 0.9968 3.95 
BT-4 y = −4.2441x + 992.84 0.9849 4.24 
BT-5 y = −4.4716x + 981.08 0.9933 4.47 
BT-6 y = −3.5449x + 934.33 0.9466 3.54 
BT-7 y = −3.2515x + 986.83 0.9684 3.25 
BT-8 y = −2.7932x + 996.16 0.9735 2.79 

Phenol reduction and kinetics at initial concentrations

As described earlier in the ‘Sulfate bio-reduction in batch cultures’ section, phenol was added at increasing concentrations starting from 10 to 300 mg l−1. The phenol utilization capability of the mixed microbial consortia is well reflected in Figure 2, where phenol could be completely degraded when present at an initial concentrations starting from 0 to 100 mg l−1 in BT-1 to BT-5. However, in the successive batch tests (phenol concentration: 150, 225 and 300 mg l−1), phenol concentration of 41.6 ± 0.58, 137 ± 0.86 and 237 ± 2.12 mg l−1, respectively, still remained in the system after 8 days of batch study. In the case of batch cultures having the highest phenol concentrations of 225 and 300 mg l−1, the phenol concentration measured between 168 and 192 hours did not decrease markedly and remained relatively constant. The phenol degradation in all the cultures followed zero order kinetics and the values of reaction rate coefficients increased up to the one containing an initial phenol concentration of 100 mg l−1 after which it decreased (Table 3). Several researchers also observed that phenol reduction under aerobic conditions followed zero order kinetics in which the reaction rate coefficient decreased with increasing phenol after a certain concentration which proved inhibitory (Lim et al. 2013). It was observed that the degradation rates achieved in this study was higher than those reported by Kumar et al. (2011). However Wang et al. (1988) reported faster phenol biodegradation rates, i.e. 0.5–7 mg l−1h−1, while using a phenol-enriched methanogenic culture under anaerobic conditions in batch cultures. Such higher degradation rates were possible since the culture derived from a continuous fermentor was previously acclimated with 4,000 mg l−1 phenol for 30 days. Rosenkranz et al. (2013) obtained a phenol degradation rate as high as 1.47 ± 0.02 mg l−1h−1 at a concentration of 200 mg l−1 phenol under anaerobic conditions while using an inoculum, obtained from a tobacco wastewater treatment plant in glass bottles, after it had been acclimated with increasing phenol concentrations up to 200 mg l−1 for over 100 days. The degradation rates obtained in this study thus show good potential abilities of the mixed microbial consortia for phenol degradation.

Table 3

Kinetics of phenol reduction with various reaction rate coefficients

Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-2 y = −0.2288x + 10.836 0.9978 0.22 
BT-3 y = −0.2508x + 32.792 0.9646 0.25 
BT-4 y = −0.3214x + 49.486 0.9569 0.32 
BT-5 y = −0.5319x + 99.043 0.9932 0.53 
BT-6 y = −0.5224x + 128.77 0.9155 0.52 
BT-7 y = −0.4957x + 227.47 0.9652 0.49 
BT-8 y = −0.326x + 303.71 0.9926 0.32 
Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-2 y = −0.2288x + 10.836 0.9978 0.22 
BT-3 y = −0.2508x + 32.792 0.9646 0.25 
BT-4 y = −0.3214x + 49.486 0.9569 0.32 
BT-5 y = −0.5319x + 99.043 0.9932 0.53 
BT-6 y = −0.5224x + 128.77 0.9155 0.52 
BT-7 y = −0.4957x + 227.47 0.9652 0.49 
BT-8 y = −0.326x + 303.71 0.9926 0.32 
Figure 2

Time series plot of phenol reduction at different initial phenol concentrations.

Figure 2

Time series plot of phenol reduction at different initial phenol concentrations.

COD reduction and kinetics at different phenol concentrations

COD was measured as total COD (COD due to lactate and phenol). The time series profile for COD reduction is shown in Figure 3. The COD utilization reduced as the phenol concentration was increased beyond 100 mg l−1. COD reduction efficiencies reached as high as 75 ± 2% in batch culture containing phenol concentration of 100 mg l−1 after which it decreased to a minimum of 55% in batch culture containing phenol concentration of 300 mg l−1. This observation leads to the fact that, probably due to inhibition caused by higher concentration of phenol, sulfate reducers were unable to utilize the available substrate. The kinetics study for COD reduction showed that it followed zero order kinetics with maximum degradation rate observed in the case of batch culture containing phenol concentration of 100 mg l−1, which declined with increasing phenol concentrations (Table 4). Liu et al. (2002) used a sequencing aerobic sludge blanket reactor to determine the utilization of acetate in presence of phenol by an acetate-fed aerobic granular sludge. They observed that as the ratio of initial concentration of phenol to initial concentration of biomass was increased from 0 to 0.19 mg phenol/mg MLSS, the zero order rate coefficient of acetate decreased from 1.15 to 0.38 mg l−1min−1. It therefore proves that addition of phenol beyond 100 mg l−1 has a significant inhibitory effect on substrate utilization required for sulfate reduction.

