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Performance of Halomonas sp. to reduce hexavalent chromium in batch and continuous fixed film reactor
Accepted Manuscript Title: Performance of Halomonas sp. to reduce hexavalent chromium in batch and continuous fixed film reactor Authors: S. Murugavelh, Kaustubha Mohanty PII: DOI: Reference:
S2213-3437(18)30158-1 https://doi.org/10.1016/j.jece.2018.03.037 JECE 2278
To appear in: Received date: Revised date: Accepted date:
20-9-2017 16-3-2018 17-3-2018
Please cite this article as: Murugavelh S., Kaustubha Mohanty, Performance of Halomonas sp.to reduce hexavalent chromium in batch and continuous fixed film reactor, Journal of Environmental Chemical Engineering https://doi.org/10.1016/j.jece.2018.03.037 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.
Revised manuscript JECE-D-17-01824R3 Performance of Halomonas sp. to reduce hexavalent chromium in batch and continuous
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fixed film reactor
S. Murugavelh * and Kaustubha Mohanty*
CO2 Research and Green Technologies Centre, VIT, Vellore 632014, Tamilnadu , India
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Department of Chemical Engineering, Indian Institute of Technology Guwahati,
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Guwahati – 781039, Assam, India.
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* Corresponding author: Email- [email protected] (S. Murugavelh)
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Tel. - +91-0416-2242504; Fax-+91-0416-2243092
Abstract
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The performance of fixed film bioreactor on bioreduction of Cr(VI) was evaluated. The reactor
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was operated under batch and continuous mode. Influent contained glucose as the single carbon source. The maximum specific growth rate obtained was 7.16 mg L-1. The half saturation
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constant for COD was 475 mg L-1. The yield coefficient was found to be 0.209. A maximum biomass of 457 h-1 was obtained for Cr(VI) free cells. Both the reactor performed better under an hydraulic retention time of 24 h. Near complete reduction of Cr(VI) was reported for an initial Cr(VI) concentration of 10 and 20 mg L-1. The maximum number of suspended cells reported 1
was 4.3 X 1014 at an Cr(VI) loading rate of 240 mg L-1day-1. The maximum amount of attached and suspended cells reported was 2013 and 238 mg. A maximum COD reduction of 84.1% was
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reported. The lag period for growth of cells under a Cr(VI) load ranging up to 40 mg L-1 was 3 h.
Keywords: Bioreduction; Halomonas sp.; Cr(VI), Fixed film reactor, wastewater treatment.
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1. Introduction
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Chromium is the commonly used metal in industries like steel production, wood preservation,
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leather tanning, electroplating, paint pigments etc. [1-3]. Chromium is available in the
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environment as Cr(VI) and Cr(III) [4,5]. The toxicity of the Cr(VI) depends on the oxidation state [6]. Chromium causes irritation on skin and respiratory tracts in humans. Chromium was
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also reported as mutagenic and carcinogenic to humans and animals [7]. Improper disposal of
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effluent and sub products of industries using chromium have increased the chromium pollution in the surrounding. The WHO and USEPA have limited the level of Cr(VI) in water at 50 µg L[6] . In order to meet the limits of the WHO, USEPA an economical method is needed to treat
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the chromium contaminated wastewater [8]. The bacterial reduction of Cr(VI) is a potential alternate to the conventional methods [9]. Many researchers have reported the bacterial reduction
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of Cr(VI). Shen and Wang, 1994, studied reduction of Cr(VI) using E.coli. Wang and Shen [10] used
Bacillus sp and Pseudomonas fluorescens LB 300 for reduction for an initial Cr(VI)
concentration of 27 mg L-1 . Philip et al., [11], reported Bacillus coagulans as a potential organism for the reduction of Cr(VI) . In particular Halomonas sp. was reported for its potential 2
in rapid reduction of Cr(VI) for an initial concentration range upt 40 mg L-1 [12]. Previous study showed that yeast extract and glucose are the essential carbon source for the growth and reduction of the Cr(VI) by Halomonas sp. [12]. Most of the chromium reduction studies reported
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was conducted under batch conditions [13, 14]. In batch operations microbial cells are guaranteed to receive the nutrients for the growth [15]. In continuous mode the intermittent seeding of the reactor is difficult and the chances of loss of limiting substrate which can lead to loss of metabolic activity [14]. In the recent years the application of continuous reactors for the reduction of Cr(VI) is gaining importance. Chirwa and Wang [16] were the first to study the
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application of fixed film reactor in the Cr(VI) bioreduction process. Shen and Wang [13], studied
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chromium reduction in a two stage reactor. The present work reports the bioreduction capacity of
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Halomonas sp. in batch and continuous mode under limited carbon energy. Limited carbon
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creates a competitive interaction for the microbe towards the glucose substrate and the Cr(VI) [1]. The biokinetic parameter which helps in determining the maximum specific growth rate, half
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saturation constant are also evaluated. 2. Materials and Methods
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2.1. Bacterial culture
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The Halomonas sp. was purchased from Institute of Microbial Technology, Chandigarh, India and grown at 37 ºC in a liquid medium at pH 7. The subcultures of the Halomonas sp. was
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prepared and stored at 4 ºC for future use. 2.2. Media The growth media for the Halomonas sp. was prepared by dissolving 5 g of yeast extract, 5 g of peptone and 1 g of glucose in 1 L of deionized water. The feed to the fixed film reactor was 3
prepared by dissolving, 0.03 g of K2HPO4, 0.03 g of KH2PO4, 0.01 g of MgSO4, 5 g of yeast extract, 5 g of peptone and 1 g of glucose in 1 L of deionized water. The media was sterilized by autoclaving (Indfos, India) at 120 °C for 15 min. Control culture is reported as the growth of the
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microbe was measured without spiking the growth media with chromium. All reagents were AR grade purchased from Merck India Ltd. 2.3. Cr(VI) stock solution
The stock solution of Cr(VI) was prepared by dissolving 2.82 g of K2Cr2O7 in 1 L of
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deionized water. The feed to the reactor containing various concentrations of Cr(VI) was
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prepared by diluting the stock solution in the growth media. All media was adjusted to pH 7
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using 0.1 N HCl.
at 540 nm using UV spectrophotometer (SpectraScan
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Cr(VI) concentration was analyzed
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2.4. Analytical procedure
(DPC)
method [17]. Total chromium was
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ThermoFisher Germany) Diphenyl carbazide
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measured using atomic absorption spectrophotometer (Varian AA240 FS) COD was determined using HACH COD digester (Model DRB 200 USA) following the
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standard methods APHA, AWWA (1994), The Dissolved oxygen concentration was measured using a DO meter (VSI-14 ATC, India).
