Arsenic toxicity to Chlorella pyrenoidosa and its phycoremediation

Acta Ecologica Sinica 36 (2016) 256–268

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Arsenic toxicity to Chlorella pyrenoidosa and its phycoremediation M.S. Podder ⁎, C.B. Majumder Department of Chemical Engineering, Indian Institute of Technology, Roorkee, Roorkee, 247667, India

a r t i c l e

i n f o

Article history: Received 11 May 2015 Received in revised form 19 April 2016 Accepted 20 April 2016 Keywords: Arsenic Arsenic contaminated water Phycoremediation Chlorella pyrenoidosa Monod model

a b s t r a c t Provision of clean drinking water remains a global requirement, so for arsenic-affected areas where various physico-chemical methods are used. These methods require regeneration of media requiring non-stop arsenic checking and it requires skilled operation. Otherwise arsenic contaminated water has to be discarded into the environment. This study examined the probability of using living microalgae Chlorella pyrenoidosa for phycoremediation of arsenic contaminated water (either As(III) or As(V). DO and pH cycles in presence of arsenic (either As(III) or As(V)) ions in the culture did not significantly differ from the control (pure media) indicating that the algae were still growing and photosynthesizing in presence of arsenic ions. As(V) was more noxious than As(III), particularly at pH 7.0, but it was opposite at pH 9.0. Phycoremediation efficiency of As(V) at pH 9.0 by algal cells was greater than that As(III). Monod model has been employed for demonstrating growth kinetics of microalgae in pure media containing various concentrations of nitrate ions. Maximum specific growth rate and saturation constant have been found to be 0.146 d−1 and 8.29E–4 g/L, respectively. With the increase in concentration of phosphate in growth medium the growth of microalgae increased. Media with 1.0 g/L NaCl indicated the highest algal growth. Addition of NaHCO3 (1 g/L) resulted in higher maximum biomass concentration. © 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Arsenic exists naturally in terrestrial and aquatic and environments because of geochemical reactions, weathering of rocks and soil. Arsenic, one of the major contaminants in waste releases is entered into the aquatic system through anthropogenic sources of activities such as mining, electricity generation by burning coal, copper smelting and use of insecticides, pigments and pesticides [1,2]. In natural waters, it typically exists as As(III) and As(V). As(III) is 60 times much more toxic than As(V), because of its higher cellular uptake [3]. Long-term exposure to inorganic arsenic compounds can cause several diseases, for example hyperpigmentation, disorders of the peripheral vascular system and central nervous system, skin, liver, bladder, prostate, lung cancer and ultimately death [1,4,5]. In drinking water the maximum contaminant level (MCL) of arsenic has been revised to 10 μg/L from 50 μg/L by the World Health Organization (WHO) in 1993 [6] and the European Commission in 2003 [7]. However, many countries including Bangladesh and China have kept the drinking water standard as 50 μg/L as the prior WHO guideline. United State Environmental Protection Agency [8] has also recommended the MCL of arsenic in drinking water 10 μg/L. In drinking water the MCL of arsenic ⁎ Corresponding author. E-mail addresses: [email protected] (M.S. Podder), [email protected] (C.B. Majumder).

http://dx.doi.org/10.1016/j.chnaes.2016.04.012 1872-2032/© 2016 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

has been set at 7 μg/L by National Health and Medical Research Committee (NHMRC), Australia [9]. Various physico-chemical treatment techniques like precipitation, adsorption, electrocoagulation, ion-exchange and solar distillation systems have exhibited favourable outcomes in remediation of water [1,10–12]. But most of these techniques generate arsenicladen discards as water, which needs safe discarding so as to escape leaching of arsenic from brine into soil [13]. Bioremediation is an emergent form of technology in that microbes are utilized for removing the pollutants which deal with a low cost and ecologically appreciated method for the alleviation of arsenic compounds poisonousness in the environment [2]. Phycoremediation is the use of plants (including macroalgae or microalgae or cynanobacteria or lower plants) and associated microflora for the elimination or biotransformation of contaminants, including heavy metals and nutrients etc. from wastewater and CO2 from waste air [14–17]. The main assumption is that the microalgae will transform some of the pollutants into non-hazardous materials and so the treated water can be recycled or safely discarded [18]. As microalgae utilize CO2 as a carbon source, they can grow photoautotrophically without the adding an organic carbon source. Microalgae propose a cheap and competent approach for removing extra nutrients and other pollutants in tertiary wastewater treatment, when creating possibly valuable biomass, owing to a high inorganic nutrient uptake capacity [19,20]. Algae deliberated as green-cell factories are not only good scavengers of poisonous chemicals but are also engaged in oxygenation of the