Table 4

Kinetics of COD reduction with various reaction rate coefficients

Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-1 y = −5.5167x + 1,416.6 0.9765 5.51 
BT-2 y = −5.6252x + 1,366 0.9741 5.62 
BT-3 y = −6.0654x + 1,502.7 0.9901 6.06 
BT-4 y = −6.3061x + 1,563.7 0.9793 6.30 
BT-5 y = −6.347x + 1,582.8 0.9877 6.34 
BT-6 y = −5.594x + 1,781.7 0.9732 5.59 
BT-7 y = −5.4489x + 1,869.4 0.9254 5.44 
BT-8 y = −5.3301x + 2,062.5 0.9154 5.33 
Batch test Equation of plot Coefficient of determination (R2Reaction rate coefficients (mg l−1 h−1
BT-1 y = −5.5167x + 1,416.6 0.9765 5.51 
BT-2 y = −5.6252x + 1,366 0.9741 5.62 
BT-3 y = −6.0654x + 1,502.7 0.9901 6.06 
BT-4 y = −6.3061x + 1,563.7 0.9793 6.30 
BT-5 y = −6.347x + 1,582.8 0.9877 6.34 
BT-6 y = −5.594x + 1,781.7 0.9732 5.59 
BT-7 y = −5.4489x + 1,869.4 0.9254 5.44 
BT-8 y = −5.3301x + 2,062.5 0.9154 5.33 
Figure 3

Time series plot of COD reduction at different initial phenol concentrations.

Figure 3

Time series plot of COD reduction at different initial phenol concentrations.

Biomass growth at different phenol concentrations

The growth of biomass against time is represented in Figure 4. The absence of a lag phase in the time series plot reveals that the mixed microbial mass could readily utilize phenol for growth. The highest growth rate of biomass was observed in batch culture containing phenol concentration of 100 mg l−1. In this case, the biomass concentration reached as high as 1,630 ± 4.2 mg l−1 from initial value of 417 ± 10 mg l−1, showing almost 75% increase. However, the percentage growth of biomass over time declined in the successive batch tests and reached a minimum in the culture containing 300 mg l−1. It could be also inferred from Figure 5 that phenol concentration between 10 and 150 mg l−1 did not impose any significant repression on the biomass growth. However, at concentration of 225 mg l−1, a prominent lag phase was observed which co-related with the delay in the utilization of phenol by the bacterial culture. Thus, it could be concluded that phenol concentration above 150 mg l−1 exerted a significant toxic effect on the culture growth.

Figure 4

Time series plot of biomass growth at different initial phenol concentrations.

Figure 4

Time series plot of biomass growth at different initial phenol concentrations.

Figure 5

Surface plots for (a) phenol and sulfate reduction reaction rate coefficients, and (b) phenol and COD reduction reaction rate coefficients with varied concentrations of phenol.

Figure 5

Surface plots for (a) phenol and sulfate reduction reaction rate coefficients, and (b) phenol and COD reduction reaction rate coefficients with varied concentrations of phenol.

Sulfate, phenol and COD reduction at different phenol concentrations

To discuss the nature of sulfate, phenol and COD reduction in batch cultures at varied initial phenol concentrations, a surface plot was fitted between these variables, as shown in Figure 5(a) and 5(b) under two different cases. The nature of degradation was explained by fitting a polynomial as mentioned below.

For case (a): Phenol reduction reaction rate coefficient at varied initial phenol concentrations, with corresponding COD reduction reaction rate coefficient:  
formula
(1)
f(x, y), is the phenol reduction reaction rate coefficient (mg l−1 h−1), with x and y representing different initial phenol concentrations starting from 0 to 300 mg l−1 and COD reduction reaction rate coefficient (mg l−1 h−1), respectively; pij, where i, j = 0, 1, 2 is the individual variable coefficient.  
formula
For case (b): Phenol reduction reaction rate coefficient at varied initial phenol concentrations, with corresponding sulfate reduction reaction rate coefficient:  
formula
(2)
where f(x, y), is the phenol reduction reaction rate coefficient (mg l−1 h−1), with x and y representing different initial phenol concentrations starting from 0 to 300 mg l−1 and sulfate reduction reaction rate coefficient (mg l−1 h−1), respectively; pij, where i = 0, 1, 2 and j = 0, 1 is the individual variable coefficient.  
formula

It was observed that the phenol reduction reaction rate coefficient increased with the corresponding sulfate and COD reduction reaction rate coefficients, with increasing phenol concentrations in the batch culture up to a concentration of 100 mg l−1. However, a further increase in phenol concentration decreased the reaction rate coefficients, thus proving inhibitory.

Bio-kinetic modeling and estimation of bio-kinetic coefficients

In order to establish the effect of phenol on the biomass growth abilities, specific growth rates of the culture at different initial phenol concentrations were calculated as per the following relationship:  
formula
where X is biomass concentration (mg l−1) at time t (h) and μ is the specific growth rate (h−1).