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Glucose was measured following the standard Dinitro salicylic method [18]. Viable cells are measured by plating 100 µL of the effluent in PYG agar. The plates were incubated for 24 h and the colonies were counted. The morphology of the culture was monitored to check for the
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contamination. Bacteria density was measured by UV spectrophotometer and the results obtained at 540 nm was expressed as mgL-1 of bacterial density. The suspended cells were calculated by plating the diluted sample (10 1) on PYG agar. Colonies
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were counted after incubation, microscopic examination of the culture was performed. The attached cells were by measured by centrifuging the glass beads drawn under sterile condition in 9 mL of 0.85% NaCl for 10 mins. The sediments of the centrifugation was reported as attached cell growth, the supernatant was plated on PYG agar and incubated for 24 h.
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2.5. Reactor setup
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The fixed film reactor was fabricated to operate under completely mixed and aerated conditions.
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The schematic diagram of the reactor set up was shown in Fig. 1.
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Fig. 1. Schematic diagram of fixed film reactor
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The fixed film reactor was 42 cm long with an internal diameter of 7 cm. The reactor was made
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from pyrex glass. The reactor bed was packed with 19241 glass beads of diameter 3 mm to provide an external surface area of 1631.6 cm2 for the growth of the Halomonas sp. The reactor
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was operated at 30 °C (room temperature). The feed was provided to the reactor in up flow mode. An aquarium pump was used to aerate the fixed film reactor. The effluent from the fixed film reactor was recycled at a ratio of 100 : 1 to maintain the microbial volume inside the reactor. An aeration chamber of 25 cm length and internal diameter of 2.5 cm was used for recycling the 6
effluent. The aeration chamber was aerated by another aquarium pump. Previously calibrated peristaltic pump (ENPD- 100 Express), Enertech India was used to feed the reactor with Cr(VI) spiked media. The pumps were calibrated to maintain a HRT of 24, 12, 6 h respectively for batch
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studies and 24 h for the continuous study. The media from the feed tank was plated in PYG agar to check for the contamination. 2.6. Reactor start up and operation
The reactor column, media tanks, glass beads, silicone tubes were sterilized by autoclaving for
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120 °C for 15 min. The fixed bed reactor was assembled in a laminar flow hood (Aeromech,
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India). The reactor was fed with influent media containing 10 mg L-1 of Cr(VI). The reactor was
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operated under HRT of 24 h without inoculum. The effluent from the reactor was analysed for
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Cr(VI) and total Cr. 100 µL of the effluent was plated in PYG agar to check for contamination.
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No growth was visible, which indicated the reactor set up was sterile. The influent and effluent
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Cr(VI) was found to be same. This indicated that there was no abiotic Cr(VI) reduction. For batch studies the reactor was inoculated with 10 mL of overnight grown culture of
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Halomonas sp. Once visible growth was obtained in the glass beads, the reactor was fed with
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Cr(VI) with an lowest initial concentration of 10 mg L-1 . The broth from the reactor was collected and pour plated on to PYGA . The cell count was found to be 9 X 1014 cell mL-1. The
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same concentration of biomass was used for all concentration of Cr(VI) studied. Initially the batch time was maintained at 12 h. Later the Cr(VI) reduction was studied under different batch time of 6 h and 3h respectively.
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For continuous study the reactor was inoculated with 10 mL of pregrown culture of Halomonas sp. and operated under 24 h HRT. Once significant growth was visualized on the glass beads, the reactor was fed with 10 mg L-1 of Cr(VI) and the concentration of Cr(VI) was steadily
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increased. 3. Results and Discussion
The reactor was operated under batch and continuous condition for a wide range of influent Cr (VI) concentration (10 – 100 mg L-1). The role of glucose as sole carbon source on growth of
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the Halomonas sp. was studied and reported in this section. The COD reduction by Halomonas
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sp. was also reported. The batch studies were performed for different batch times ranging from 3
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to 24 h. The biological activity in the fixed film reactor was also reported.
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3.1. Batch studies
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Batch studies was performed to estimate the COD and Cr(VI) bioreduction by Halomonas sp.
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An initial biomass concentration of 9 X 1014 cells of Halomonas sp. was inoculated in the bioreactor. The inhibitory effect of the influent Cr(VI) on the growth of the Halomonas sp. was
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reported in Fig. 2. It was observed that the initial lag for the control culture was less than 3 h. The lag period was in the range of 3 h for growth media spiked with intial Cr(VI) ranging upto
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40 mg L-1. An initial Cr(VI) concentration of 50 mg L-1 in the growth media reported a lag
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period of 12 h. Other concentration of Cr(VI) studied had a greater inhibitory effect on the growth of the
Halomonas sp. as the lag period was found to increase beyond
Cr(VI)
concentration of 50 mg L-1. 60, 70 and 80 mg L-1 of Cr(VI) in the growth media resulted in a prolonged lag period of 18 h. 90 and 100 mg L-1 completely inhibited the growth of the bacteria 8
as the biomass obtained was negligible. The resulted showed that the increase in influent Cr(VI) concentration inhibited the growth of the bacteria. A maximum biomass concentration of 457 mg
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L-1 was reported for control culture.
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Fig. 2. Growth of Halomonas sp. in the presence of different concentration of Cr(VI) COD was reported as the substrate concentration and calculated as 3000 mg L-1 for the total
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media composition. It was observed that the Halomonas sp. was able to reduce the COD
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significantly (Fig 3). It was observed that COD was reduced to a final concentration of 524 mg L-1 and 654 mg L-1 respectively in the presence of 10 and 20 mg L-1 of Cr(VI). The COD reduction was found to decrease when the concentration of Cr(VI) in the media increased . Presence of 30, 40 and 50 mg L-1 of Cr(VI) in the media resulted in final COD of 721, 791 and 921 mg L-1 from an initial COD of 3000 mg L-1. The COD reduction was very less when the 9
concentration of Cr(VI) in the media was increased beyond 60 mg L-1. The decrease in the removal of COD in the presence of the higher concentration of Cr(VI) was possibly due to the inhibitory effect of the Cr(VI). Substrate utilization is a metabolic process. Cr(VI) is a toxic
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substance , presence of the Cr(VI) affects the growth of the bacteria as the biomass concentration decreased with increase in Cr(VI) concentration, this in turn affected the substrate utilization
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ability of the Halomonas sp.
Fig. 3. COD removal in batch studies
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The Cr(VI) reduction was studied in eleven phases. The current study was conducted in phases , the concentration of the inlet Cr(VI) was increased from 10 mgL-1 (Phase I) to 100 mg L-1 (Phase X). The
influent Cr(VI) concentration to the reactor was varied from 10 to 100 mg L-1. It was observed
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that the batch time did not had any significant effect on the reduction for initial Cr(VI) concentration ranging up to 30 mg L-1 (Table 1). The reduction of Cr(VI) reported with 12 h and 6 h for an initial Cr(VI) concentration was less. It was due to the fact that the reduction was a metabolism dependant process and the organism needed sufficient time for the growth and there by reduction of Cr(VI). The percentage reduction of Cr(VI) was found to decrease with increase
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in Cr(VI) concentration beyond 50 mg L-1.