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atmosphere and CO2 sequestration, thus creating them a better applicant among bioremediation systems. Arsenic-affected rural regions need methods which should not only be lucrative but also simple to operate and maintain [21]. Out of the existing physico-chemical techniques, most need expert operations and observing. Less expensive sewage treatment techniques, run in some developing countries, include the usage of algal ponds having living forms of algae which aid in stabilisation of sewage in rural areas [22]. As an emerging technology, investigation on phycoremediation has to be advanced for overcoming complex environmental issues [23]. The cultivation of algae in wastewater offers the combined advantages of mitigation of greenhouse gases, treatment of the wastewaters and simultaneously producing algal biomass. This biomass produced after wastewater treatment can be exploited for multiple uses such as production of bioenergy (biogas and biofuels), fertilizer, bio-ore for precious heavy metals, pharmaceuticals, cosmetics and other valuable chemicals [24,25]. In copper smelting wastewater concentration of arsenic is as high as 1979 mg/L [11]. The presence of metal ions, like lead, copper, zinc, iron, cadmium and bismuth, nickel, chromium restrict the solubility of arsenic due to the formation of sparingly soluble metal arsenates, poses a serious threat towards man and the flora and fauna of our ecosystem contaminating the natural water tables (ground and surface water) in the vicinity. Owing to its toxicity, it should be removed or recovered from wastewater before discarding. Although some researchers have reported on the biosorption of heavy metals using mango leaf powder and rice husk, they have dealt with low concentrations of arsenic, maximum being 1000 mg/L [26]. Treatment of such a wastewater with the reported techniques includes a dilution step, which requires the use of large amounts of water and, probably, extra space which would be a major limitation. Moreover, it is felt that recovery of arsenic from microalgae for reuse may be more beneficial and cost-effective when dealing with high adsorbate concentrations. Although a number of pioneering works have been reported on treatment of arsenic containing wastewater by various techniques, only a few studies on a microbial route for detoxification of arsenic has been reported so far. Furthermore, since Chlorella pyrenoidosa grows more in polluted water [27] and the biomass can be considered as a prospective source for biofuel production [28], the potential of the living microalgae C. pyrenoidosa has been assessed in terms of its arsenic (either As(III) or As(V)) removal efficiency. As far as it is known, this is the first ever try for exhibiting quantitatively the effect of arsenic (either As(III) or As(V)) on the growth and phycoremediation of microalgae C. pyrenoidosa. The objectives of the current research were 1) to find the effects of initial concentrations of arsenic (either As(III) or As(V)) onto the growth and phycoremediation properties of the microalgae C. pyrenoidosa, 2) to investigate the influence of initial nitrate concentration onto the growth of C. pyrenoidosa in absence and presence of arsenic ions (either As(III) or As(V), 3) to determine the kinetic parameters for growth of microalgae C. pyrenoidosa biomass in pure media containing various concentration of nitrate ions using the Monod equation and 4) to find the impact of bicarbonate concentrations and salinity onto the growth properties of the microalgae C. pyrenoidosa in pure media.

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Table 1 Major elements. Composition

Quantity (per litre)

NaNO3 K2HPO4 MgSO4.7H2O CaCl2.2H2O Citric acid Na2EDTA Na2CO3 Ferric ammonium citrate Trace metal mix A5

1.5 g 0.04 g 0.075 0.036 0.006 g 0.001 g 0.02 g 0.006 g 1.0 mL

2.2. Microalgae and culture medium C. pyrenoidosa was collected from Department of Biotechnology, IIT Roorkee and grown in BG11 culture medium (without adding carbon source) [29,30]. The compositions of the BG11 medium are given in Tables 1 and 2 as follows: 2.3. Preparation of living cells of C. pyrenoidosa The algal strain was first grown in the round bottom flask tightly closed with cotton plug for keeping the batch reactor sterile. The algal inoculum was prepared by aseptically transferring a loop full of algal culture from the nutrient agar plates to the flask consisting of growth medium (sterilized at 121 °C temperature, 15 psi pressure for at least 15 min), incubated at 28 °C for 7 days in a thermostatically controlled environmental chamber (PGC-292, Plant Growth Chamber, Ambala, India). The pH of the growth medium was adjusted to 9.0 with 1 N NaOH and 1 N HCl before inoculation. Then the algal strain was grown on a petri plates consisting of agar medium containing the nutrient components (Table 1) and agar (12.0 g/L). The algae were inoculated from the plates on another fresh agar plate after the incubation of cultures at 28 °C for 7 days in agar plates and stored at 4 °C till required for further studies. 2.4. Preparation of arsenic contaminated water Arsenic stock solution was made by using salts of NaAsO2 and Na2HAsO4, 7H2O in double distilled water, purchased from Himedia Laboratories Pvt. Ltd. Mumbai India (APHA, 1998). BG11 media composition was also added into the solution. Then the prepared growth media was subjected to autoclave sterilization at 15 psi pressure and at 121 °C temperature for 15 min. The pH of the phycoremediation medium was adjusted to the requisite value by dropwise addition of sterile 1 N HCl and 1 N NaOH solution. 2.5. Experimental set-up Experiments were arranged to investigate the growth and phycoremediation properties of growing microalgae C. pyrenoidosa in different concentrations of arsenic-enriched water in a thermostatically controlled environmental chamber (PGC-292, Plant Growth Chamber, Ambala, India) (Fig. 1). BG11 medium was served as control. The pH

2. Materials and methods 2.1. Materials All the chemicals and reagents were of analytical reagent grade and used without additional purification. The stock solutions of As(III) and As(V) were prepared by dissolving NaAsO2 and Na2HAsO4 ∙ 7H2O purchased from Himedia Laboratories Pvt. Ltd. Mumbai India in double distilled water. All required chemicals utilized in the experiments, were bought from Himedia Laboratories Pvt. Ltd. Mumbai India.