The observation made on biomass growth due to phenol inhibition was modeled using a suitable substrate inhibition model described in literature. During the course of batch studies, it was observed that the specific growth rate increased with addition of up to 100 mg l−1 phenol and then decreased in the successive batch tests. These models were analyzed in MATLAB 7.0 to give an idea of the extent and effect of phenol concentration on the mixed microbial culture. The experimental specific growth rate and predicted values using various models are presented in Figure 6. Table 5 gives the detailed values of various bio-kinetic coefficients obtained using the models along with the root mean square error (RMSE) between the predicted and experimental values. The Haldane model (R2 = 0.81, RMSE = 0.001321) was able to describe the bio-kinetic study; however, the Edward model proved to be a better fit (R2 = 0.85, RMSE = 0.001011). The values of the inhibitory constants given in Table 5 showed a good tolerance of the mixed microbial culture to grow in presence of phenol, and hence proceed to sulfate reduction in the process.

Table 5

Bio-kinetic parameters for various models

Model Model description μmax (h−1Ks (mg l−1Ki (mg l−1K (mg l−1R2 RMSE 
Haldane  0.04 58.22 36.25 --- 0.81 0.001321 
Edward  0.052 58.22 88.05 --- 0.85 0.001011 
Webb  0.026 53.67 48.5 150.4 0.63 0.002544 
Aiba  0.03 56.45 52.15 --- 0.786 0.002325 
Yano  0.045 48.65 100.6 103.2 0.82 0.00135 
Andrews  0.040 52.872 112.34 --- 0.808 0.00187 
Model Model description μmax (h−1Ks (mg l−1Ki (mg l−1K (mg l−1R2 RMSE 
Haldane  0.04 58.22 36.25 --- 0.81 0.001321 
Edward  0.052 58.22 88.05 --- 0.85 0.001011 
Webb  0.026 53.67 48.5 150.4 0.63 0.002544 
Aiba  0.03 56.45 52.15 --- 0.786 0.002325 
Yano  0.045 48.65 100.6 103.2 0.82 0.00135 
Andrews  0.040 52.872 112.34 --- 0.808 0.00187 

µg: specific growth rate (hr−1), µm: maximum specific growth rate (hr−1), S: substrate concentration (mg l−1), Ks: half velocity constant (mg l−1), Ki: substrate inhibition constant (mg l−1), K: Webb constant and Yano constant (mg l−1) respectively.

Figure 6

Bio-kinetic analysis using various bio-kinetic models at different initial phenol concentrations.

Figure 6

Bio-kinetic analysis using various bio-kinetic models at different initial phenol concentrations.

Bacterial images and Gram staining

The mixed bacterial mass was observed in FESEM Σ (Sigma, Carl Zeiss) Germany in the Central Instruments Facility, IIT Guwahati. A cluster of bacterial cells, mostly rod shaped, was observed at different magnifications, whose size ranged from 1.312 × 0.43 μm to 1.53 × 0.54 μm (Figure 7(a)–7(c)). Gram staining of the culture was done and is shown in Figure 7(d). The bacteria retained the stain and appeared purple when viewed under an oil immersion microscope and hence proved to be Gram-negative. For identification of organisms present in the mixed bacterial mass, the culture was subjected for TRFLP analysis. The TRFLP analysis is a culture-independent, rapid, sensitive and reproducible method of assessing diversity of complex communities without the need for any genomic sequence information. The presence of SRB such as Desulfarculus, Pseudomonas, Desulfovibrio and Acetobacter species was noticed in the bacterial mass as shown in Figure 8.

Figure 7

Images of mixed microbial culture using FESEM at a magnification of (a) 39 KX, (b) 69.45 KX, (c) 14.01KX. (d) Gram staining image.

Figure 7

Images of mixed microbial culture using FESEM at a magnification of (a) 39 KX, (b) 69.45 KX, (c) 14.01KX. (d) Gram staining image.

Figure 8

TRFLP analysis of bacterial sample.

Figure 8

TRFLP analysis of bacterial sample.

CONCLUSION

Sulfate reduction behavior was investigated using mixed microbial consortia in presence of phenol. As observed from kinetic studies, higher concentrations of phenol affected the process of sulfate and COD reduction. The highest sulfate, phenol and COD reduction efficiencies were observed at 100 mg l−1 phenol concentration. The substrate inhibition due to phenol was explained using various models, among which the Edward model described the phenomenon with maximum specific growth rate of 0.052 h−1 and substrate inhibition constant of 88.05 mg l−1 The bio-kinetic constants evaluated showed good tolerance of SRB to varied phenol doses. The outcome of this study suggests that further research on sulfate reduction with prior acclimation using phenol can be carried out. However, the applicability of the microbial culture in a suitable large-scale reactor system for treating phenol- and sulfate-containing wastewater should be studied, which would help in its better application.

ACKNOWLEDGEMENTS

This research was supported by Council of Scientific and Industrial Research (CSIR), India (Project No. 22(0609)/12/EMR-II). The authors are thankful to the Head, Central Instruments Facility (CIF) of IIT Guwahati for allowing the analysis of samples.

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