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III
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IV
V
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Effluent Cr(VI) concentration (mg L-1) 0.06 0.09 0.121 0.14 1.29 2.32 1.46 2.52 4.71 2.91 4.26 8.44 9.14 14.16 21.82 29.48 37.41 46.89
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Days 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7
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Phase
Batch time (h) 24 12 6 24 12 6 24 12 6 24 12 6 24 12 6 24 12 6
Influent Cr(VI) concentration (mg L-1) 10 10 10 20 20 20 30 30 30 40 40 40 50 50 50 60 60 60
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Table 1. Performance of the fixed film bioreactor operated in batch mode Effluent Cr(III) concentration (mg L-1) 9.94 9.91 9.879 19.86 18.71 17.68 28.54 27.48 25.29 37.09 35.74 31.56 40.86 35.84 28.18 30.52 22.59 13.11
DO (mg L-1) 3.71 2.74 2.72 2.8 2.5 2.1 2.7 2.5 2.1 2.9 2.7 2.8 2.1 2.1 2.1 3.3 3.1 3.5
Reduction (%) 99.4 99.1 98.79 99.3 93.55 88.4 95.13 91.6 84.3 92.72 89.35 78.9 81.72 71.68 56.36 50.86 37.65 21.85 11
VIII
IX
X
70 70 70 80 80 80 90 90 90 100 100 100 10 10 10
46.38 59.14 63.88 68.66 74.32 76.89 80.88 83.14 83.88 94.32 94.31 94.32 0.06 0.08 0.08
23.62 10.86 6.12 11.34 5.68 3.11 9.12 6.86 6.12 5.68 5.69 5.68 9.94 9.92 9.92
3.7 3.5 3.7 3.7 3.4 3.4 3.1 3.4 3.7 2.7 2.7 2.7 2.7 2.7 2.7
33.74 15.51 8.74 14.17 7.1 3.88 10.13 7.62 6.8 5.68 5.69 5.68 99.4 99.2 99.2
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24 12 6 24 12 6 24 12 6 24 12 6 24 12 6
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1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7
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A minimum of 5.68 % reduction of Cr(VI) concentration was reported for an initial Cr(VI)
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concentration of 100 mg L-1. 60, 70, 80 mg L-1 of initial Cr(VI) in the influent resulted in 50, 33, 14 % bioreduction of Cr(VI). Since the biological activity in the reactor decreased , the
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reactor was inoculated with A maximum of 99.94 % of Cr(VI) reduction was reported for an
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initial Cr(VI) concentration of 10 mg L-1. It was observed that when the Cr(VI) concentration in
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the reactor was decreased from 100 mg L-1 to 10 mg L-1the Cr(VI) reduction recovered drastically. The results suggest that higher metabolic activity has significant effect on the
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reduction of Cr(VI).
The metabolic activity of the cells are drastically affected by the presence of Cr(VI) in the media.
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The number of attached colonies was also found to be maximum (2013 mg) for Cr(VI) (Table 2). As the Cr(VI) was increased further the number of colonies decreased drastically. Only 21 and 3 CFU/mL were observed when the Cr(VI) concentration was 90 and 100 mg L-1 respectively. The attached and suspended colonies reported for the inlet Cr(VI) concentration of 90 and 100 mg L12
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was 41, 60 and 6 , 4 mg respectively. The viable cells observed with further increase in the
Cr(VI) loading was zero.
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cells Attached cells (CFU mL-1) 2013 1028 1016 941 719 708 296 60 4 Not detectable amount Not detectable amount
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Total suspended CFU mL-1) 238 234 166 134 96 96 94 41 6 6
Viable Cell count (CFU mL-1) 4.3X 10 14 6.1 X 10 11 3.4x 10 9 2X 10 6 1X104 1X10 2 2X101 21 3 Not detectable amount Not detectable amount
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Biomass distribution (Phase) I II III IV V VI VII VIII IX X
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Table 2. Biological activity in the bioreactor operated in batch mode
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3.2. Continuous operation
The reactor was operated with HRT of 24 h. An initial influent Cr(VI) concentration of
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10 mg L-1 was fed to the reactor. The percentage reduction obtained with an initial Cr(VI) concentration of 10 and 20 mg L-1 was near 100%. When the concentration of Cr(VI) in the
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influent was increased to 30 and 40 mg L-1 the effluent Cr(VI) concentration was found to be 2.94 and 3.49 mg L-1. When the concentration of Cr(VI) was increased beyond 40 mg L-1 the percentage reduction was drastically affected . The effluent Cr(VI) was found to increase with increase in Cr(VI) concentration. A maximum of 96.12 mg L-1 of effluent Cr(VI) concentration 13
was reported for an initial Cr(VI) concentration of 100 mg L-1(Fig. 4) . The drop in percentage reduction with increased influent Cr(VI) was due to the loss of biological activity of the cells. Increase in the Cr(VI) concentration affected the growth of the Halomonas sp. which resulted in
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the decreased percentage reduction of Cr(VI).
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Fig. 4. Cr(VI) reduction in continuous reactor The COD removal was also dependant on the influent Cr(VI) concentration. A maximum of 94.7 % of COD removal was attained when the initial Cr(VI) was 10 mg L-1. The COD removal percentage decreased with increase in Cr(VI) concentration. The COD removal attained on day
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3, day 4 and day 5 was found to be 78, 75, 73 % respectively (Fig. 5). COD reduction attained on day 9 and 10 are found to be 10.32 and 2 % respectively. The decrease in the COD removal after day 6 was due to the fact that the Cr(VI) influent concentration in on days 6 to 10 was in the
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range of 60 to 100 mg L-1. The organism was unable to sustain under such a high chromium load
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and that affected the substrate utilization (COD) of the Halomonas sp.
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Fig. 5. COD removal in continuous reactor Cr(VI) reduction was studied in fifteen phases. The HRT for the phase I to X was maintained at 24 h. The HRT for the phase XI to XV was maintained at 12 h. The DO was found to be 3.1 ±
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0.4 . Complete reduction of Cr(VI) was obtained for 10 and 20 mg L-1 of Cr(VI). The percentage reduction obtained was 91.275 and 90.2 for phase III and IV (Table 3). The percentage reduction was found to decrease drastically from phase VI as the influent Cr(VI) concentration was above
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60 mg L-1. The effluent Cr(VI) concentration matched with the influent Cr(VI) in the IX and X phases. The reason for no significant Cr(VI) reduction in phase IX and X was due to the loss of biological activity in the reactor.