Table 2 Trace metal mix A5. Composition

Quantity (per 100 mL)

H3BO3 MnCl2.4H2O ZnSO4.7H2O Na2MoO4.2H2O CuSO4.5H2O Co(NO3)2.6H2O

2.86 mg 1.81 mg 0.22 mg 0.39 mg 0.08 g 0.05g

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Fig. 1. Front view of plant growth chamber (Not to scale).

of the medium was adjusted by dropwise addition of sterile 1 N HCl and 1 N NaOH. An optimum aliquot (10%, volume of inoculum/volume of growth medium) (0.182 g/L) of preculture was harvested aseptically during the exponential growth phase (OD value ∼ 0.515 at 680 nm) and it was transferred to the fresh media (100 mL) supplemented with arsenic (either As(III) or As(V)) of requisite quantity for different studies in 250 mL Erlenmeyer flasks. After the inoculation of microalgae into the fresh growth medium, cultures were grown in an environmental chamber. Thermostatically controlled environmental chamber was sustained at 28 °C temperature. Illumination was supplied by continuous cool white fluorescent lamps at 2000 lx (Philips 40 W, cool daylight, 6500 K) with a dark/light period of 12:12 h (75 μmol photons/m2/ s). The experiments were performed for 16 days and all tests were done in duplicate (Fluid Particle Research Lab). The inoculated flasks were incubated in an environmental chamber under the above mentioned experimental conditions. The cultures were aerated with 2% CO 2 in air at a flow rate of approximately 150 mL/ min twice in a day for 30 min by bubbling through the growth medium using an air pump. Aeration of cultures with CO2 enriched air is necessary to achieve high productivities for phototrophic microalgae and also helps in mixing and pH buffering [31]. Then the cultures were manually agitated for another 20 min to prevent algal cells sedimentation on the flasks bottom and to enhance the distribution of CO2 in the growth medium [32]. In the checking of the experimental flasks, DO cycling and pH cycling (made by the alternating 12 h illumination and darkness) (DO and pH were measured using DO probe of HQ 40d, HACH, India and pH probe of HQ 40d, HACH, India, respectively) sustained without alteration after adding arsenic (either As(III) or As(V)) to the flasks. A thorough kinetic study was performed by culturing a living microalgae of C. pyrenoidosa in a simulated solution of different concentration arsenic ions (either As(III) or As(V)). Phycoremediation studies were also

performed in the batch reactors to determine the effect of contact time and initial arsenic (either As(III) or As(V)) concentrations on the removal of arsenic (As(III)/As(V)) from synthetic wastewater. To optimize the conditions for the maximum growth of microalgae C. pyrenoidosa in presence of various ions (such bicarbonate ions and sodium chloride), experiments were carried in arsenic free media. Experiments were also performed for studying the growth C. pyrenoidosa in the variable NaNO3 concentration from 0.05 to 2.0 g/L in the absence and presence of arsenic ions (50 mg/L of either As(III) or As(V)) under same experimental conditions. The growth of the microalgae C. pyrenoidosa in the pure media containing different concentration of NaNO3 ∙ 4H2O salt has been modelled employing Monod equation and kinetic parameters have been evaluated. Samples (5 mL) were withdrawn at certain time intervals and then centrifuged (Remi Instruments ltd., Mumbai India) at 5000 rpm for 5 min and the supernatant fraction was analyzed for residual concentration of either As(III) or As(V) ions. Absorbance (OD) of the samples collected intermittently from the flask was also measured at 680 nm and the microalgal biomass has been determined in terms of dry biomass. Arithmetic mean of results of two similar experiments was utilized to estimate data. 2.6. Metal analysis The analysis of total arsenic was conducted with a ThermoFisher Scientific iCE 3000 Series AA graphite furnace atomic absorption (GFAA) spectrometer, with Zeeman background correction (GF-AAS). A 0.5 nm slit-width and a wavelength of 193.7 nm were chosen. An arsenic high-intensity hollow-cathode lamp was utilized for determining As b20 μg/L. Pyrolytic graphite coated tubes with forked pyrolytic platforms were utilized. Argon (Ar) was utilized as a protective gas all through. To determine the dissolved arsenic, 8.0 mL of a centrifuged sample was added to 1 mL

M.S. Podder, C.B. Majumder / Acta Ecologica Sinica 36 (2016) 256–268

of 1% HNO3 and 1.0 mL of chemical matrix modifier (50 g/L of nickel nitrate solution) in a flask. The prepared sample injected was 20 μL. All measurements were conducted at least in duplicate and on the basis of integrated absorbance. The procedure of detailed analysis for this fast and easy to operate method was defined by Michon et al. [33]. The phycoremediation % of arsenic is evaluated utilizing the following equation: Phycoremediation% ¼

