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Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa
Accepted Manuscript Title: Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa Authors: S. Dayana Priyadharshini, A.K. Bakthavatsalam PII: DOI: Reference:
S0957-5820(17)30186-6 http://dx.doi.org/doi:10.1016/j.psep.2017.06.006 PSEP 1086
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
31-1-2017 1-4-2017 6-6-2017
Please cite this article as: Priyadharshini, S.Dayana, Bakthavatsalam, A.K., Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa S.Dayana Priyadharshini 1, A.K.Bakthavatsalam*, [email protected] 1, [email protected] *, Fax no :+91-431-2501081* 1,* National Institute of Technology –Tiruchirappalli-620015, Tamilnadu, India. (* corresponding author) Highlights
Degradation of coal gasification effluent by C. pyrenoidosa
Cultivation of C. pyrenoidosa in high phenolic concentrations
Experimentation conducted under ambient conditions
91% degradation of phenolic effluent with 20% concentration during 7 days treatment
Abstract Batch experiments were carried out to investigate the degradation effect of Chlorella pyrenoidosa (KX686118) on the phenolic effluent of a Coal Gasification (CG) plant. The major pollutant present in the effluent is phenol (C6H5OH). The effect of C. pyrenoidosa on phenol degradation was analyzed by inoculating 1gram of wet biomass into four different phenolic effluent concentrations viz. 20, 40, 60 and 80% i.e. the total phenols concentration of 282±1, 564±1.5, 846±2.2 and 1128±2mgL-1 respectively. The experiments were performed under ambient temperature of 30±5°C at pH 8.0. The results indicated that 1gram of wet biomass per Liter of effluent could degrade more than 90% of phenol and other contaminants, for effluent concentrations up to 60%. Bio kinetic coefficients viz., k-reaction rate constant and Km-halfsaturation constant were determined using Michaelis-Menten rate expression and found as k = 50mg of phenol g-1(algae)day-1, Km = 347mgL-1. The highest carbon fixation rate of 0.25gL-1 day-1 was obtained with a 60% effluent concentration.
Abbreviations BOD-Biological Oxygen Demand; COD-Chemical Oxygen Demand; CG-Coal Gasification; mgL-1–milligram per Liter; GC-Gas Chromatography; IS-Indian Standards; MIBK-Methyl isobutyl ketone; mM-milli Moles; O&G-Oil & Grease; PCP-Pentachlorophenol; PG-Producer Gas; TDS-Total Dissolved Solids; TS-Total Solids; TSS-Total Suspended Solids; µgL-1-micro gram per Liter. Keywords Phenol; Biodegradation; Chlorella pyrenoidosa; Bio-kinetics; Wastewater treatment; Coal gasification 1.0 Introduction Amongst various industrial pollutants, phenolic compounds constitute a family of pollutants toxic to plants, animals and aquatic life. These compounds get released to the water bodies by many industries like chemical, oil refineries, pharmaceutical, coke, producer gas, paper and pulp etc. (Stoilova et al., 2006). CG wastewater is generated during coal gas cleaning and production of chemicals from coal (Li et al., 2011; Wang et al., 2010). Wastewater of CG contains a large number of toxic compounds such as phenols, thiocyanate, polycyclic aromatic hydrocarbons, heterocyclic compounds, long chain hydrocarbons, ammonia, cyanide and a variety of refractory and toxic compounds (Lim et al., 2002; Wang et al., 2011). Various conventional processes to treat CG wastewater include chemical neutralization, activated sludge process, sand filtration, solvent extraction, ozonation and anaerobic treatment. Removal of organic and inorganic pollutants from the CG wastewater is a complex process as several contaminants need to be treated simultaneously and effectively. Activated sludge process is a prevalent biological treatment method for wastewater from coal processing, but it is inefficient in removing ammonia due to the presence of toxic and inhibitory compounds. High hydraulic residence time could be adopted in treating coal process wastewater to achieve simultaneous biological oxidation of organic and nitrogenous compounds in single sludge
systems due to the nitrification inhibition. A high sludge recycle ratio may also be adopted in activated sludge systems to maintain a high biomass concentration in the process plant. However, high sludge recycling often leads to the growth of filamentous bacteria, which promotes sludge bulking, scum formation and increased sludge wasting (Li et al., 2011). Moreover, nitrifying bacteria get washed out easily in treating coal process wastewater in the activated sludge process due to the fast growth of competitive microorganisms at higher temperatures under an increased concentration of phenol and thiocyanate (Kim et al., 2007). Anaerobic process is another suitable treatment for the CG wastewater, but the process is seriously inhibited due to its lower efficiency (Wang et al., 2010). Solvent extraction is also one of the treatments available for CG wastewater. MIBK was used as an extracting solvent for the treatment of CG wastewater having 5000mgL-1 phenol and 20,000 mgL-1 COD and as it could reduce 93% of the phenols and 80% of COD (Yang et al., 2006). Although some more treatment processes, e.g., chemical coagulation, ion exchange, and active carbon adsorption, could achieve higher removal efficiencies of organic matter and color, these are accompanied by increased costs of sludge treatment, disposal, and activated carbon regeneration (Chang et al., 2008). 1.1 Treatment of phenolic compounds using algae and cyanobacteria Microalgae are used for wastewater treatment because of their ability to remove chemical and organic contaminants, heavy metals and pathogens. Microalgae can process hazardous compounds in wastewater since they produce the oxygen required for degradation of pollutants such as polycyclic aromatic hydrocarbons (PAHs), phenols and organic solvents n (Brennan and Owende, 2010; Cuellar-Bermudez et al., 2015; Razzak et al., 2013). Microalgae have found applications in metal sorption, CO2 sequestration, value added products and biofuels. Microalgae are found abundantly in both marine and fresh water environment. The simple cellular structure
and wide distribution in water bodies make the algae a distinctly suitable biological agent for wastewater treatment (Yu et al., 2014). The microalgae utilize the sunlight and the organic pollutants in the waste water for growth. Kshirsagar, 2013 reported that the C. vulgaris reduced 80.6, 70.9, 78.1 and 62.7% of COD, BOD, nitrate and phosphate of wastewater respectively with 15 days of treatment. While using S. quadricauda, the removal efficiencies of COD, BOD, nitrate and phosphate of wastewater were 71, 89.2, 70.3 and 81.3%, respectively up to 15 days. C. pyrenoidosa could degrade 97% of 800mgL-1 phenol with the algal concentration of 4gL-1 within 4 days of treatment (Dayana Priyadharshini and Bakthavatsalam, 2016). O. Danica grew heterotrophically on phenol and p-cresol up to a concentration of 4mM and bio-accumulates the carbon in the nucleic acid and lipid content of cells (Semple, 1997; Semple and Cain, et al., 1996). Among the different biological members, cyanobacteria and microalgae are highly adaptive through many eons, and can grow autotrophically, heterotrophically or mixotrophically (Subashchandrabose et al., 2013). The cultivation of A. platensis in olive oil mill wastewater treated with sodium hypochlorite led to a higher biomass of 1.6gL-1and removed phenolic pollutants present in the wastewater (Markou et al., 2012). Thus, phycoremediation of phenolic compounds proves to be a more efficient and effective alternative to the conventional degradation methods, due to the added advantage of eliminating the formation of toxic byproducts. As of date, biodegradation of GG wastewater using algae is not available in published literature. The potential of C. pyrenoidosa in effective degradation of phenolic wastewater in coal gasification plant has not been investigated so far. This research work aims to evaluate the effect of fresh water microalga C. pyrenoidosa on wastewater containing phenolic compounds generated by a CG plant. The alga chosen for this work is natively isolated from the target
effluent treatment plant. To verify the practical applicability of the C. pyrenoidosa, the kinetics underlying the phenol degradation in coal gasification effluent was studied. 2.0 Source of effluent sample The effluent was collected from a CG plant located at Trichy, India. Coal is gasified using air and steam in this plant. The gasifiers used for gas production are of single stage with rotating bottom parts, water cooled jacket, coal charging and ash removal system. Water is used for cooling and scrubbing the hot raw gas coming out from the bottom seal of the pre-cooler. The diagram of the gas cooling system of the producer gas plant is shown in Fig.1. Phenolic effluent used in this study is from coal based producer gas plant, in which, about 200m3/day of water is used for CG cleaning system. 150m3/day of water comes out of the PG plant as phenolic effluent. Due to stripping, various phenolic compounds get dissolved in water, in which phenol (C6H5OH) is the major contaminant. In addition, the effluent also carries oil and grease. The effluent discharged from the coal gasification plant is found to have a total phenolic content of 1475.3± 67.8mgL-1 (as per IS3025 part no.43). The effluent treatment plant of the industry works with a combination of chemical and biological treatment methods, wherein the primary and the secondary chemical treatments of the effluent are carried out using alum and polymer. The phenolic effluent is pumped from the collection sump to the tar oil separator with a retention time of 18 hours and then fed to the primary chemical treatment plant. The chemical treatment consumes 0.78 kg of polymer and 102.5kg of alum per day. The sludge produced by the chemical treatment plant per day is 45kg on wet basis. In tertiary treatment process, the chemically treated effluent is mixed with sewage and mixed liquor suspended solids. They are fed to biological treatment plant with a retention time of 25h. The sludge generated from the biological treatment plant is 27kg/day on wet basis. High retention time, removal and disposal
of sludge produced during degradation are the major disadvantages of the existing treatment facility. The discharge limits of effluents with phenolic compounds (as C6H5OH) as specified by Environment (Protection) Rules-1986 laid by the Central Pollution Control Board (CPCB) – Tamilnadu, India are 1.0, 5.0 and 5.0 mgL-1 for inland surface water, public sewers and marine coastal area respectively. Due to the hazardous effects of phenolic compounds and the formation of toxic byproducts during chemical treatment, it becomes necessary to remove the phenol present in the effluent discharged.