Table 3. Performance of the fixed film bioreactor operated in continuous mode Effluent Cr(III) concentration (mg L-1) 9.933 19.91 27.06 36.51 38.74 27.54 18.08 10.86 3.89 4.84 9.94 19.92 35.39 8.82 3.88
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Effluent Cr(VI) concentration (mg L-1) 0.067 0.089 2.94 3.49 11.26 32.46 51.92 69.14 86.11 95.16 0.06 0.08 4.61 81.18 96.12
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HRT (h) 24 24 24 24 24 24 24 24 24 24 12 12 12 12 12
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Days 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-12 12-14 14-16 16-20 20-24
DO (mg Reduction L-1) (% ) 2.7 99.33 2.7 99.55 2.7 90.2 3.1 91.27 3.1 77.48 3.1 45.9 2.5 25.82 2.5 13.57 2.5 4.32 2.1 4.84 2.1 99.4 2.1 99.6 3.3 88.47 3.3 9.8 3.1 3.88
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Phase I II III IV V VI VII VIII IX X XI XII XIII XIV XV
Influent Cr(VI) concentration (mg L-1) 10 20 30 40 50 60 70 80 90 100 10 20 40 90 100
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It can be observed from the Table 4 that the cell count was near Colony forming units ( CFU) for Phase IX and X. It was observed that the biological activity regained (2 × 10 12 ) CFU when the Cr(VI) loading rate was reduced to 240 mg L-1 day -1
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Viable cell count (CFU mL1 ) 6.2 X 1016 3.8X 1014 3.4x 108 1.6X 102 1X102 241 210 21 3 3 2X10 12 3X 109 1X102 21 2
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Attached cells (CFU mL-1) 2542 2568 1949 1962 708 321 247 84 16 3 2439 2439 1841 116 12
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Total suspended cells (CFU mL-1) 269 249 212 208 184 93 84 33 8 8 247 238 169 84 12
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Cr(VI) loading rate Biomass distribution (mg day-1) I 240 II 480 III 720 IV 960 V 1200 VI 1440 VII 1680 VIII 1920 IX 2160 X 2400 XI 240 XII 960 XIII 1920 XIV 2160 XV 2400
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Table 4. Biological activity in the bioreactor operated in continuous mode
Glucose was the sole carbon used for the growth and reduction of Cr(VI) by Halomonas sp.
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Complete reduction of Cr(VI) was obtained when the glucose concentration was in the range of 1000 mg L-1(Fig. 6). When the glucose concentration was increased the cumulative Cr(VI)
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concentration reduced was found to increase. It was evident that the glucose concentration is not a limiting factor in the reduction of Cr(VI) in the bioreactor.
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Fig. 6. Relationship between cumulative Cr(VI) reduced and cumulative glucose utilized
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2.7. Determination of biokinetic parameter
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The maximum specific growth rate, half saturation constant was calculated using Monod’s equation. The inhibition constant KI was determined by using the equation (2) [11].
max S
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KS S
max S
KI KS S K I Cr (VI )
(1)
(2)
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where µ, µmax are the specific growth rate (h-1) and maximum specific growth rate (h-1), Ks is the half saturation constant of COD (mg L-1), S is the COD (mg L-1), Cr(VI) is the hexavalent concentration (mg L-1), KI is the inhibition constant (mg L-1). The yield coefficient was
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calculated from the ratio of the specific growth rate to the substrate utilization rate. The kinetic parameters obtained are tabulated (Table 5). The maximum specific growth rate obtained was 7.16 mg L-1. The half saturation constant for COD was 475 mg L-1. The yield coefficient was found to be 0.209. The inhibition constant was found to be 4.07 mg L-1. KI specifies the concentration of chromium in which the growth rate of the microbe was found to
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decrease.
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Table 5. Biokinetic parameter
Model value (h-1)
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Parameter
7.16 475 3 0.20 0.005
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µmax ( Ks KI YT kd
Reference
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95
Fixed film
Elangovan 2009
10
100
Fixed film packed bed Shen and Wang 1995 reactor
5.6
100
Batch reactor suspended Murugavelh
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Concentration of the Cr(VI) Percentage removal Type of reactor mgL-1 (%)
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Table 6 : Comparison of the Cr(VI) using various reactor
and
Philp
and
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growth
Mohanty 2012
10
100
Packed-bed bioreactor
Chirwa and Wang 1997
50
81
Fixed film
Current study
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The comparison of Cr(VI) reduction by other microorganisms reported in literature summarized in Table 6. In the current study it was observed that a
is
maximum of 81 %
bioreduction of Cr(VI) was obtained with at an initial chromium concentration of 50mg L-1 at pH 6. A fixed film reactor provides an advantage that the contact time needed and the retention
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time are maintained.
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4. Conclusion
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Fixed film reactor for the growth of the Halomonas sp. and sequential reduction of the CR (VI)
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has been reported. Halomonas sp. showed an optimal growth in the batch and continuous reactor.
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It was observed that the limited carbon supply helped in better reduction as a maximum of 81.5% of Cr(VI) reduction was obtained for an initial Cr(VI) concentration of 50 mg L-1. Complete
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reduction of Cr(VI) was reported for an initial Cr(VI) concentration of 10 and 20 mg L-1. Glucose did not limit the bioreduction process. Influent Cr(VI) concentration is the limiting
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factor in the batch and continuous reactor. The continuous reactor was operated for 24 days. The DO concentration was found to be in the range of 3.1±0.4 mg L-1. The COD reduction obtained
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was 84.1 %. The biokinetic parameter was evaluated, the maximum specific growth rate reported was 7.16 mg L-1, and the KI reported was 4.07 mg L-1. The increase in the influent Cr(VI) concentration increased the lag period of the Halomonas sp. The results obtained showed that the fixed film reactor has a great potential in the reduction of Cr(VI). 20
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solutions by Eichhornia crassipes, Chem. Eng. 117 (2006) 71-77.
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8. M.T. Monatanes, R.S. Tovar, M.S. Roux, The effectiveness of the stabilization/solidification process on the leachability and toxicity of the tannery sludge chromium, J. Environ. Manage. 143 (2014) 71-79.
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9. M.A. Rege, J.N. Petersen, D.L. Johnstone, D. Turick, R. Yonge, W.A. Apel, Bacterial reduction of hexavalent chromium by Enterobacter cloacae strain HO1 grown on sucrose, Biotechnology letters, 19 (1997) 691- 694.
10. Y.T. Wang, H. Shen, Modelling Cr(VI) reduction by pure bacterial cultures , Wat. Res. 31
Cr(VI) reduction by Bacillus coagulans isolated
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11. L. Philip, L. Iyengar, C. Venkobachar,
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(1997) 727-732.
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from contaminated soils, J. Environ. Eng. 124 (1998) 1165-1170.