ðC 0 −C e Þ  100 C0

ð1Þ

2.7. Biomass analysis

The pellet was subjected to Fourier transform infrared spectrometer (NICHOLET 6700, coupled with OMNIC software version 6.2) spectrum between wave number 4000–400 cm−1 [35]. The Infrared spectra of the unloaded and metal loaded C. pyrenoidosa biomass were obtained using FTIR. 2.10. Modelling of growth kinetics and determination of kinetic parameters for growth of algal biomass in pure media The classical Monod equation was suggested for modelling the growth kinetics of the current microalgae C. pyrenoidosa as follows [34]:

μ¼

The growth of algae was checked by measuring the absorbance (optical density) of small volume of samples taken out from the flask periodically. The optical density was measured at 680 nm, against double distilled water, which served as reagent. The values of optical density were measured by serially diluting 7 d cultures with known OD 680 values. By oven drying an aliquot in a preweighed aluminium foil container to a constant weight at 60 ° C and measuring their mass [34] and then correlated with the cell concentrations, in terms of dry weight of cells per litre of suspension (g/L) by appropriate calibration curves. A correlation for converting OD680 values to algal dry weight was established from the calibration curve. These correlations were utilized all through the research for determining the biomass concentration of algae.

259

μ max C A ðK S þ C A Þ

ð3Þ

2.11. Statistical analysis Each set of experiments were conducted in duplicates. Experimentally obtained results were checked for statistical significance by Oneway Anova test coupled with Tukey's test using utilizing professional graphics software package OriginPro (8.5.1 version). Now the role of arsenic on the growth of microalgae in presence of phosphate is tested with ANOVA (Analysis of Variance) calculations. For ANOVA calculations we assume the Null hypothesis as. A1 = A2 = 0

2.8. Growth of algal biomass The percentage increase in dry biomass can be calculated by using the following equation: % increase in dry biomass ¼

w1 −w2  100 w2

ð2Þ

2.9. Characterization of microalgae The 7 days culture of C. pyrenoidosa, grown in BG11 media in absence and presence of 2000 mg/L arsenic (either As(III) or As(V)) ions, was centrifuged at 5000 g for 5 min. The supernatant was discarded and the cells were washed two times with phosphate buffer solution. The washed cells were resuspended in glutaraldehyde (2% v/v) for 2 h. The resuspended cells were centrifuged at 5000 g for 5 min. The supernatant was discarded and the cells were treated with serial dilution of ethanol. The samples were serially diluted to 30%, 50, 70%, 90% and finally 100%. Every dilution of ethanol was applied on the cells for 10 min individually. The cells were allowed to evaporate ethanol under atmospheric conditions. The cells were subjected to gold sputtering using Sputter Coater, Edwards S150, which provides conductivity to the samples, under vacuum followed by degassing. Finally, the cells were characterized by scanning electron microscopy (Fe-SEM) and EDX. The measurements of SEM were done for observing the surface morphologies of the microalgae (SEM; LEO electron Microscopy, England) [35]. The images were taken with an accelerator voltage = 15 kV and an emission current = 100 μA by the Tungsten filament. The microalgal cells of C. pyrenoidosa grown in BG11 media were centrifuged at 5000 g for 5 min. The cells obtained after centrifugation were washed twice with phosphate buffer solution and dried in oven at 353 K for 24 h. The dried biomass was finally grounded in mortar to obtain fine powder. The finely powdered cell biomass was mixed with photometric potassium bromide (KBr) to make a pellet of 1% (w/w).

Where A1 represents the influence of nitrate without arsenic (either As(III) or As(V)) and A2 represents the influence of nitrate with arsenic (either As(III) or As(V)). In this model it has been assumed that there is no influence of adding arsenic on the growth of microalgae and to test this hypothesis the following data were considered for ANOVA calculation. Supposing there is no influence of arsenic (either As(III) or As(V)) on the growth of microalgae and the reduced specific growth rate is happened by chance. For this investigation the ANOVA table is constructed for As(III) and As(V) and F value is estimated for both the species and compared with the F table value. For the growth A1 and A2 the five different samples were analysed for their specific growth rate. The specific growth rate data using phosphate with and without arsenic (either As(III) or As(V)) six different concentration solutions of phosphate and arsenic (either As(III) or As(V)).

Fig. 2. Data collected for three cultures: DO for first with no arsenic (control) and second with 50 mg/L As(III) and third with 50 mg/L As(V) (dark/light period: 12:12 h).

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establish a natural pH buffering system in water as defined as follows [39,40]:

3. Results and discussion 3.1. Calibration curve

H2 0 þ CO2 ↔H2 CO3

ð5Þ

H2 CO3 ↔Hþ þ HCO− 3

ð6Þ

2− þ Hþ þ HCO− 3 ↔2H þ CO3

ð7Þ

Correlation for converting OD680 values to microalgal dry weight was established from the calibration curves. The correlations utilized for the microalgae C. pyrenoidosa can be expressed by Eq. (4) as follows: Cellconcentration ðg=LÞ ¼ 4:57OD680−0:16