3.0 Materials and Methods 3.1 Materials All chemicals used in this study were of the highest purity grade purchased from SigmaAldrich/ Merck- India. 3.2 Methods 3.2.1 Culture of C. pyrenoidosa A culture of targeted algal species was isolated from the effluent treatment plant of a CG plant located at Trichy and characterized at molecular level (18S rRNA) as Chlorella pyrenoidosa. It is a eukaryotic fresh water green alga. C. pyrenoidosa can grow autotrophically, heterotrophically or mixotrophically. C. pyrenoidosa exhibited fast growth under ambient conditions (Temperature-30±5°C and light intensity 10±5MJ/m2) with high phenol content. The nutrients used for the cultivation of C. pyrenoidosa are potassium bicarbonate and urea in the concentration level of 2gL-1of fresh water and 1gL-1 of fresh water respectively and sterilized using an autoclave. A rectangular plastic transparent trough with a capacity of 40 L was used for the cultivation of seed culture under ambient condition and its growth was monitored by
analyzing absorbance at 600nm using UV-VIS Spectrophotometer (UV-VIS Spectroquant Pharo 300 Spectroquant, Merck Millipore). The pH of the culture was maintained in the range of 7.08.0. The culture was kept in a closed room with sufficient natural light and air. No artificial lamps were used for lighting. Seed culture could always be maintained in logarithmic phase. 3.2.2 Characterization and quantification of phenolic effluent The conventional parameters like pH, color, Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), Total Dissolved Solids (TDS) and Total Suspended Solids (TSS) were analyzed using standard procedures (Table-S1). The types of phenolic compounds present in the effluent were analyzed using gas chromatography. 3.2.2.1 Analysis of phenolic compounds through gas chromatography The phenolic compounds present in the effluent were characterized by using gas chromatography (Shimadzu GC 2010) with area normalization. The phenolic compounds were extracted from the effluent using ethyl acetate as a solvent, dried over anhydrous sodium sulphate and analyzed using the following operating conditions: Restek Rtx-5 capillary column (60mX0.32mm), Injector: 250°C, injection volume: 1µl (split-1:100), Flame Ionization Detector: 250°C, carrier gas: N2, oven temperature: 100°C(hold 2min) to 230°C at 20°C/min (hold 35min) (Ganesamoorthy et al., 2014). To identify the types of phenol present in the effluent, 11 phenolic standards were selected based on the list enumerated by the Environmental Protection Agency (EPA) (Nollet and Gelder 2013). The selected standard phenolic compounds were phenol, 2-chlorophenol, 2methylphenol, 2-nitrophenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4,6-trichlorophenol, 2,4-dinitrophenol, 4-nitrophenol and pentachlorophenol. 3.2.3. Analysis of Carbon, Hydrogen and Nitrogen contents of algal biomass
Carbon, Hydrogen and Nitrogen contents of the algal pellet were analyzed before and after treatment using CHNS/O Analyzer (EA2400 Series II PerkinElmer). The algal pellets were washed thoroughly with distilled water and were air dried for 24h in a hot air oven and then analyzed. Phenols adsorbed on the algal cells were analyzed and compared with three trials. 3.2.4 Statistical analysis Analysis of variance was performed using Excel one way ANOVA-F-Test (95% significance level). The method was used to determine the significant difference, if any, in observation among the trials and further between the batches containing different concentrations of effluent. All the results were averaged out of three trials.
3.2.5 Experimental setup A 15-day old culture was centrifuged at 7930g for 10min to separate the algal biomass without disturbing the cells. The wet biomass of culture was washed completely to accomplish the complete removal of salts present on the cells. Wet biomass of C. pyrenoidosa was inoculated into four different concentrations of effluent viz. 20, 40, 60 and 80% i.e. the total phenol concentration of 282±1, 564±1.5, 846±2.2 and 1128±2 mgL-1 respectively. The algal concentration of samples was maintained initially at 1g of algae (wet biomass)/L of effluent. The degradation study was carried out in Erlenmeyer flasks with a capacity of 1L. The effluent devoid of algae, and the algae devoid of effluent served as controls. Samples and controls were maintained at the same condition as it was for the algal cultivation over a period of 7 days. No external nutrients were added to the samples and controls. The physical changes in the algae were monitored using a fluorescent inverted microscope (Nikon DS-Fi2). Bio-accumulated phenol was analyzed by extracting the phenol from the treated algal pellet separated by
centrifugation, washed with the solvent ethyl acetate and pelletized. The extractant was analyzed using gas chromatography with the same operating conditions adopted for the raw effluent. 6.2.6 Determination of biomass productivity and specific growth rate Biomass productivity (P) (gL-1day-1) was calculated from the variation in biomass concentration (gL-1) within a cultivation time (day) according to the following eq. no. (1):
Specific growth rate (µmax) (day-1) was calculated from the following eq. no. (2):
where, X1 and X0 were the biomass concentration (gL-1) on days t1 and t0, respectively. Biomass concentration (gL-1) is used to quantify the values of cell density in all the experiments. 3.2.7 Measurement of carbon dioxide biofixation rate According to the method described by Tang et al., 2011 and Yun et al., 1997, the carbon dioxide biofixation rate (
) (gL-1 day-1) was calculated using the following eq. no. (3):
where, CC is the carbon content of the microalgal cell (%, w/w),which was measured using a CHNS/O Analyzer (EA2400 Series II PerkinElmer). MC is the atomic weight of carbon. the molecular weight of CO2. 4.0 Results and Discussion
is
4.1 Isolation and molecular identification of phenol-tolerant microalgae Phenol-tolerant single algal species were isolated from the effluent treatment plant of CG plant (Fig. 2). The isolate DEE01 was identified on the basis of 18S rRNA gene sequence. BLAST analysis suggests the isolate DEE01 to be a microalga Chlorella pyrenoidosa based on homology with 98% sequence similarities with Chlorella pyrenoidosa KJ868082.1. The 18S rRNA gene sequence of microalgae DEE01 was submitted to NCBI GenBank, and an Accession No. KX686118 was obtained. 4.2 Characteristics of phenolic effluent 4.2.1. Initial characteristics of phenolic effluent The characteristics of raw effluent are reported in Table-1. Total phenol concentration of the raw effluent was 1475.3±67.8mgL-1. All the experiments were repeated three times and statistically analyzed for the significant difference between the samples. The types of phenolic compounds present in the effluent were identified by gas chromatography. The gas chromatogram (Fig. 3) showed that the raw effluent before the treatment contained 9 different compounds. The results were compared with the gas chromatogram of the standard known phenolic compounds. Out of the different compounds, phenol (C6H5OH) and 2,4,6trichlorophenol (the two priority phenolic pollutants listed by EPA) were confirmed by showing the same retention times at the same operating conditions. The area normalization of 9 compounds indicated that phenol (C6H5OH) is the major constituent and comprised more than 50% of contaminants of the raw effluent. 4.3 Effect of phenolic effluent on the growth of C. pyrenoidosa
The effect of phenolic effluent on the growth of C. pyrenoidosa is shown in Fig. 4. The growth curves show that there is no lag phase either in control or in low concentration (20%) of effluent. However, a lag phase could be observed in samples starting from 40 to 80% of effluent concentrations (Fig. 4) due to an adaptation period required by algae when exposed to a higher concentration of phenol (Semple and Cain, 1996). The increase in lag is subsequent to the removal of pollutant by algal species (Scragg 2006). Lag phase is followed by exponential growth phase and phenol utilization by the C. pyrenoidosa. After the exponential growth period, phenol gets depleted, and hence the microalgae enter the stationary phase. The growth of biomass even after the depletion of phenol could be attributed to the biotransformation of phenol into its metabolic intermediates, which served as growth substrates until phenol gets fully utilized. Li et al. 2010 reported similar results of appearance of lag phase with an increasing phenol concentration, simultaneous phenol biotransformation during the exponential phase, appearance of a stationary phase concomitant with phenol depletion, and an increase in biomass even after the complete phenol utilization by Pseudomonas putida LY1. Growth curve shows that the specific growth rate of 0.06day−1of control (devoid of effluent) is found to be comparatively lower to the specific growth rate, observed in the presence of phenol (Table-2). This higher specific growth rate is due to the utilization of phenol as organic carbon source by C. pyrenoidosa. The specific growth rate increased with an increase in phenol concentration up to 60% of effluent. The highest value of 0.13day−1 was attained at an effluent concentration of 60% (Table-2). However, the growth rate was found to decline with an increase in the phenol concentration beyond 60%, indicating an inhibition effect of phenol present in the effluent. The utilization of phenol as an organic carbon source by algae is due to the phenol getting metabolized into CO2, contributing to the growth of microalgae and organic end products
like pyruvate (Semple and Cain, 1996; Lika and Papadakis, 2009). In addition, the growth of algae present in control was comparatively lower than the growth in phenolic effluent, indicating that the increase in phenol concentration led to an increase in algal concentration (Table-2). Further no change in structure of the algae could be noticed due to the degradation, analyzed using fluorescent inverted microscope. During the degradation study, the increase in pH was observed throughout the experiment. It is due to the utilization of CO2 released by photosynthesis. An increase in pH led to the disinfection of bacteria and fungi. 4.4 Degradation of phenol present in the effluent The gas chromatogram of samples (Fig. 5) showed that the 1gL-1 of wet biomass of C. pyrenoidosa degraded phenol (C6H5OH) and other contaminants present in the effluent completely for all concentrations up to 60%. During the phenol degradation, an unidentified GC peak, concomitant with the disappearance of phenol and other pollutants, more polar than the parent compound, could be observed in the chromatograms of all samples (Fig. 5). The GC-MS analysis (Fig. 6) of phenolic effluent identified the peak raised during degradation as that of benzene methanol. The concentration of phenol in the effluent treated with algae was found to be progressively reducing to a minimum, with a corresponding increase in biomass growth. Further, to nullify the effect of abiotic factors in phenol degradation, a change in phenol concentration was analyzed for the control, indicating nil change. This proves that the C. pyrenoidosa is the sole source of the degradation of phenolic compounds. The other pollutants not taken up for characterization in this study were also found to get degraded by C. pyrenoidosa in all the effluent samples. No intracellular or cell surface adsorbed phenol (C6H5OH) was detected through the GC analysis in the microalgal pellet ethyl acetate extract. C. pyrenoidosa reduced the total phenol content to a maximum of 91% with 20% effluent (Fig. 7) Table-3.