12. S. Murugavelh, K. Mohanty, Bioreduction of hexavalent chromium by free cells and ell free
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extracts of Halomonas sp., Chem. Engg. J. 203 (2012) 415–422.
13. H. Shen, Y.T. Wang, Hexavalent chromium removal in two stage bioreactor system J.
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Environ.Eng. 121 (1995) 798-804.
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14. R. Elangovan, L.Philip, Performance evaluation of various bioreactors for the removal of
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Cr(VI) and organic matter from industrial effluent, Biochem. Eng. J. 44 (2009) 174-186.
15. S. Murugavelh,
K. Mohanty,
Isolation , Identification an characterization of Cr(VI)
reducing Bacillus cereus from Chromium contaminated soil . Chem. Engg. J. 203 (2013) 415– 422. 22
16. E.M.N Chirwa, Y.T. Wang, Hexavalent chromium reduction by Bacillus sp. in a packed bed bioreactor, Environ.Sci. Technol. 31(1997) 1446-1451. 17. APHA, AWWA, Standard methods for the examination of water and wastewater, (1994) 19th
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ed., Washington, DC.
18. G.L. Miler, Use of DNS for the determination of reducing sugar, Anal.Chem. 31 (1972) 426-
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428.
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S2213-3437(18)30158-1 https://doi.org/10.1016/j.jece.2018.03.037 JECE 2278
To appear in: Received date: Revised date: Accepted date:
20-9-2017 16-3-2018 17-3-2018
Please cite this article as: Murugavelh S., Kaustubha Mohanty, Performance of Halomonas sp.to reduce hexavalent chromium in batch and continuous fixed film reactor, Journal of Environmental Chemical Engineering https://doi.org/10.1016/j.jece.2018.03.037 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.
Revised manuscript JECE-D-17-01824R3 Performance of Halomonas sp. to reduce hexavalent chromium in batch and continuous
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fixed film reactor
S. Murugavelh * and Kaustubha Mohanty*
CO2 Research and Green Technologies Centre, VIT, Vellore 632014, Tamilnadu , India
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Department of Chemical Engineering, Indian Institute of Technology Guwahati,
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Guwahati – 781039, Assam, India.
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* Corresponding author: Email- [email protected] (S. Murugavelh)
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Tel. - +91-0416-2242504; Fax-+91-0416-2243092
Abstract
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The performance of fixed film bioreactor on bioreduction of Cr(VI) was evaluated. The reactor
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was operated under batch and continuous mode. Influent contained glucose as the single carbon source. The maximum specific growth rate obtained was 7.16 mg L-1. The half saturation
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constant for COD was 475 mg L-1. The yield coefficient was found to be 0.209. A maximum biomass of 457 h-1 was obtained for Cr(VI) free cells. Both the reactor performed better under an hydraulic retention time of 24 h. Near complete reduction of Cr(VI) was reported for an initial Cr(VI) concentration of 10 and 20 mg L-1. The maximum number of suspended cells reported 1
was 4.3 X 1014 at an Cr(VI) loading rate of 240 mg L-1day-1. The maximum amount of attached and suspended cells reported was 2013 and 238 mg. A maximum COD reduction of 84.1% was
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reported. The lag period for growth of cells under a Cr(VI) load ranging up to 40 mg L-1 was 3 h.
Keywords: Bioreduction; Halomonas sp.; Cr(VI), Fixed film reactor, wastewater treatment.
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1. Introduction
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Chromium is the commonly used metal in industries like steel production, wood preservation,
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leather tanning, electroplating, paint pigments etc. [1-3]. Chromium is available in the
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environment as Cr(VI) and Cr(III) [4,5]. The toxicity of the Cr(VI) depends on the oxidation state [6]. Chromium causes irritation on skin and respiratory tracts in humans. Chromium was
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also reported as mutagenic and carcinogenic to humans and animals [7]. Improper disposal of
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effluent and sub products of industries using chromium have increased the chromium pollution in the surrounding. The WHO and USEPA have limited the level of Cr(VI) in water at 50 µg L[6] . In order to meet the limits of the WHO, USEPA an economical method is needed to treat
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1
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the chromium contaminated wastewater [8]. The bacterial reduction of Cr(VI) is a potential alternate to the conventional methods [9]. Many researchers have reported the bacterial reduction
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of Cr(VI). Shen and Wang, 1994, studied reduction of Cr(VI) using E.coli. Wang and Shen [10] used
Bacillus sp and Pseudomonas fluorescens LB 300 for reduction for an initial Cr(VI)
concentration of 27 mg L-1 . Philip et al., [11], reported Bacillus coagulans as a potential organism for the reduction of Cr(VI) . In particular Halomonas sp. was reported for its potential 2
in rapid reduction of Cr(VI) for an initial concentration range upt 40 mg L-1 [12]. Previous study showed that yeast extract and glucose are the essential carbon source for the growth and reduction of the Cr(VI) by Halomonas sp. [12]. Most of the chromium reduction studies reported
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was conducted under batch conditions [13, 14]. In batch operations microbial cells are guaranteed to receive the nutrients for the growth [15]. In continuous mode the intermittent seeding of the reactor is difficult and the chances of loss of limiting substrate which can lead to loss of metabolic activity [14]. In the recent years the application of continuous reactors for the reduction of Cr(VI) is gaining importance. Chirwa and Wang [16] were the first to study the
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application of fixed film reactor in the Cr(VI) bioreduction process. Shen and Wang [13], studied
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chromium reduction in a two stage reactor. The present work reports the bioreduction capacity of
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Halomonas sp. in batch and continuous mode under limited carbon energy. Limited carbon
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creates a competitive interaction for the microbe towards the glucose substrate and the Cr(VI) [1]. The biokinetic parameter which helps in determining the maximum specific growth rate, half
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saturation constant are also evaluated. 2. Materials and Methods
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2.1. Bacterial culture
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The Halomonas sp. was purchased from Institute of Microbial Technology, Chandigarh, India and grown at 37 ºC in a liquid medium at pH 7. The subcultures of the Halomonas sp. was
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prepared and stored at 4 ºC for future use. 2.2. Media The growth media for the Halomonas sp. was prepared by dissolving 5 g of yeast extract, 5 g of peptone and 1 g of glucose in 1 L of deionized water. The feed to the fixed film reactor was 3
prepared by dissolving, 0.03 g of K2HPO4, 0.03 g of KH2PO4, 0.01 g of MgSO4, 5 g of yeast extract, 5 g of peptone and 1 g of glucose in 1 L of deionized water. The media was sterilized by autoclaving (Indfos, India) at 120 °C for 15 min. Control culture is reported as the growth of the
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microbe was measured without spiking the growth media with chromium. All reagents were AR grade purchased from Merck India Ltd. 2.3. Cr(VI) stock solution
The stock solution of Cr(VI) was prepared by dissolving 2.82 g of K2Cr2O7 in 1 L of
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deionized water. The feed to the reactor containing various concentrations of Cr(VI) was
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prepared by diluting the stock solution in the growth media. All media was adjusted to pH 7
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using 0.1 N HCl.
at 540 nm using UV spectrophotometer (SpectraScan
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Cr(VI) concentration was analyzed
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2.4. Analytical procedure
(DPC)
method [17]. Total chromium was
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ThermoFisher Germany) Diphenyl carbazide
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measured using atomic absorption spectrophotometer (Varian AA240 FS) COD was determined using HACH COD digester (Model DRB 200 USA) following the
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standard methods APHA, AWWA (1994), The Dissolved oxygen concentration was measured using a DO meter (VSI-14 ATC, India).