ð4Þ

3.2. Level of DO and pH during growth of C. pyrenoidosa Fig. 2 shows the level of dissolved oxygen (DO) during the growth of the microalgae C. pyrenoidosa after the addition of arsenic (either As(III) or As(V)) to the flasks. It shows that the DO cycling was continued without change after adding arsenic (either As(III) or As(V)) to the flasks. It specified that even with an addition of arsenic (either As(III) or As(V)), the algae were still photosynthesizing and generating oxygen at comparatively low levels than the control. No noteworthy variance was found for the treatment with arsenic (either As(III) or As(V)) ions. Related trend was described by Murray et al. [36] using Chlorella vulgaris in absence and presence of 1000 mg/L As(V). 3.3. Level of pH during growth of C. pyrenoidosa Fig. 3 shows the level of pH during the growth of the microalgae C. pyrenoidosa after the addition of arsenic (either As(III) or As(V)) to the flasks. It shows that the pH cycling was continued without change after adding arsenic (either As(III) or As(V)) to the flasks. The pH continued to increase with the increase in oxygen and fall as this was utilized up in respiration in absence and presence of arsenic (either As(III) or As(V)). Throughout the algal growth, fluxes of organic and inorganic species occur between various compartments within the cells and between the cells and surrounding medium. In a closed system, a steady reduction in nutrients is complemented by an alteration in alkalinity and pH. Alterations in alkalinity and pH are the net fluxes of H+ across the plasma membrane in addition to influx of various dissociation states of weak electrolytes for example NH+ 4 /NH 3 and CO2/HCO− 3 [37,38]. The pH of the medium is clearly affected by all alkaline and acidic components. When CO2 is dissolved in water, it combines with water to form carbonic acid, which disassociates to bicarbonate 2− (HCO − 3 ), then to carbonate ions (CO 3 ) for reaching equilibrium. The chemical equilibria between protons and inorganic carbon

Fig. 3. Data collected for three cultures: pH for first with no arsenic (control) and second with 50 mg/L As(III) and third with 50 mg/L As(V) (dark/light period: 12:12 h).

The coefficients K0, K1, K2, respectively for the above reactions are dependent on pressure, salinity and temperature. As indicated in the above reactions, concentration of H+ rises while CO2 dissolves in water. But sufficient H+ can reverse reaction (7) on the way to the left side, occasioning in decrease of carbonate ions (CO2− 3 ) [40]. While microalgae accomplish photosynthetic CO2 fixation, the inorganic carbon equilibria in the surrounding medium are shifted in the left direction and the pH rises. Contrariwise algal respiration (dark respirations) generates CO2 and shifts the equilibria towards the right and pH reduces. In a closed system, a dense culture can intensely impact pH in the medium. Algae have a restricted pH range within which the growth is probable. For avoiding extreme pH impacts, CO2 can be efficiently resupplied (or removed in dark) by aeration. An alternate approach of pH regulation of culture is the addition of acid (e.g. HCl) in the light and base (e.g. NaOH) in the dark throughout the growth. Furthermore various types of pH buffers can be added to the culture medium for keeping the pH comparatively constant [41]. In the current investigation, acid (1 N HCl) and base (1 N NaOH) were used for maintaining the pH of the growth medium. Similar trend was found by Murray et al. [36] using Chlorella vulgaris in absence and presence of 1000 mg/L As(V). From the above study it can be concluded that DO and pH cycles did not significantly differ due to the absence or presence of arsenic (either As(III) or As(V)) ions in the culture. So, the fixation and generation of CO2 by microalgae was not adversely affected by the presence of As(III) or As(V).

3.4. Effect of initial arsenic concentration on growth properties of C. pyrenoidosa Algal growth curves in the absence and presence of increasing concentrations of arsenic (either As(III) or As(V)) (50, 100, 500, 1000, 1500 and 2000 mg/L) in the BG11 growth media at an initial optimized pH value of 9.0 are shown in Fig. 4. Volume of media, inoculum size was kept constant at 100 mL and 10% (v/v), respectively. Growth of the microalgae C. pyrenoidosa was not inhibited by concentrations of arsenic ions (either As(III) or As(V)) up to 1000 mg/L and the lag phase was approximately 4 days and the stationary phase starts after 14 days. While concentrations of As(III) and As(V) were at N1000 mg/L, microalgal growth was noticeably inhibited with the lag phase increased to 4 days and the stationary phase starts after 14 days (Fig. 4). Thus to get an overall idea, experiments have been continued up to 16 days. Remarkably throughout the time span of 1–3 d, when exposed to 50–2000 mg/L of arsenic ions (either As(III) or As(V)), the growth rate was 1.47–12% lower than that in arsenic free medium, demonstrating that microalgal growth was encouraged by low concentrations of arsenic ions (either As(III) or As(V)) because of hormesis. For evaluating the poisonous effect of different inorganic arsenic species (As(III) and As(V)) on C. pyrenoidosa, the influence of rising concentrations of arsenic (either As(III) or As(V)) on the microalgal growth rate inhibition after 16th day was investigated.