4.5 Determination of batch kinetic coefficients The Michaelis–Menten kinetic model is used for the determination of kinetic coefficients viz. Km-half-saturation constant (Michaelis constant) and k-reaction rate constant by considering the non-inhibitory reaction. The model is described by the equation (4). The rate of substrate removal (υ) is determined by relating the maximum rate of substrate removal (Vmax) and the substrate concentration (S) as follows.
where, Vmax represents the maximum rate achieved by the system, when the substrate concentration is at its saturation. The Michaelis constant Km (half-saturation constant) is the substrate concentration at which the reaction rate is half of Vmax. The initial substrate concentration and the initial substrate removal rate are considered for the batch operation. Equation (4) is rewritten as:
where,
is
the
. Equation (5) is rewritten as:
maximum
initial
rate
of
substrate
removal
i.e.
where, k is the reaction rate constant (day−1), X0 is the initial biomass concentration. The specific rate of substrate removal
can be calculated by dividing both sides of equation (6) by the initial
biomass concentration (equation (7)).
Equation (7) can be linearized in double reciprocal form (equation (8)) and its plot of vs.
yields
a
linear
line
with
a
slope
of
and
y-axis
intercept
of
.
The experimental data given in Table-4 were plotted in the form of
vs.
(Fig. 8).
From the slope and the intercept of this plot (best fit), the kinetic coefficients of phenol removal by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination = 0.93. 4.6 Effect of phenolic effluent on carbon, hydrogen and nitrogen contents and CO2 biofixation rate of C. pyrenoidosa Carbon, hydrogen and nitrogen contents of the treated biomass were analyzed using CHNS/O Analyzer (EA2400 Series II PerkinElmer) to determine the carbon utilized by the algae from the phenolic effluent. The results showed an increase in the carbon content of the algal biomass treated with the phenolic effluent when compared to algae cultivated devoid of effluent (Table-2) (Fig. 9). The highest carbon, hydrogen and nitrogen content of the algae cultivated in the 60% of effluent were 30.3±0.17 % (C), 2.1±0.14% (H) and 3.7±0.01 % (N).
The CO2 biofixation rate RCO2 (g L-1 day-1) was calculated by equation (3), and the results are shown in (Fig. 10). C. pyrenoidosa showed the maximum CO2 biofixation rate when the effluent concentration was 60%. The maximum CO2 biofixation rate of C. pyrenoidosa was 0.25 g L-1 day-1 with the effluent concentration of 60%. The algae cultivated in control (algae devoid of effluent) showed a lower carbon fixation rate, indicating that the increase in carbon fixation rate of algal samples cultivated in effluent samples is due to the utilization of phenolic compounds. 4.7 Discussion Physico and chemical methods of phenol degradation are effective only if the effluent volume is small. The limiting factor of these methods is cost and sludge disposal. Due to the problems associated with the sludge disposal and the washing out of nitrifying bacteria, the biodegradation of phenol using bacteria is incapable of adaptation on a continuous basis at a large scale. The effluent used for the treatment is raw effluent without any prior treatment. The major contaminant present in the effluent is phenol (C6H5OH). Phenolic compounds and other contaminants present in the effluent are the source of carbon and energy for the growth of C. pyrenoidosa in the degradation process. Hence, the increase in the cell mass is a function of the exhaustion of phenolic compounds. The results demonstrated that the C. pyrenoidosa had a high ability to degrade phenolic compounds as well as the other contaminants present in the effluent with concentrations up to 60% within 7 days of reaction time. The results obtained from CHN analysis and carbon fixation rate suggested that C. pyrenoidosa would be able to fix the utilized carbon content inside the cell. The highest carbon fixation rate was found to be 0.25g L-1day-1 with a 60% effluent concentration. The results of this work suggest that the growth of algae in phenol rich effluent offers a new option of applying phycoremediation in the treatment of
phenolic effluent to manage the pollution load. The cultivation of C. pyrenoidosa for the treatment does not require much complex cultivation strategies. The nutritional requirement of the algae is very low and can easily be scaled up for higher volumes. Kinetic coefficients of phenol degradation by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination = 0.93. Rather than attempting to treat the effluent at the same time, phycoremediation offers the alternative use of algal biomass for other beneficial uses such as biomass production and biofuel production. 4.8 Practical applicability of the phycoremediation of phenolic effluent The effluent discharge from CG plant is 150m3/day. The existing industrial treatment facility of the target producer gas plant which operates largely on chemical treatment method requires 7X300m3 capacity tanks for treatment of effluent. The suggested algae treatment method (with 1gL-1) after dilution would require 240m3 capacity tanks X 7 numbers (for a treatment time of 7 days) that would reduce the phenol concentration to 20mgL-1. To reach the legal limit of 5mgL-1 additional chemical methods would continue and consume less chemicals resulting in reduced operating costs. Further, referring to Dayana Priyadharshini and Bakthavatsalam,2016 with 4gL-1 of algae feed and 4 days of treatment time, the phenol concentration in the effluent gets reduced to 20mgL-1 (for a treatment time of 4 days). Accordingly, 4 tanks of capacity with 240 m3 capacity would reduce the industrial effluent to 20mgL-1. Hence totally 4 tanks of 240m3 for treatment and 1 tank of 250m3 for algal cultivation is required against 7*300m3 tanks installed in the existing treatment plant. It must be noted that the 5 tanks required for the algae based treatment plant need to be of photobioreactor (with adequate sunlight penetration for reaction). Accordingly, the tank dimension would be one of shallow depth with large surface
area. However the final configuration and tank capacity assessment would call for more detailed design and engineering. 5.0 Conclusion The experiment was carried out for the removal of phenol from the raw effluent of coal based producer gas plant using eukaryotic microalgae C. pyrenoidosa, under ambient conditions and without any prior treatment. Phenol is identified as the major contaminant present in the effluent in addition to other contaminants. C. pyrenoidosa degrades phenol to the maximum of 91% with 20% effluent and the maximum degradation rate and the maximum specific growth rate were observed with 60% of effluent concentration. The kinetic coefficients of phenol degradation by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination=0.93. The various CG process plants producing effluent containing high phenol concentration will benefit with appropriately designed photobioreactor and achieve public acceptance by using this environmentally friendly biotreatment method.
Acknowledgement Authors thank National Institute of Technology, Trichy and the World Bank funded Technical Education Quality Improvement Program (TEQIP). This research did not receive any specific grant from the funding agencies in the public, commercial, or not-for-profit sectors.