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Glucose was measured following the standard Dinitro salicylic method [18]. Viable cells are measured by plating 100 µL of the effluent in PYG agar. The plates were incubated for 24 h and the colonies were counted. The morphology of the culture was monitored to check for the
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contamination. Bacteria density was measured by UV spectrophotometer and the results obtained at 540 nm was expressed as mgL-1 of bacterial density. The suspended cells were calculated by plating the diluted sample (10 1) on PYG agar. Colonies
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were counted after incubation, microscopic examination of the culture was performed. The attached cells were by measured by centrifuging the glass beads drawn under sterile condition in 9 mL of 0.85% NaCl for 10 mins. The sediments of the centrifugation was reported as attached cell growth, the supernatant was plated on PYG agar and incubated for 24 h.
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2.5. Reactor setup
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The fixed film reactor was fabricated to operate under completely mixed and aerated conditions.
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The schematic diagram of the reactor set up was shown in Fig. 1.
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Fig. 1. Schematic diagram of fixed film reactor
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The fixed film reactor was 42 cm long with an internal diameter of 7 cm. The reactor was made
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from pyrex glass. The reactor bed was packed with 19241 glass beads of diameter 3 mm to provide an external surface area of 1631.6 cm2 for the growth of the Halomonas sp. The reactor
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was operated at 30 °C (room temperature). The feed was provided to the reactor in up flow mode. An aquarium pump was used to aerate the fixed film reactor. The effluent from the fixed film reactor was recycled at a ratio of 100 : 1 to maintain the microbial volume inside the reactor. An aeration chamber of 25 cm length and internal diameter of 2.5 cm was used for recycling the 6
effluent. The aeration chamber was aerated by another aquarium pump. Previously calibrated peristaltic pump (ENPD- 100 Express), Enertech India was used to feed the reactor with Cr(VI) spiked media. The pumps were calibrated to maintain a HRT of 24, 12, 6 h respectively for batch
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studies and 24 h for the continuous study. The media from the feed tank was plated in PYG agar to check for the contamination. 2.6. Reactor start up and operation
The reactor column, media tanks, glass beads, silicone tubes were sterilized by autoclaving for
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120 °C for 15 min. The fixed bed reactor was assembled in a laminar flow hood (Aeromech,
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India). The reactor was fed with influent media containing 10 mg L-1 of Cr(VI). The reactor was
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operated under HRT of 24 h without inoculum. The effluent from the reactor was analysed for
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Cr(VI) and total Cr. 100 µL of the effluent was plated in PYG agar to check for contamination.
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No growth was visible, which indicated the reactor set up was sterile. The influent and effluent
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Cr(VI) was found to be same. This indicated that there was no abiotic Cr(VI) reduction. For batch studies the reactor was inoculated with 10 mL of overnight grown culture of
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Halomonas sp. Once visible growth was obtained in the glass beads, the reactor was fed with
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Cr(VI) with an lowest initial concentration of 10 mg L-1 . The broth from the reactor was collected and pour plated on to PYGA . The cell count was found to be 9 X 1014 cell mL-1. The
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same concentration of biomass was used for all concentration of Cr(VI) studied. Initially the batch time was maintained at 12 h. Later the Cr(VI) reduction was studied under different batch time of 6 h and 3h respectively.
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For continuous study the reactor was inoculated with 10 mL of pregrown culture of Halomonas sp. and operated under 24 h HRT. Once significant growth was visualized on the glass beads, the reactor was fed with 10 mg L-1 of Cr(VI) and the concentration of Cr(VI) was steadily
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increased. 3. Results and Discussion
The reactor was operated under batch and continuous condition for a wide range of influent Cr (VI) concentration (10 – 100 mg L-1). The role of glucose as sole carbon source on growth of
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the Halomonas sp. was studied and reported in this section. The COD reduction by Halomonas
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sp. was also reported. The batch studies were performed for different batch times ranging from 3
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to 24 h. The biological activity in the fixed film reactor was also reported.
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3.1. Batch studies
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Batch studies was performed to estimate the COD and Cr(VI) bioreduction by Halomonas sp.
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An initial biomass concentration of 9 X 1014 cells of Halomonas sp. was inoculated in the bioreactor. The inhibitory effect of the influent Cr(VI) on the growth of the Halomonas sp. was
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reported in Fig. 2. It was observed that the initial lag for the control culture was less than 3 h. The lag period was in the range of 3 h for growth media spiked with intial Cr(VI) ranging upto
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40 mg L-1. An initial Cr(VI) concentration of 50 mg L-1 in the growth media reported a lag
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period of 12 h. Other concentration of Cr(VI) studied had a greater inhibitory effect on the growth of the
Halomonas sp. as the lag period was found to increase beyond
Cr(VI)
concentration of 50 mg L-1. 60, 70 and 80 mg L-1 of Cr(VI) in the growth media resulted in a prolonged lag period of 18 h. 90 and 100 mg L-1 completely inhibited the growth of the bacteria 8
as the biomass obtained was negligible. The resulted showed that the increase in influent Cr(VI) concentration inhibited the growth of the bacteria. A maximum biomass concentration of 457 mg
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L-1 was reported for control culture.
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Fig. 2. Growth of Halomonas sp. in the presence of different concentration of Cr(VI) COD was reported as the substrate concentration and calculated as 3000 mg L-1 for the total
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media composition. It was observed that the Halomonas sp. was able to reduce the COD
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significantly (Fig 3). It was observed that COD was reduced to a final concentration of 524 mg L-1 and 654 mg L-1 respectively in the presence of 10 and 20 mg L-1 of Cr(VI). The COD reduction was found to decrease when the concentration of Cr(VI) in the media increased . Presence of 30, 40 and 50 mg L-1 of Cr(VI) in the media resulted in final COD of 721, 791 and 921 mg L-1 from an initial COD of 3000 mg L-1. The COD reduction was very less when the 9
concentration of Cr(VI) in the media was increased beyond 60 mg L-1. The decrease in the removal of COD in the presence of the higher concentration of Cr(VI) was possibly due to the inhibitory effect of the Cr(VI). Substrate utilization is a metabolic process. Cr(VI) is a toxic
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substance , presence of the Cr(VI) affects the growth of the bacteria as the biomass concentration decreased with increase in Cr(VI) concentration, this in turn affected the substrate utilization
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ability of the Halomonas sp.