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261

Fig. 4. Growth of C. pyrenoidosa in absence and presence of increasing concentrations of biomass concentration of As(III) and As(V) (initial pH: 9.0; inoculum size (% v/v): 10; initial arsenic concentration: 50 mg/L; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

The maximum biomass concentration of microalgae C. pyrenoidosa in absence and presence of arsenic (either As(III) and As(V)) are presented in Fig. 4. During the growth of microalgae C. pyrenoidosa in pure media, maximum biomass concentration increased from 0.146 g/ L to 2.527 g/L. The maximum biomass concentration and specific growth rate of microalgae C. pyrenoidosa in presence of arsenic (either As(III) or As(V)) showed a declining trend, signifying that toxicity of media augmented with increasing arsenic species concentration. With the increase in concentration of As(III) in the media from 50 to 2000 mg/L, maximum biomass concentration reduced from 2.362 g/L to 1.631 g/L and the specific growth rate also reduced from 0.142 to 0.134 d−1 because of toxicity of media by As(III) ions. Increasing the concentration of As(V) from 50 to 2000 mg/L, also led to decreased maximum biomass concentration from 2.321 g/L to 1.855 g/L and specific growth rate from 0.14 to 0.133 d−1 also because of toxicity of media by As(V) ions. The maximum growth rate of algae in arsenic enriched water is about 2/3 of the growth in arsenic free conditions. Various literature described that freshwater algae are more sensitive to As(V), while marine microalgae are more sensitive to As(III) [42,43]. The toxicity of As(III) and As(V) was found to be similar for the microalgae C. pyrenoidosa because the maximum biomass concentration of C. pyrenoidosa in the growth media was almost analogous in the presence of both As(III) and As(V) (pH 9.0). As(V) and As(III) showed similar toxicity to freshwater algae Stichococcus bacillaris at pH 8.2 with levels of phosphate between 0.03 and 0.3 mg P/L [44].

Equal toxicity of As(III) and As(V) was assessed from the 72 h growth inhibition tests for another freshwater microalgal species Chlorella sp. (pH 7.6), while As(V) was more toxic than As(III) for Monoraphidium arcuatum (pH 7.6) [43]. 3.5. Effect of initial As(III) and As(V) concentration on phycoremediation of C. pyrenoidosa The effect of initial metal ion concentration on phycoremediation using C. pyrenoidosa was carried out by varying arsenic (either As(III) or As(V)) concentrations (50, 100, 200, 500, 800, 1000, 1200, 1500, 1800 and 2000 mg/L) in the growth media, at an initial optimized pH value of 9.0. For all the two arsenic species (As(III) and As(V)), phycoremediation capacity of C. pyrenoidosa reduced as the arsenic (As(III)/As(V)) concentration was increased from 50 to 2000 mg/L are exposed in Fig. 5. The phycoremediation % and phycoremediation level of the As(III) and As(V) by the microalgal species at different initial arsenic (As(III)/As(V)) concentrations were determined as given in Table 3. The phycoremediation % decreased with the increase in arsenic (As(III)/As(V)) concentration from 50 to 2000 mg/L. It was supported by the decrease in microalgal growth. The reduction in phycoremediation % of As(III) and As(V) was caused by the toxicity of As(III) and As(V) at higher concentrations which decreased the biomass concentration. The maximum biomass concentration was found to

Table 3 Comparison of As(III) and As(V) phycoremediation, at different levels of As(III) and As(V) concentration. Arsenic species

C0 (mg/L)

Removal (%)

Arsenic conc. (mg/L)

As(III)

50 100 200 500 800 1000 1200 1500 1800 2000 50 100 200 500 800 1000 1200 1500 1800 2000

62.609 66.087 71.304 76.522 80.000 76.522 71.304 67.826 62.609 57.391 85.077 83.038 81.000 77.362 76.519 72.700 69.385 66.044 62.442 59.883

31.304 66.087 142.609 382.609 640.000 765.217 855.652 1017.391 1126.957 1147.826 42.538 83.038 162.000 386.808 612.154 727.000 832.615 990.654 1123.962 1197.654

As(V)

Fig. 5. Effect of initial concentration on phycoremediation of As(III) and As(V) of C. pyrenoidosa (initial pH: 9.0; inoculum size (% v/v): 10; contact time: 168 h; temperature: 28 °C) (Error bars represent means ± standard errors from the mean of duplicate experiments).

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decrease from 2.362 g/L to 1.631 g/L with the increase in As(III) concentration from 50 mg/L to 2000 mg/L. Varying the concentration of As(III) from 50 to 2000 mg/L resulted in an increased phycoremediation levels from 31.304 mg/L to 1147.826 mg/L. Similarly, the maximum biomass concentration was found to decrease from 2.321 g/L to 1.855 g/L with the increase in As(V) concentration from 50 mg/L to 2000 mg/L. Varying the concentration of As(V) from 50 to 2000 mg/L also resulted in an increased phycoremediation levels from 42.538 mg/L to 1197.654 mg/L. The removal of As(V) is higher than As(III) for all concentrations due to more production of phytochelatins (PC2–3) in cells after exposure to As(V) than to As(III) [44]. So the removal of As(V) was higher than As(III) for all concentrations [44].