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Scragg, A.H., 2006. The effect of phenol on the growth of Chlorella vulgaris and Chlorella VT1, Enzyme Microb. Tech. 39, 796-799. Subashchandrabose, S.R., Ramakrishnan, B., Megharaj, M., Venkateswarlu, K., Naidu, R., 2013. Mixotrophic cyanobacteria and microalgae as distinctive biological agents for organic pollutant degradation, Environment International. 51, 59–72. Tang, D., Han, W., Li, P., Miao, X., Zhong, J., 2011. CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels, Bioresource Technol. 102, 3071–3076. Wang, Wei., Han, H., Yuan, M., Li, H., 2010. Enhanced anaerobic biodegradability of real coal gasification wastewater with methanol addition, J. Environ. Sci. 22(12), 1868-1874. Wang, W., Han, H., Yuan, M., Li, H., Fang, F., Wang, K., 2011. Treatment of coal gasification wastewater by a two-continuous UASB system with step-feed for COD and phenols removal, Bioresource Technol. 102, 5454-5460. Yun, Y.S., Lee, S.B., Park, J.M., Lee, C.I., Yang, J.W., 1997. Carbon dioxide fixation by algal cultivation using wastewater nutrients, J. Chem. Tech. Biotechnol. 69, 451- 455. Yang, C., Qian, Y., Zhang, L., Feng, J., 2006. Solvent extraction process development and onsite trial-plant for phenol removal from industrial coal-gasification wastewater, Che. Eng. J. 117,179-185. Yu, X., Zuo, J., Tang, X., Li, R., Li, Z.X., Zhang, F., 2014. Toxicity evaluation of pharmaceutical wastewaters using the algae Scenedesmus obliquus and the bacteria Vibrio fischeri, J. Hazard Mater. 266, 68-74.
Fig. 1 Schematic diagram of gas cooling system of coal based producer gas plant
ESP-Electrostatic Precipitator
Fig. 2 Culture of C. pyrenoidosa (KX686118)
Fig. 3 Analysis of types of phenol and other contaminants by Gas Chromatography
Phenol (C6H5OH)
2,4,6-Trichlorophenol
Fig. 4 Effect of phenolic effluent on the growth of C. pyrenoidosa
Biomass concentration (g/L)
20%
40%
60%
80%
Control
3 2.5 2
1.5 1 0
1
2
3
4
Time (days)
5
6
7
20, 40, 60 and 80%- Effluent concentration
Fig. 5 Reduction of phenol (C6H5OH) peak and other pollutants peaks compared to fig. 3
8
*Unidentified peak rose after the treatment period
♦- peak corresponds to phenol (C6H5OH)
Fig.6 GC-MS analysis of the effluent unidentified peak of fig. 5
24
Fig. 7 Reduction of total phenol (as per IS 3025 Part no. 43)
25
Concentration of phenol (mg/L)
Initial concentration of phenol
Final concentration of phenol
1200
1000 800 600 400 200
0 20
40 60 Concentration of effluent (% )
80
Fig. 8 Effect of concentration of phenol on specific growth rate of C. pyrenoidosa
26
0.05 y = 6.9343x + 0.0201 R² = 0.9275
0.04
1/υx
0.03 0.02 0.01 0
0
0.001
0.002 1/S 0
0.003
0.004
k-50 mg of phenol g-1 (algae) day-1; Km-347 mgL-1; υx –Specific phenol removal rate; S0- Initial phenol concentration
Carbon, hydrogen, nitrogen content (% )
Fig. 9 Analysis of Carbon, Hydrogen and Nitrogen (% w/w) Carbon
Hydrogen
Nitrogen
40
60
40
35 30 25
20 15 10
5 0
0
20
Concentration of effluent (% )
27
80
Carbon (% )
Carbon
Carbon fixation rate
35
0.3
30
0.25
25
0.2
20
0.15
15
0.1
10
0.05
5 0
0 0
20 40 60 Concentration of effluent (% )
28
80
Carbon fixation rate (gL-1 day-1 )
Fig. 10 Effect of C. pyrenoidosa on carbon and carbon fixation rate
29
Table-1 Characteristics of raw effluent
Parameter Total suspended solids (mgL-1) Total dissolved solids (mgL-1) Biological oxygen demand (mgL-1) Chemical oxygen demand (mgL-1) Oil & grease (mgL-1) Phenolic concentration (mgL-1) pH Color
30
Mean ± SD 359.75 ±107.7 1515.5 ±195.1 152.5 ±12.3 2106 ±139.9 197.25 ±23.5 1475.3±67.8 8-9.2 Dark Brown
Table-2 Biomass productivity (P), Maximum sp. growth rate (µmax), CO2 biofixation rate (RCO2), CHN analysis of C. pyrenoidosa under different effluent concentrations (mean ± standard deviation).
Concentration P of effluent (g L-1 day-1) (%) 0 0.07 20 0.09 40 0.15 60 0.22 80 0.03
µmax -1
(day ) 0.06 0.07 0.10 0.13 0.03
CO2 biofixation rate (RCO2) (g L-1day-1) 0.02±0.01 0.09±0.03 0.15±0.01 0.25±0.04 0.01±0.01
31
Carbon (%)
Hydrogen (%)
Nitrogen (%)
7.3±0.21 26.7±0.32 28.4±0.11 30.3±0.17 10.4±0.20
1±0.01 1.6±0.11 1.7±0.07 2.1±0.14 0.87±0.1
0.6±0.001 3.2±0.004 3.5±0.007 3.7±0.01 0.24±0.05
Table-3 Amount of total phenol available after treatment
Batches
Initial concentration of total phenol (mgL-1)
Concentration of total phenol after treatment (mgL-1)
Percentage Degradation (%)
20
282±1.7
26±3.2
90.78± 3.5
40
564±1.2
39±2.6
93.80±2.8
60
846±2
84±2.5
91.14±2.5
80
1128±2.4
792±1.34
29.8±1.11
(%)
32
Table-4 Specific phenol removal rate
υx (mgL of phenol g-1 (algae) day-1)
1/ υx
1/S0
282
Final concentration of algae (gL-1) 1.65
22.17
0.045
0.0035
564
2.02
33.03
0.030
0.0017
846
2.55
31.82
0.031
0.0011
1128
1.2
40
0.025
0.0009
Initial concentration of phenol (mgL-1)
-1
Treatment time -7 days υx - specific phenol removal rate S0-initial substrate concentration
33
Notations Notation X0 X1 t0 t1 µmax Cc P
v Vmax S Km k
Name Initial Concentration of biomass Final Concentration of biomass Initial reaction time Final reaction time Specific growth rate Carbon dioxide biofixation rate Carbon content Biomass productivity Molecular weight of carbon dioxide Molecular weight of carbon Rate of substrate removal Maximum rate of substrate removal Substrate concentration Saturation constant, Michaelis constant Rate of the reaction
34
Units gL-1 gL-1 day day day-1 gL-1day-1 %(w/w) gL-1day-1 mgL-1day-1 mgL-1day-1 mgL-1 mgL-1 mg of phenol g-1 (algae) day-1
S0957-5820(17)30186-6 http://dx.doi.org/doi:10.1016/j.psep.2017.06.006 PSEP 1086
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
31-1-2017 1-4-2017 6-6-2017
Please cite this article as: Priyadharshini, S.Dayana, Bakthavatsalam, A.K., Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phycoremediation of phenolic effluent of a coal gasification plant by Chlorella pyrenoidosa S.Dayana Priyadharshini 1, A.K.Bakthavatsalam*, [email protected] 1, [email protected] *, Fax no :+91-431-2501081* 1,* National Institute of Technology –Tiruchirappalli-620015, Tamilnadu, India. (* corresponding author) Highlights
Degradation of coal gasification effluent by C. pyrenoidosa
Cultivation of C. pyrenoidosa in high phenolic concentrations
Experimentation conducted under ambient conditions
91% degradation of phenolic effluent with 20% concentration during 7 days treatment
Abstract Batch experiments were carried out to investigate the degradation effect of Chlorella pyrenoidosa (KX686118) on the phenolic effluent of a Coal Gasification (CG) plant. The major pollutant present in the effluent is phenol (C6H5OH). The effect of C. pyrenoidosa on phenol degradation was analyzed by inoculating 1gram of wet biomass into four different phenolic effluent concentrations viz. 20, 40, 60 and 80% i.e. the total phenols concentration of 282±1, 564±1.5, 846±2.2 and 1128±2mgL-1 respectively. The experiments were performed under ambient temperature of 30±5°C at pH 8.0. The results indicated that 1gram of wet biomass per Liter of effluent could degrade more than 90% of phenol and other contaminants, for effluent concentrations up to 60%. Bio kinetic coefficients viz., k-reaction rate constant and Km-halfsaturation constant were determined using Michaelis-Menten rate expression and found as k = 50mg of phenol g-1(algae)day-1, Km = 347mgL-1. The highest carbon fixation rate of 0.25gL-1 day-1 was obtained with a 60% effluent concentration.