Fig. 3. COD removal in batch studies
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The Cr(VI) reduction was studied in eleven phases. The current study was conducted in phases , the concentration of the inlet Cr(VI) was increased from 10 mgL-1 (Phase I) to 100 mg L-1 (Phase X). The
influent Cr(VI) concentration to the reactor was varied from 10 to 100 mg L-1. It was observed
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that the batch time did not had any significant effect on the reduction for initial Cr(VI) concentration ranging up to 30 mg L-1 (Table 1). The reduction of Cr(VI) reported with 12 h and 6 h for an initial Cr(VI) concentration was less. It was due to the fact that the reduction was a metabolism dependant process and the organism needed sufficient time for the growth and there by reduction of Cr(VI). The percentage reduction of Cr(VI) was found to decrease with increase
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in Cr(VI) concentration beyond 50 mg L-1.
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III
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IV
V
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Effluent Cr(VI) concentration (mg L-1) 0.06 0.09 0.121 0.14 1.29 2.32 1.46 2.52 4.71 2.91 4.26 8.44 9.14 14.16 21.82 29.48 37.41 46.89
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Days 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7
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Phase
Batch time (h) 24 12 6 24 12 6 24 12 6 24 12 6 24 12 6 24 12 6
Influent Cr(VI) concentration (mg L-1) 10 10 10 20 20 20 30 30 30 40 40 40 50 50 50 60 60 60
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Table 1. Performance of the fixed film bioreactor operated in batch mode Effluent Cr(III) concentration (mg L-1) 9.94 9.91 9.879 19.86 18.71 17.68 28.54 27.48 25.29 37.09 35.74 31.56 40.86 35.84 28.18 30.52 22.59 13.11
DO (mg L-1) 3.71 2.74 2.72 2.8 2.5 2.1 2.7 2.5 2.1 2.9 2.7 2.8 2.1 2.1 2.1 3.3 3.1 3.5
Reduction (%) 99.4 99.1 98.79 99.3 93.55 88.4 95.13 91.6 84.3 92.72 89.35 78.9 81.72 71.68 56.36 50.86 37.65 21.85 11
VIII
IX
X
70 70 70 80 80 80 90 90 90 100 100 100 10 10 10
46.38 59.14 63.88 68.66 74.32 76.89 80.88 83.14 83.88 94.32 94.31 94.32 0.06 0.08 0.08
23.62 10.86 6.12 11.34 5.68 3.11 9.12 6.86 6.12 5.68 5.69 5.68 9.94 9.92 9.92
3.7 3.5 3.7 3.7 3.4 3.4 3.1 3.4 3.7 2.7 2.7 2.7 2.7 2.7 2.7
33.74 15.51 8.74 14.17 7.1 3.88 10.13 7.62 6.8 5.68 5.69 5.68 99.4 99.2 99.2
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24 12 6 24 12 6 24 12 6 24 12 6 24 12 6
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1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7 1 to 3 3 to 5 5 to 7
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A minimum of 5.68 % reduction of Cr(VI) concentration was reported for an initial Cr(VI)
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concentration of 100 mg L-1. 60, 70, 80 mg L-1 of initial Cr(VI) in the influent resulted in 50, 33, 14 % bioreduction of Cr(VI). Since the biological activity in the reactor decreased , the
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reactor was inoculated with A maximum of 99.94 % of Cr(VI) reduction was reported for an
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initial Cr(VI) concentration of 10 mg L-1. It was observed that when the Cr(VI) concentration in
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the reactor was decreased from 100 mg L-1 to 10 mg L-1the Cr(VI) reduction recovered drastically. The results suggest that higher metabolic activity has significant effect on the
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reduction of Cr(VI).
The metabolic activity of the cells are drastically affected by the presence of Cr(VI) in the media.
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The number of attached colonies was also found to be maximum (2013 mg) for Cr(VI) (Table 2). As the Cr(VI) was increased further the number of colonies decreased drastically. Only 21 and 3 CFU/mL were observed when the Cr(VI) concentration was 90 and 100 mg L-1 respectively. The attached and suspended colonies reported for the inlet Cr(VI) concentration of 90 and 100 mg L12
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was 41, 60 and 6 , 4 mg respectively. The viable cells observed with further increase in the
Cr(VI) loading was zero.
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cells Attached cells (CFU mL-1) 2013 1028 1016 941 719 708 296 60 4 Not detectable amount Not detectable amount
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Total suspended CFU mL-1) 238 234 166 134 96 96 94 41 6 6
Viable Cell count (CFU mL-1) 4.3X 10 14 6.1 X 10 11 3.4x 10 9 2X 10 6 1X104 1X10 2 2X101 21 3 Not detectable amount Not detectable amount
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Biomass distribution (Phase) I II III IV V VI VII VIII IX X
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Table 2. Biological activity in the bioreactor operated in batch mode
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3.2. Continuous operation
The reactor was operated with HRT of 24 h. An initial influent Cr(VI) concentration of
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10 mg L-1 was fed to the reactor. The percentage reduction obtained with an initial Cr(VI) concentration of 10 and 20 mg L-1 was near 100%. When the concentration of Cr(VI) in the
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influent was increased to 30 and 40 mg L-1 the effluent Cr(VI) concentration was found to be 2.94 and 3.49 mg L-1. When the concentration of Cr(VI) was increased beyond 40 mg L-1 the percentage reduction was drastically affected . The effluent Cr(VI) was found to increase with increase in Cr(VI) concentration. A maximum of 96.12 mg L-1 of effluent Cr(VI) concentration 13
was reported for an initial Cr(VI) concentration of 100 mg L-1(Fig. 4) . The drop in percentage reduction with increased influent Cr(VI) was due to the loss of biological activity of the cells. Increase in the Cr(VI) concentration affected the growth of the Halomonas sp. which resulted in
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the decreased percentage reduction of Cr(VI).
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Fig. 4. Cr(VI) reduction in continuous reactor The COD removal was also dependant on the influent Cr(VI) concentration. A maximum of 94.7 % of COD removal was attained when the initial Cr(VI) was 10 mg L-1. The COD removal percentage decreased with increase in Cr(VI) concentration. The COD removal attained on day
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3, day 4 and day 5 was found to be 78, 75, 73 % respectively (Fig. 5). COD reduction attained on day 9 and 10 are found to be 10.32 and 2 % respectively. The decrease in the COD removal after day 6 was due to the fact that the Cr(VI) influent concentration in on days 6 to 10 was in the
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range of 60 to 100 mg L-1. The organism was unable to sustain under such a high chromium load
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and that affected the substrate utilization (COD) of the Halomonas sp.