Abbreviations BOD-Biological Oxygen Demand; COD-Chemical Oxygen Demand; CG-Coal Gasification; mgL-1–milligram per Liter; GC-Gas Chromatography; IS-Indian Standards; MIBK-Methyl isobutyl ketone; mM-milli Moles; O&G-Oil & Grease; PCP-Pentachlorophenol; PG-Producer Gas; TDS-Total Dissolved Solids; TS-Total Solids; TSS-Total Suspended Solids; µgL-1-micro gram per Liter. Keywords Phenol; Biodegradation; Chlorella pyrenoidosa; Bio-kinetics; Wastewater treatment; Coal gasification 1.0 Introduction Amongst various industrial pollutants, phenolic compounds constitute a family of pollutants toxic to plants, animals and aquatic life. These compounds get released to the water bodies by many industries like chemical, oil refineries, pharmaceutical, coke, producer gas, paper and pulp etc. (Stoilova et al., 2006). CG wastewater is generated during coal gas cleaning and production of chemicals from coal (Li et al., 2011; Wang et al., 2010). Wastewater of CG contains a large number of toxic compounds such as phenols, thiocyanate, polycyclic aromatic hydrocarbons, heterocyclic compounds, long chain hydrocarbons, ammonia, cyanide and a variety of refractory and toxic compounds (Lim et al., 2002; Wang et al., 2011). Various conventional processes to treat CG wastewater include chemical neutralization, activated sludge process, sand filtration, solvent extraction, ozonation and anaerobic treatment. Removal of organic and inorganic pollutants from the CG wastewater is a complex process as several contaminants need to be treated simultaneously and effectively. Activated sludge process is a prevalent biological treatment method for wastewater from coal processing, but it is inefficient in removing ammonia due to the presence of toxic and inhibitory compounds. High hydraulic residence time could be adopted in treating coal process wastewater to achieve simultaneous biological oxidation of organic and nitrogenous compounds in single sludge
systems due to the nitrification inhibition. A high sludge recycle ratio may also be adopted in activated sludge systems to maintain a high biomass concentration in the process plant. However, high sludge recycling often leads to the growth of filamentous bacteria, which promotes sludge bulking, scum formation and increased sludge wasting (Li et al., 2011). Moreover, nitrifying bacteria get washed out easily in treating coal process wastewater in the activated sludge process due to the fast growth of competitive microorganisms at higher temperatures under an increased concentration of phenol and thiocyanate (Kim et al., 2007). Anaerobic process is another suitable treatment for the CG wastewater, but the process is seriously inhibited due to its lower efficiency (Wang et al., 2010). Solvent extraction is also one of the treatments available for CG wastewater. MIBK was used as an extracting solvent for the treatment of CG wastewater having 5000mgL-1 phenol and 20,000 mgL-1 COD and as it could reduce 93% of the phenols and 80% of COD (Yang et al., 2006). Although some more treatment processes, e.g., chemical coagulation, ion exchange, and active carbon adsorption, could achieve higher removal efficiencies of organic matter and color, these are accompanied by increased costs of sludge treatment, disposal, and activated carbon regeneration (Chang et al., 2008). 1.1 Treatment of phenolic compounds using algae and cyanobacteria Microalgae are used for wastewater treatment because of their ability to remove chemical and organic contaminants, heavy metals and pathogens. Microalgae can process hazardous compounds in wastewater since they produce the oxygen required for degradation of pollutants such as polycyclic aromatic hydrocarbons (PAHs), phenols and organic solvents n (Brennan and Owende, 2010; Cuellar-Bermudez et al., 2015; Razzak et al., 2013). Microalgae have found applications in metal sorption, CO2 sequestration, value added products and biofuels. Microalgae are found abundantly in both marine and fresh water environment. The simple cellular structure
and wide distribution in water bodies make the algae a distinctly suitable biological agent for wastewater treatment (Yu et al., 2014). The microalgae utilize the sunlight and the organic pollutants in the waste water for growth. Kshirsagar, 2013 reported that the C. vulgaris reduced 80.6, 70.9, 78.1 and 62.7% of COD, BOD, nitrate and phosphate of wastewater respectively with 15 days of treatment. While using S. quadricauda, the removal efficiencies of COD, BOD, nitrate and phosphate of wastewater were 71, 89.2, 70.3 and 81.3%, respectively up to 15 days. C. pyrenoidosa could degrade 97% of 800mgL-1 phenol with the algal concentration of 4gL-1 within 4 days of treatment (Dayana Priyadharshini and Bakthavatsalam, 2016). O. Danica grew heterotrophically on phenol and p-cresol up to a concentration of 4mM and bio-accumulates the carbon in the nucleic acid and lipid content of cells (Semple, 1997; Semple and Cain, et al., 1996). Among the different biological members, cyanobacteria and microalgae are highly adaptive through many eons, and can grow autotrophically, heterotrophically or mixotrophically (Subashchandrabose et al., 2013). The cultivation of A. platensis in olive oil mill wastewater treated with sodium hypochlorite led to a higher biomass of 1.6gL-1and removed phenolic pollutants present in the wastewater (Markou et al., 2012). Thus, phycoremediation of phenolic compounds proves to be a more efficient and effective alternative to the conventional degradation methods, due to the added advantage of eliminating the formation of toxic byproducts. As of date, biodegradation of GG wastewater using algae is not available in published literature. The potential of C. pyrenoidosa in effective degradation of phenolic wastewater in coal gasification plant has not been investigated so far. This research work aims to evaluate the effect of fresh water microalga C. pyrenoidosa on wastewater containing phenolic compounds generated by a CG plant. The alga chosen for this work is natively isolated from the target
effluent treatment plant. To verify the practical applicability of the C. pyrenoidosa, the kinetics underlying the phenol degradation in coal gasification effluent was studied. 2.0 Source of effluent sample The effluent was collected from a CG plant located at Trichy, India. Coal is gasified using air and steam in this plant. The gasifiers used for gas production are of single stage with rotating bottom parts, water cooled jacket, coal charging and ash removal system. Water is used for cooling and scrubbing the hot raw gas coming out from the bottom seal of the pre-cooler. The diagram of the gas cooling system of the producer gas plant is shown in Fig.1. Phenolic effluent used in this study is from coal based producer gas plant, in which, about 200m3/day of water is used for CG cleaning system. 150m3/day of water comes out of the PG plant as phenolic effluent. Due to stripping, various phenolic compounds get dissolved in water, in which phenol (C6H5OH) is the major contaminant. In addition, the effluent also carries oil and grease. The effluent discharged from the coal gasification plant is found to have a total phenolic content of 1475.3± 67.8mgL-1 (as per IS3025 part no.43). The effluent treatment plant of the industry works with a combination of chemical and biological treatment methods, wherein the primary and the secondary chemical treatments of the effluent are carried out using alum and polymer. The phenolic effluent is pumped from the collection sump to the tar oil separator with a retention time of 18 hours and then fed to the primary chemical treatment plant. The chemical treatment consumes 0.78 kg of polymer and 102.5kg of alum per day. The sludge produced by the chemical treatment plant per day is 45kg on wet basis. In tertiary treatment process, the chemically treated effluent is mixed with sewage and mixed liquor suspended solids. They are fed to biological treatment plant with a retention time of 25h. The sludge generated from the biological treatment plant is 27kg/day on wet basis. High retention time, removal and disposal
of sludge produced during degradation are the major disadvantages of the existing treatment facility. The discharge limits of effluents with phenolic compounds (as C6H5OH) as specified by Environment (Protection) Rules-1986 laid by the Central Pollution Control Board (CPCB) – Tamilnadu, India are 1.0, 5.0 and 5.0 mgL-1 for inland surface water, public sewers and marine coastal area respectively. Due to the hazardous effects of phenolic compounds and the formation of toxic byproducts during chemical treatment, it becomes necessary to remove the phenol present in the effluent discharged.
3.0 Materials and Methods 3.1 Materials All chemicals used in this study were of the highest purity grade purchased from SigmaAldrich/ Merck- India. 3.2 Methods 3.2.1 Culture of C. pyrenoidosa A culture of targeted algal species was isolated from the effluent treatment plant of a CG plant located at Trichy and characterized at molecular level (18S rRNA) as Chlorella pyrenoidosa. It is a eukaryotic fresh water green alga. C. pyrenoidosa can grow autotrophically, heterotrophically or mixotrophically. C. pyrenoidosa exhibited fast growth under ambient conditions (Temperature-30±5°C and light intensity 10±5MJ/m2) with high phenol content. The nutrients used for the cultivation of C. pyrenoidosa are potassium bicarbonate and urea in the concentration level of 2gL-1of fresh water and 1gL-1 of fresh water respectively and sterilized using an autoclave. A rectangular plastic transparent trough with a capacity of 40 L was used for the cultivation of seed culture under ambient condition and its growth was monitored by
analyzing absorbance at 600nm using UV-VIS Spectrophotometer (UV-VIS Spectroquant Pharo 300 Spectroquant, Merck Millipore). The pH of the culture was maintained in the range of 7.08.0. The culture was kept in a closed room with sufficient natural light and air. No artificial lamps were used for lighting. Seed culture could always be maintained in logarithmic phase. 3.2.2 Characterization and quantification of phenolic effluent The conventional parameters like pH, color, Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), Total Dissolved Solids (TDS) and Total Suspended Solids (TSS) were analyzed using standard procedures (Table-S1). The types of phenolic compounds present in the effluent were analyzed using gas chromatography. 3.2.2.1 Analysis of phenolic compounds through gas chromatography The phenolic compounds present in the effluent were characterized by using gas chromatography (Shimadzu GC 2010) with area normalization. The phenolic compounds were extracted from the effluent using ethyl acetate as a solvent, dried over anhydrous sodium sulphate and analyzed using the following operating conditions: Restek Rtx-5 capillary column (60mX0.32mm), Injector: 250°C, injection volume: 1µl (split-1:100), Flame Ionization Detector: 250°C, carrier gas: N2, oven temperature: 100°C(hold 2min) to 230°C at 20°C/min (hold 35min) (Ganesamoorthy et al., 2014). To identify the types of phenol present in the effluent, 11 phenolic standards were selected based on the list enumerated by the Environmental Protection Agency (EPA) (Nollet and Gelder 2013). The selected standard phenolic compounds were phenol, 2-chlorophenol, 2methylphenol, 2-nitrophenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4,6-trichlorophenol, 2,4-dinitrophenol, 4-nitrophenol and pentachlorophenol. 3.2.3. Analysis of Carbon, Hydrogen and Nitrogen contents of algal biomass
Carbon, Hydrogen and Nitrogen contents of the algal pellet were analyzed before and after treatment using CHNS/O Analyzer (EA2400 Series II PerkinElmer). The algal pellets were washed thoroughly with distilled water and were air dried for 24h in a hot air oven and then analyzed. Phenols adsorbed on the algal cells were analyzed and compared with three trials. 3.2.4 Statistical analysis Analysis of variance was performed using Excel one way ANOVA-F-Test (95% significance level). The method was used to determine the significant difference, if any, in observation among the trials and further between the batches containing different concentrations of effluent. All the results were averaged out of three trials.
3.2.5 Experimental setup A 15-day old culture was centrifuged at 7930g for 10min to separate the algal biomass without disturbing the cells. The wet biomass of culture was washed completely to accomplish the complete removal of salts present on the cells. Wet biomass of C. pyrenoidosa was inoculated into four different concentrations of effluent viz. 20, 40, 60 and 80% i.e. the total phenol concentration of 282±1, 564±1.5, 846±2.2 and 1128±2 mgL-1 respectively. The algal concentration of samples was maintained initially at 1g of algae (wet biomass)/L of effluent. The degradation study was carried out in Erlenmeyer flasks with a capacity of 1L. The effluent devoid of algae, and the algae devoid of effluent served as controls. Samples and controls were maintained at the same condition as it was for the algal cultivation over a period of 7 days. No external nutrients were added to the samples and controls. The physical changes in the algae were monitored using a fluorescent inverted microscope (Nikon DS-Fi2). Bio-accumulated phenol was analyzed by extracting the phenol from the treated algal pellet separated by
centrifugation, washed with the solvent ethyl acetate and pelletized. The extractant was analyzed using gas chromatography with the same operating conditions adopted for the raw effluent. 6.2.6 Determination of biomass productivity and specific growth rate Biomass productivity (P) (gL-1day-1) was calculated from the variation in biomass concentration (gL-1) within a cultivation time (day) according to the following eq. no. (1):
Specific growth rate (µmax) (day-1) was calculated from the following eq. no. (2):
where, X1 and X0 were the biomass concentration (gL-1) on days t1 and t0, respectively. Biomass concentration (gL-1) is used to quantify the values of cell density in all the experiments. 3.2.7 Measurement of carbon dioxide biofixation rate According to the method described by Tang et al., 2011 and Yun et al., 1997, the carbon dioxide biofixation rate (
) (gL-1 day-1) was calculated using the following eq. no. (3):
where, CC is the carbon content of the microalgal cell (%, w/w),which was measured using a CHNS/O Analyzer (EA2400 Series II PerkinElmer). MC is the atomic weight of carbon. the molecular weight of CO2. 4.0 Results and Discussion
is
4.1 Isolation and molecular identification of phenol-tolerant microalgae Phenol-tolerant single algal species were isolated from the effluent treatment plant of CG plant (Fig. 2). The isolate DEE01 was identified on the basis of 18S rRNA gene sequence. BLAST analysis suggests the isolate DEE01 to be a microalga Chlorella pyrenoidosa based on homology with 98% sequence similarities with Chlorella pyrenoidosa KJ868082.1. The 18S rRNA gene sequence of microalgae DEE01 was submitted to NCBI GenBank, and an Accession No. KX686118 was obtained. 4.2 Characteristics of phenolic effluent 4.2.1. Initial characteristics of phenolic effluent The characteristics of raw effluent are reported in Table-1. Total phenol concentration of the raw effluent was 1475.3±67.8mgL-1. All the experiments were repeated three times and statistically analyzed for the significant difference between the samples. The types of phenolic compounds present in the effluent were identified by gas chromatography. The gas chromatogram (Fig. 3) showed that the raw effluent before the treatment contained 9 different compounds. The results were compared with the gas chromatogram of the standard known phenolic compounds. Out of the different compounds, phenol (C6H5OH) and 2,4,6trichlorophenol (the two priority phenolic pollutants listed by EPA) were confirmed by showing the same retention times at the same operating conditions. The area normalization of 9 compounds indicated that phenol (C6H5OH) is the major constituent and comprised more than 50% of contaminants of the raw effluent. 4.3 Effect of phenolic effluent on the growth of C. pyrenoidosa
The effect of phenolic effluent on the growth of C. pyrenoidosa is shown in Fig. 4. The growth curves show that there is no lag phase either in control or in low concentration (20%) of effluent. However, a lag phase could be observed in samples starting from 40 to 80% of effluent concentrations (Fig. 4) due to an adaptation period required by algae when exposed to a higher concentration of phenol (Semple and Cain, 1996). The increase in lag is subsequent to the removal of pollutant by algal species (Scragg 2006). Lag phase is followed by exponential growth phase and phenol utilization by the C. pyrenoidosa. After the exponential growth period, phenol gets depleted, and hence the microalgae enter the stationary phase. The growth of biomass even after the depletion of phenol could be attributed to the biotransformation of phenol into its metabolic intermediates, which served as growth substrates until phenol gets fully utilized. Li et al. 2010 reported similar results of appearance of lag phase with an increasing phenol concentration, simultaneous phenol biotransformation during the exponential phase, appearance of a stationary phase concomitant with phenol depletion, and an increase in biomass even after the complete phenol utilization by Pseudomonas putida LY1. Growth curve shows that the specific growth rate of 0.06day−1of control (devoid of effluent) is found to be comparatively lower to the specific growth rate, observed in the presence of phenol (Table-2). This higher specific growth rate is due to the utilization of phenol as organic carbon source by C. pyrenoidosa. The specific growth rate increased with an increase in phenol concentration up to 60% of effluent. The highest value of 0.13day−1 was attained at an effluent concentration of 60% (Table-2). However, the growth rate was found to decline with an increase in the phenol concentration beyond 60%, indicating an inhibition effect of phenol present in the effluent. The utilization of phenol as an organic carbon source by algae is due to the phenol getting metabolized into CO2, contributing to the growth of microalgae and organic end products
like pyruvate (Semple and Cain, 1996; Lika and Papadakis, 2009). In addition, the growth of algae present in control was comparatively lower than the growth in phenolic effluent, indicating that the increase in phenol concentration led to an increase in algal concentration (Table-2). Further no change in structure of the algae could be noticed due to the degradation, analyzed using fluorescent inverted microscope. During the degradation study, the increase in pH was observed throughout the experiment. It is due to the utilization of CO2 released by photosynthesis. An increase in pH led to the disinfection of bacteria and fungi. 4.4 Degradation of phenol present in the effluent The gas chromatogram of samples (Fig. 5) showed that the 1gL-1 of wet biomass of C. pyrenoidosa degraded phenol (C6H5OH) and other contaminants present in the effluent completely for all concentrations up to 60%. During the phenol degradation, an unidentified GC peak, concomitant with the disappearance of phenol and other pollutants, more polar than the parent compound, could be observed in the chromatograms of all samples (Fig. 5). The GC-MS analysis (Fig. 6) of phenolic effluent identified the peak raised during degradation as that of benzene methanol. The concentration of phenol in the effluent treated with algae was found to be progressively reducing to a minimum, with a corresponding increase in biomass growth. Further, to nullify the effect of abiotic factors in phenol degradation, a change in phenol concentration was analyzed for the control, indicating nil change. This proves that the C. pyrenoidosa is the sole source of the degradation of phenolic compounds. The other pollutants not taken up for characterization in this study were also found to get degraded by C. pyrenoidosa in all the effluent samples. No intracellular or cell surface adsorbed phenol (C6H5OH) was detected through the GC analysis in the microalgal pellet ethyl acetate extract. C. pyrenoidosa reduced the total phenol content to a maximum of 91% with 20% effluent (Fig. 7) Table-3.