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Fig. 5. COD removal in continuous reactor Cr(VI) reduction was studied in fifteen phases. The HRT for the phase I to X was maintained at 24 h. The HRT for the phase XI to XV was maintained at 12 h. The DO was found to be 3.1 ±
15
0.4 . Complete reduction of Cr(VI) was obtained for 10 and 20 mg L-1 of Cr(VI). The percentage reduction obtained was 91.275 and 90.2 for phase III and IV (Table 3). The percentage reduction was found to decrease drastically from phase VI as the influent Cr(VI) concentration was above
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60 mg L-1. The effluent Cr(VI) concentration matched with the influent Cr(VI) in the IX and X phases. The reason for no significant Cr(VI) reduction in phase IX and X was due to the loss of biological activity in the reactor.
Table 3. Performance of the fixed film bioreactor operated in continuous mode Effluent Cr(III) concentration (mg L-1) 9.933 19.91 27.06 36.51 38.74 27.54 18.08 10.86 3.89 4.84 9.94 19.92 35.39 8.82 3.88
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Effluent Cr(VI) concentration (mg L-1) 0.067 0.089 2.94 3.49 11.26 32.46 51.92 69.14 86.11 95.16 0.06 0.08 4.61 81.18 96.12
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HRT (h) 24 24 24 24 24 24 24 24 24 24 12 12 12 12 12
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Days 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-12 12-14 14-16 16-20 20-24
DO (mg Reduction L-1) (% ) 2.7 99.33 2.7 99.55 2.7 90.2 3.1 91.27 3.1 77.48 3.1 45.9 2.5 25.82 2.5 13.57 2.5 4.32 2.1 4.84 2.1 99.4 2.1 99.6 3.3 88.47 3.3 9.8 3.1 3.88
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Phase I II III IV V VI VII VIII IX X XI XII XIII XIV XV
Influent Cr(VI) concentration (mg L-1) 10 20 30 40 50 60 70 80 90 100 10 20 40 90 100
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It can be observed from the Table 4 that the cell count was near Colony forming units ( CFU) for Phase IX and X. It was observed that the biological activity regained (2 × 10 12 ) CFU when the Cr(VI) loading rate was reduced to 240 mg L-1 day -1
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Viable cell count (CFU mL1 ) 6.2 X 1016 3.8X 1014 3.4x 108 1.6X 102 1X102 241 210 21 3 3 2X10 12 3X 109 1X102 21 2
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Attached cells (CFU mL-1) 2542 2568 1949 1962 708 321 247 84 16 3 2439 2439 1841 116 12
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Total suspended cells (CFU mL-1) 269 249 212 208 184 93 84 33 8 8 247 238 169 84 12
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Cr(VI) loading rate Biomass distribution (mg day-1) I 240 II 480 III 720 IV 960 V 1200 VI 1440 VII 1680 VIII 1920 IX 2160 X 2400 XI 240 XII 960 XIII 1920 XIV 2160 XV 2400
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Table 4. Biological activity in the bioreactor operated in continuous mode
Glucose was the sole carbon used for the growth and reduction of Cr(VI) by Halomonas sp.
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Complete reduction of Cr(VI) was obtained when the glucose concentration was in the range of 1000 mg L-1(Fig. 6). When the glucose concentration was increased the cumulative Cr(VI)
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concentration reduced was found to increase. It was evident that the glucose concentration is not a limiting factor in the reduction of Cr(VI) in the bioreactor.
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Fig. 6. Relationship between cumulative Cr(VI) reduced and cumulative glucose utilized
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2.7. Determination of biokinetic parameter
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The maximum specific growth rate, half saturation constant was calculated using Monod’s equation. The inhibition constant KI was determined by using the equation (2) [11].
max S
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KS S
max S
KI KS S K I Cr (VI )
(1)
(2)
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where µ, µmax are the specific growth rate (h-1) and maximum specific growth rate (h-1), Ks is the half saturation constant of COD (mg L-1), S is the COD (mg L-1), Cr(VI) is the hexavalent concentration (mg L-1), KI is the inhibition constant (mg L-1). The yield coefficient was
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calculated from the ratio of the specific growth rate to the substrate utilization rate. The kinetic parameters obtained are tabulated (Table 5). The maximum specific growth rate obtained was 7.16 mg L-1. The half saturation constant for COD was 475 mg L-1. The yield coefficient was found to be 0.209. The inhibition constant was found to be 4.07 mg L-1. KI specifies the concentration of chromium in which the growth rate of the microbe was found to
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decrease.
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Table 5. Biokinetic parameter
Model value (h-1)
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Parameter
7.16 475 3 0.20 0.005
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µmax ( Ks KI YT kd
Reference
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95
Fixed film
Elangovan 2009
10
100
Fixed film packed bed Shen and Wang 1995 reactor
5.6
100
Batch reactor suspended Murugavelh
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Concentration of the Cr(VI) Percentage removal Type of reactor mgL-1 (%)
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Table 6 : Comparison of the Cr(VI) using various reactor
and
Philp
and
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growth
Mohanty 2012
10
100
Packed-bed bioreactor
Chirwa and Wang 1997
50
81
Fixed film
Current study
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The comparison of Cr(VI) reduction by other microorganisms reported in literature summarized in Table 6. In the current study it was observed that a
is
maximum of 81 %
bioreduction of Cr(VI) was obtained with at an initial chromium concentration of 50mg L-1 at pH 6. A fixed film reactor provides an advantage that the contact time needed and the retention
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time are maintained.
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4. Conclusion
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Fixed film reactor for the growth of the Halomonas sp. and sequential reduction of the CR (VI)
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has been reported. Halomonas sp. showed an optimal growth in the batch and continuous reactor.
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It was observed that the limited carbon supply helped in better reduction as a maximum of 81.5% of Cr(VI) reduction was obtained for an initial Cr(VI) concentration of 50 mg L-1. Complete
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reduction of Cr(VI) was reported for an initial Cr(VI) concentration of 10 and 20 mg L-1. Glucose did not limit the bioreduction process. Influent Cr(VI) concentration is the limiting
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factor in the batch and continuous reactor. The continuous reactor was operated for 24 days. The DO concentration was found to be in the range of 3.1±0.4 mg L-1. The COD reduction obtained
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was 84.1 %. The biokinetic parameter was evaluated, the maximum specific growth rate reported was 7.16 mg L-1, and the KI reported was 4.07 mg L-1. The increase in the influent Cr(VI) concentration increased the lag period of the Halomonas sp. The results obtained showed that the fixed film reactor has a great potential in the reduction of Cr(VI). 20
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