4.5 Determination of batch kinetic coefficients The Michaelis–Menten kinetic model is used for the determination of kinetic coefficients viz. Km-half-saturation constant (Michaelis constant) and k-reaction rate constant by considering the non-inhibitory reaction. The model is described by the equation (4). The rate of substrate removal (υ) is determined by relating the maximum rate of substrate removal (Vmax) and the substrate concentration (S) as follows.
where, Vmax represents the maximum rate achieved by the system, when the substrate concentration is at its saturation. The Michaelis constant Km (half-saturation constant) is the substrate concentration at which the reaction rate is half of Vmax. The initial substrate concentration and the initial substrate removal rate are considered for the batch operation. Equation (4) is rewritten as:
where,
is
the
. Equation (5) is rewritten as:
maximum
initial
rate
of
substrate
removal
i.e.
where, k is the reaction rate constant (day−1), X0 is the initial biomass concentration. The specific rate of substrate removal
can be calculated by dividing both sides of equation (6) by the initial
biomass concentration (equation (7)).
Equation (7) can be linearized in double reciprocal form (equation (8)) and its plot of vs.
yields
a
linear
line
with
a
slope
of
and
y-axis
intercept
of
.
The experimental data given in Table-4 were plotted in the form of
vs.
(Fig. 8).
From the slope and the intercept of this plot (best fit), the kinetic coefficients of phenol removal by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination = 0.93. 4.6 Effect of phenolic effluent on carbon, hydrogen and nitrogen contents and CO2 biofixation rate of C. pyrenoidosa Carbon, hydrogen and nitrogen contents of the treated biomass were analyzed using CHNS/O Analyzer (EA2400 Series II PerkinElmer) to determine the carbon utilized by the algae from the phenolic effluent. The results showed an increase in the carbon content of the algal biomass treated with the phenolic effluent when compared to algae cultivated devoid of effluent (Table-2) (Fig. 9). The highest carbon, hydrogen and nitrogen content of the algae cultivated in the 60% of effluent were 30.3±0.17 % (C), 2.1±0.14% (H) and 3.7±0.01 % (N).
The CO2 biofixation rate RCO2 (g L-1 day-1) was calculated by equation (3), and the results are shown in (Fig. 10). C. pyrenoidosa showed the maximum CO2 biofixation rate when the effluent concentration was 60%. The maximum CO2 biofixation rate of C. pyrenoidosa was 0.25 g L-1 day-1 with the effluent concentration of 60%. The algae cultivated in control (algae devoid of effluent) showed a lower carbon fixation rate, indicating that the increase in carbon fixation rate of algal samples cultivated in effluent samples is due to the utilization of phenolic compounds. 4.7 Discussion Physico and chemical methods of phenol degradation are effective only if the effluent volume is small. The limiting factor of these methods is cost and sludge disposal. Due to the problems associated with the sludge disposal and the washing out of nitrifying bacteria, the biodegradation of phenol using bacteria is incapable of adaptation on a continuous basis at a large scale. The effluent used for the treatment is raw effluent without any prior treatment. The major contaminant present in the effluent is phenol (C6H5OH). Phenolic compounds and other contaminants present in the effluent are the source of carbon and energy for the growth of C. pyrenoidosa in the degradation process. Hence, the increase in the cell mass is a function of the exhaustion of phenolic compounds. The results demonstrated that the C. pyrenoidosa had a high ability to degrade phenolic compounds as well as the other contaminants present in the effluent with concentrations up to 60% within 7 days of reaction time. The results obtained from CHN analysis and carbon fixation rate suggested that C. pyrenoidosa would be able to fix the utilized carbon content inside the cell. The highest carbon fixation rate was found to be 0.25g L-1day-1 with a 60% effluent concentration. The results of this work suggest that the growth of algae in phenol rich effluent offers a new option of applying phycoremediation in the treatment of
phenolic effluent to manage the pollution load. The cultivation of C. pyrenoidosa for the treatment does not require much complex cultivation strategies. The nutritional requirement of the algae is very low and can easily be scaled up for higher volumes. Kinetic coefficients of phenol degradation by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination = 0.93. Rather than attempting to treat the effluent at the same time, phycoremediation offers the alternative use of algal biomass for other beneficial uses such as biomass production and biofuel production. 4.8 Practical applicability of the phycoremediation of phenolic effluent The effluent discharge from CG plant is 150m3/day. The existing industrial treatment facility of the target producer gas plant which operates largely on chemical treatment method requires 7X300m3 capacity tanks for treatment of effluent. The suggested algae treatment method (with 1gL-1) after dilution would require 240m3 capacity tanks X 7 numbers (for a treatment time of 7 days) that would reduce the phenol concentration to 20mgL-1. To reach the legal limit of 5mgL-1 additional chemical methods would continue and consume less chemicals resulting in reduced operating costs. Further, referring to Dayana Priyadharshini and Bakthavatsalam,2016 with 4gL-1 of algae feed and 4 days of treatment time, the phenol concentration in the effluent gets reduced to 20mgL-1 (for a treatment time of 4 days). Accordingly, 4 tanks of capacity with 240 m3 capacity would reduce the industrial effluent to 20mgL-1. Hence totally 4 tanks of 240m3 for treatment and 1 tank of 250m3 for algal cultivation is required against 7*300m3 tanks installed in the existing treatment plant. It must be noted that the 5 tanks required for the algae based treatment plant need to be of photobioreactor (with adequate sunlight penetration for reaction). Accordingly, the tank dimension would be one of shallow depth with large surface
area. However the final configuration and tank capacity assessment would call for more detailed design and engineering. 5.0 Conclusion The experiment was carried out for the removal of phenol from the raw effluent of coal based producer gas plant using eukaryotic microalgae C. pyrenoidosa, under ambient conditions and without any prior treatment. Phenol is identified as the major contaminant present in the effluent in addition to other contaminants. C. pyrenoidosa degrades phenol to the maximum of 91% with 20% effluent and the maximum degradation rate and the maximum specific growth rate were observed with 60% of effluent concentration. The kinetic coefficients of phenol degradation by C. pyrenoidosa were determined as k=50mg of phenol g-1(algae) day-1; Km=347mgL-1 with (R2) coefficient of determination=0.93. The various CG process plants producing effluent containing high phenol concentration will benefit with appropriately designed photobioreactor and achieve public acceptance by using this environmentally friendly biotreatment method.
Acknowledgement Authors thank National Institute of Technology, Trichy and the World Bank funded Technical Education Quality Improvement Program (TEQIP). This research did not receive any specific grant from the funding agencies in the public, commercial, or not-for-profit sectors.
References Brennan, L., Owende, P., 2010. Biofuels from microalgae - A review of technologies for production, processing, and extractions of biofuels and co-products, Renew. Sust. Energ. Rev. 14(2), 557-577. Chang, E.E., Hsing, H.J., Chiang, P.C., Chen, M.Y., Shyng, J.Y., 2008. The chemical and biological characteristics of coke-oven wastewater by ozonation. J. Hazard. Mater. 156,560-567. Cuellar-Bermudez, S.P., Garcia-Perez, J.S., Rittmann, B.E., Parra-Saldivar, R., 2015. Photosynthetic bioenergy utilizing CO2: an approach on flue gases utilization for third generation biofuels, J. Clean. Prod. 98, 53-65. Dayana Priyadharshini, S., Bakthavatsalam, A.K., 2016. Optimization of phenol degradation by the microalga Chlorella pyrenoidosa using Plackett-Burman Design and Response Surface Methodology, Bioresource Technol. 207, 50-156. Ganesamoorthy, S., Jerome, P., Shanmugasundaram, K., Karvembu, R., 2014. Highly efficient homogeneous and heterogenized ruthenium catalysts for transfer hydrogenation of carbonyl compounds, RSC Adv. 4, 27955-27962. Kim, Y.M., Park, D., Lee, D.S., Park, J.M., 2007. Instability of biological nitrogen removal in a cokes wastewater treatment facility during summer, J. Hazard.Mater. 141, 27–32. Kshirsagar, A.D., 2013. Bioremediation of wastewater by using microalgae: An experimental study, Int. J. Life Sc. Biotech. & Pharm. 2(3), 339-346. Lim, B-R., Hu, H-Y., Huang, X., Fujie, K., 2002. Effect of seawater on treatment performance and microbial population in a biofilter treating coke-oven wastewater, Process Biochem. 37, 943–948.
Lika, K., Papadakis, I.A., 2009. Modeling biodegradation of phenolic compounds by microalgae, J.Sea Research. 62,135–146. Li, Y., Li, J., Wang, C., Wang, P., 2010. Growth kinetics and phenol biodegradation of psychrotrophic Pseudomonas putida LY1. Bioresource Technol. 101, 6740–6744. Li, Hui-qiang., Han, Hong-jun., Du, Mao-an., Wang, W., 2011. Removal of phenols, thiocyanate and ammonium from coal gasification wastewater using moving bed biofilm reactor, Bioresource Technol. 102, 4667-4673. Markou, G., Chatzipavlidis, I., Georgakakis, D., 2012. Cultivation of Arthrospira (Spirulina) platensis in olive-oil mill wastewater treated with sodium hypochlorite, Bioresource Technol. 112, 234-241. Nollet, L.M.L., De Gelder, L.S.P., Handbook of Water Analysis, 2nd ed. pp.411, ISBN-13: 9781439889640. Razzak, S.A., Hossain, M.M., Lucky, R.A., Bassi, A.S., De Lasa, H., 2013. Integrated CO2 capture, wastewater treatment and biofuel production by microalgae culturing a review, Renew. Sust. Energ. Rev. 27 (C), 622-653. Semple, K.T., Cain, R.B., 1996. Biodegradation of phenols by the Alga Ochromonas Danica, Appl. Environ. Microb. 62(4), 1265-1273. Semple, K.T., 1997. Biodegradation of phenols by a eukaryotic alga, Res. Microbiol. 148, 365367. Stoilova, I., Krastanov, A., Stanchev, V., Daniel, D., Gerginova, M., Alexieva, Z., 2006. Biodegradation of high amounts of phenol, catechol, 2,4-dichlorophenol and 2,6dimethoxyphenol by Aspergillus awamori cells, Enzyme Microb. Tech. 39, 1036-1041.
Scragg, A.H., 2006. The effect of phenol on the growth of Chlorella vulgaris and Chlorella VT1, Enzyme Microb. Tech. 39, 796-799. Subashchandrabose, S.R., Ramakrishnan, B., Megharaj, M., Venkateswarlu, K., Naidu, R., 2013. Mixotrophic cyanobacteria and microalgae as distinctive biological agents for organic pollutant degradation, Environment International. 51, 59–72. Tang, D., Han, W., Li, P., Miao, X., Zhong, J., 2011. CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels, Bioresource Technol. 102, 3071–3076. Wang, Wei., Han, H., Yuan, M., Li, H., 2010. Enhanced anaerobic biodegradability of real coal gasification wastewater with methanol addition, J. Environ. Sci. 22(12), 1868-1874. Wang, W., Han, H., Yuan, M., Li, H., Fang, F., Wang, K., 2011. Treatment of coal gasification wastewater by a two-continuous UASB system with step-feed for COD and phenols removal, Bioresource Technol. 102, 5454-5460. Yun, Y.S., Lee, S.B., Park, J.M., Lee, C.I., Yang, J.W., 1997. Carbon dioxide fixation by algal cultivation using wastewater nutrients, J. Chem. Tech. Biotechnol. 69, 451- 455. Yang, C., Qian, Y., Zhang, L., Feng, J., 2006. Solvent extraction process development and onsite trial-plant for phenol removal from industrial coal-gasification wastewater, Che. Eng. J. 117,179-185. Yu, X., Zuo, J., Tang, X., Li, R., Li, Z.X., Zhang, F., 2014. Toxicity evaluation of pharmaceutical wastewaters using the algae Scenedesmus obliquus and the bacteria Vibrio fischeri, J. Hazard Mater. 266, 68-74.
Fig. 1 Schematic diagram of gas cooling system of coal based producer gas plant
ESP-Electrostatic Precipitator
Fig. 2 Culture of C. pyrenoidosa (KX686118)
Fig. 3 Analysis of types of phenol and other contaminants by Gas Chromatography
Phenol (C6H5OH)
2,4,6-Trichlorophenol
Fig. 4 Effect of phenolic effluent on the growth of C. pyrenoidosa
Biomass concentration (g/L)
20%
40%
60%
80%
Control
3 2.5 2
1.5 1 0
1
2
3
4
Time (days)
5
6
7
20, 40, 60 and 80%- Effluent concentration
Fig. 5 Reduction of phenol (C6H5OH) peak and other pollutants peaks compared to fig. 3
8
*Unidentified peak rose after the treatment period
♦- peak corresponds to phenol (C6H5OH)
Fig.6 GC-MS analysis of the effluent unidentified peak of fig. 5
24
Fig. 7 Reduction of total phenol (as per IS 3025 Part no. 43)
25
Concentration of phenol (mg/L)
Initial concentration of phenol
Final concentration of phenol
1200
1000 800 600 400 200
0 20
40 60 Concentration of effluent (% )
80
Fig. 8 Effect of concentration of phenol on specific growth rate of C. pyrenoidosa
26
0.05 y = 6.9343x + 0.0201 R² = 0.9275
0.04
1/υx
0.03 0.02 0.01 0
0
0.001
0.002 1/S 0
0.003
0.004
k-50 mg of phenol g-1 (algae) day-1; Km-347 mgL-1; υx –Specific phenol removal rate; S0- Initial phenol concentration
Carbon, hydrogen, nitrogen content (% )
Fig. 9 Analysis of Carbon, Hydrogen and Nitrogen (% w/w) Carbon
Hydrogen
Nitrogen
40
60
40
35 30 25
20 15 10
5 0
0
20
Concentration of effluent (% )
27
80
Carbon (% )
Carbon
Carbon fixation rate
35
0.3
30
0.25
25
0.2
20
0.15
15
0.1
10
0.05
5 0
0 0
20 40 60 Concentration of effluent (% )
28
80
Carbon fixation rate (gL-1 day-1 )
Fig. 10 Effect of C. pyrenoidosa on carbon and carbon fixation rate
29
Table-1 Characteristics of raw effluent
Parameter Total suspended solids (mgL-1) Total dissolved solids (mgL-1) Biological oxygen demand (mgL-1) Chemical oxygen demand (mgL-1) Oil & grease (mgL-1) Phenolic concentration (mgL-1) pH Color
30
Mean ± SD 359.75 ±107.7 1515.5 ±195.1 152.5 ±12.3 2106 ±139.9 197.25 ±23.5 1475.3±67.8 8-9.2 Dark Brown
Table-2 Biomass productivity (P), Maximum sp. growth rate (µmax), CO2 biofixation rate (RCO2), CHN analysis of C. pyrenoidosa under different effluent concentrations (mean ± standard deviation).
Concentration P of effluent (g L-1 day-1) (%) 0 0.07 20 0.09 40 0.15 60 0.22 80 0.03
µmax -1
(day ) 0.06 0.07 0.10 0.13 0.03
CO2 biofixation rate (RCO2) (g L-1day-1) 0.02±0.01 0.09±0.03 0.15±0.01 0.25±0.04 0.01±0.01
31
Carbon (%)
Hydrogen (%)
Nitrogen (%)
7.3±0.21 26.7±0.32 28.4±0.11 30.3±0.17 10.4±0.20
1±0.01 1.6±0.11 1.7±0.07 2.1±0.14 0.87±0.1
0.6±0.001 3.2±0.004 3.5±0.007 3.7±0.01 0.24±0.05
Table-3 Amount of total phenol available after treatment
Batches
Initial concentration of total phenol (mgL-1)
Concentration of total phenol after treatment (mgL-1)
Percentage Degradation (%)
20
282±1.7
26±3.2
90.78± 3.5
40
564±1.2
39±2.6
93.80±2.8
60
846±2
84±2.5
91.14±2.5
80
1128±2.4
792±1.34
29.8±1.11
(%)
32
Table-4 Specific phenol removal rate
υx (mgL of phenol g-1 (algae) day-1)
1/ υx
1/S0
282
Final concentration of algae (gL-1) 1.65
22.17
0.045
0.0035
564
2.02
33.03
0.030
0.0017
846
2.55
31.82
0.031
0.0011
1128
1.2
40
0.025
0.0009
Initial concentration of phenol (mgL-1)
-1
Treatment time -7 days υx - specific phenol removal rate S0-initial substrate concentration
33
Notations Notation X0 X1 t0 t1 µmax Cc P
v Vmax S Km k
Name Initial Concentration of biomass Final Concentration of biomass Initial reaction time Final reaction time Specific growth rate Carbon dioxide biofixation rate Carbon content Biomass productivity Molecular weight of carbon dioxide Molecular weight of carbon Rate of substrate removal Maximum rate of substrate removal Substrate concentration Saturation constant, Michaelis constant Rate of the reaction
34
Units gL-1 gL-1 day day day-1 gL-1day-1 %(w/w) gL-1day-1 mgL-1day-1 mgL-1day-1 mgL-1 mgL-1 mg of phenol g-1 (algae) day-1