3.6. Effect of initial nitrate concentration on growth properties of C. pyrenoidosa in arsenic free media Macronutrient such as nitrogen plays a vital role in regulating the metabolism and finally the growth of microalgae [45]. It was described that nitrate ions usually have stimulating effect [34]. In the present investigation, NaNO3, 4H2O was used in higher amount compared to other media components and it was considered as the growth limiting substrate [34]. Thus, the influence of initial nitrate ion concentration on the growth rate of microalgae C. pyrenoidosa in both arsenic free media was explored. NaNO3 ∙ 4H2O the only source of nitrate ion in the media, was varied throughout the experiments in the range of 0.05–2.0 g/L keeping other compositions constant as prescribed in BG11 medium, at pH 9.0 and at a temperature of 28 °C for 16 days and the flask was analyzed for dry biomass. Fig. 6 shows that the relationship between specific growth rate and nitrate ion concentration for microalgae C. pyrenoidosa, respectively, often adopts the saturation kinetics form. It was seen that both biomass concentration and specific growth rate increased with rising initial nitrate ion concentration from 0.05 to 2 g/L. The effects of nitrogen reduction include reduction in oxygen evolution, carbon dioxide fixation, chlorophyll content, and tissue production [46,47]. Thus, the growth rate of C. pyrenoidosa reduced significantly when the concentration of nitrogen source reduced in the photoautotrophic media and the cells were in nitrogen limiting [48,49]. The specific growth rate improved from 0.144 to 0.146 d−1, with an increase in initial nitrate concentration from 0.05 to 2.0 g/L and corresponding biomass concentration of microalgae C. pyrenoidosa also increased from 2.344 g/L to 2.554 g/L.

Fig. 7. Concentration − time histories of dry biomass obtained experimentally during the growth study of the microalgae with concentration of NaNO3, 4H2O as a parameter (initial pH: 9.0; inoculum size (% v/v): 10; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

3.7. Modelling of growth kinetics of algal biomass in arsenic free media containing various concentration of nitrate ions The growth of the microalgae has been investigated in batch culture by varying the quantity of NaNO3∙ 4H2O in the range of 0.05–2.0 g/L. The experimental values of dry biomass obtained all through the experimentation period for different values of NaNO3∙ 4H2O in pure media have been plotted in Fig. 7. The classical Monod equation was employed for representing the growth of the microalgae C. pyrenoidosa in pure media during exponential phase only. Maximum specific growth rate and saturation constant were evaluated by nonlinear regression analysis utilizing professional graphics software package OriginPro (8.5.1 version) for fitting experimental data obtained during the batch study. The value of μmax was 0.146 d−1 and that of Ks was 8.29E − 04 g/L (correlation coefficient is equal to 0.996).

3.8. Effect of phosphate concentration on growth properties of C. pyrenoidosa in arsenic free media Macronutrient such as phosphorous also plays a major role to control the metabolism and finally the growth of microalgae [34,50]. The effect of phosphate ion concentration on the growth rate of microalgae C. pyrenoidosa in arsenic free media was explored in the phosphate concentration range of 0.005–0.05 g/L keeping other compositions constant as prescribed in the BG11 medium, at pH 9.0 and at a temperature of 28 °C. As presented in Table 4 and Fig. 8, the relationship of biomass concentration to phosphate ion concentration for microalgae C. pyrenoidosa, often adopted the saturation kinetics form. It was observed that biomass concentration increased with increasing initial phosphate ion concentration from 0.005 to 0.5 g/L. Phosphorus typically Table 4 Comparison of specific growth rate (μ) of microalgae C. pyrenoidosa at different levels of phosphate concentration.

Fig. 6. Effect of initial nitrate ion concentration on biomass concentration of C. pyrenoidosa in arsenic free media (initial pH: 9.0; inoculum size (% v/v): 10; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

S0 (g/L)

Xm (g/L)

μ (d−1)

0.005 0.01 0.02 0.03 0.04 0.05

2.321 2.403 2.449 2.485 2.527 2.568

0.143 0.144 0.145 0.145 0.146 0.146

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Fig. 8. Effect of phosphate concentration on growth of microalgae C. pyrenoidosa in arsenic free media (initial pH: 9.0; inoculum size (% v/v): 10; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

Fig. 9. Effect of bicarbonate concentration on biomass concentration of C. pyrenoidosa (initial pH: 9.0; inoculum size (% v/v): 10; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

constitutes 1% of dry weight of algae [51], but it may be essential in significant excess because not all added phosphate is bioavailable because of the formation of complexes with metal ions [52]. Immediate effects of phosphorus limitation include a decrease in the synthesis and regeneration of substrates in the Calvin-Benson cycle and a significant decrease in the rate of light utilization necessary for carbon fixation [53]. Phosphorus starvation reduces chlorophyll a and protein content [47]. Phosphate deficiency was revealed to result in accumulation of astaxanthin and an overall reduction in cell growth [54]. It was observed that the specific growth rate increased from 0.142 to 0.146 d−1, with an increase in phosphate concentration from 0.005 to 0.05 g/L and biomass concentration of microalgae C. pyrenoidosa increased from 2.321 g/L to 2.568 g/L.

growth rate (0.148 d−1) and maximum biomass concentration (2.6 g/ L) and was highest when 1 g/L bicarbonate was used. The specific growth rate and maximum biomass concentration of microalgae C. pyrenoidosa also reduced from 0.142 d−1 and 2.357 g/L to 0.141 d−1 and 2.28 g/L, respectively when the concentration of bicarbonate ion was increased from 1.5 to 2 g/L. The utilization of bicarbonate by algae tends to increase culture pH as follows [56,57]:

3.9. Effect of NaHCO3 concentration on growth properties of C. pyrenoidosa Carbon, hydrogen, and oxygen are three essential non-mineral nutrients. Abundance of hydrogen and oxygen in the media for microalgae cultures indicates that their availability is not a challenge to cellular growth or metabolism. Carbon is one of the other major nutrients that must be provided. It is essential for photosynthesis and therefore microalgal growth and reproduction. Carbon fixed by the microalgae can end up in three endpoints; it will either be used: (1) for respiration; (2) as an energy source; or (3) as a raw material in the formation of additional cells [55]. Reduced carbon fixation rate indicates a decrease in microalgal growth rate. Microalgae need an inorganic carbon source to carry out photosynthesis. Carbon can be used in the form of CO2, carbonate, or bicarbonate for autotrophic growth and in form of glucose or acetate for heterotrophic growth. So the influence of NaHCO3 concentration on the growth rate of C. pyrenoidosa in arsenic free media was explored in the NaHCO3 concentration range of 0.0–2.0 g/L keeping other compositions constant as prescribed in BG11 medium, at pH 9.0 and at a temperature of 28 °C. The growth behaviour of the microalgae C. pyrenoidosa under different bicarbonate levels are shown in Fig. 10. In this figure, the relationship between specific growth rate and bicarbonate ion concentration for microalgae C. pyrenoidosa, often adopts the saturation kinetics form. It was seen that both biomass concentration and specific growth rate reduced with rising bicarbonate ion concentration from 0.05 to 2.0 g/L. In pure BG11 growth media, the maximum biomass concentration and specific growth rate decreased from 0.146 d−1 and 2.527 g/L to 0.145 d−1 and 2.449 g/L, respectively when bicarbonate ion was increased to 0 to 1 g/L. It was found that both specific

− HCO− 3 →CO2 þ OH

ð8Þ

With the purpose of growing, microalgae need to have access to a carbon source [58]. Earlier, Lee et al. [59] utilized air enriched with CO2 as carbon source. Borowitzka [60] stated that green algae could not tolerate bicarbonate ions (i.e. the carbonated conditions) at concentrations above 0.2 M. In the present investigation, CO2 was used as main carbon source. Fig. 9 presents the effect of added NaHCO3 (0.0, 0.1 0.5, 1.0, 1.5 and 2 g/L) on yield of biomass production and the specific growth rate, respectively. The bicarbonate concentration at which maximum growth rate was obtained was at 1 g/L. Jeong et al. [61] observed that 15.3 mg/L bicarbonate salt is equivalent to 243 mg/L CO2 gas i.e. the algal strain C. pyrenoidosa exhibited its best growth at 15.882 g/L CO2 gas which is equivalent to 1 g/L bicarbonate, at which maximum biomass concentration of 3.395 g/L and specific growth rate increase of 0.1471 was verified. Lehman [62] reconfirmed the well-established view that free CO2 is the sole direct substrate for the Calvin cycle and recommended that bicarbonate serves as a vehicle for the transport of inorganic carbon into the cell. Many microalgae and cyanobacteria species can actively take up HCO− 3 from the external environment through transport across the plasma membrane into the cytosol and derive CO2 from HCO− 3 through the action of carbonic anhydrase preserving a steady state flux to ribulose-1.5-bisphosphate carboxylase oxygenase for photosynthesis. Otherwise extracellular carbonic anhydrase can catalyse the interconversion of HCO− 3 and CO2. Both mechanisms of use of HCO− 3 were described in phytoplankton [63,64]. White et al. [64] described that final cell abundances were considerably higher in cultures supplemented with 1 g/L bicarbonate (1.22 ± 0.04 × 106 cells m/L) compared to those with 0 and 2 g/L supplementation (1.07 ± 0.02 × 106 and 1.04 ± 0.03 × 106 cells/mL, respectively), which were not considerably different. Goswami et al. [65] found that microalgal strain Selenastrum sp. exhibited its best growth at 952.9 mg/L CO2 gas which is equivalent to 60 mg/L bicarbonate, at which maximum rise

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Fig. 10. Effect of salinity on biomass concentration of C. pyrenoidosa (initial pH: 9.0; inoculum size (% v/v): 10; temperature: 28 °C; incubation time: 16 d; dark/light period: 12:12 h) (Error bars represent means ± standard errors from the mean of duplicate experiments).

in cells/mL/day of 2.078 × 104 and a biomass increase of 1.102 mg/mL/d was found. 3.10. Effect of salinity on growth properties of C. pyrenoidosa C. pyrenoidosa was fit for growing in all tested concentrations of NaCl (0.0 g/L to 5.0 g/L). In the current investigation, biomass concentration was higher as compared to the control under salinity (Fig. 10). The biomass yields increased with increasing concentration of NaCl and maximum biomass was achieved in 1.0 g/L and 2.0 g/L salinity. After that increasing NaCl concentration from 3.0 to 5.0 g/L resulted in corresponding reduction in the growth of microalgae C. pyrenoidosa. This could be also perhaps because of the adaptation of the microalgae to the lower salinity (1.0 g/L and 2.0 g/L) [66]. Fábregas et al. [67] also reported reduced growth at higher salinities, which might be because of an inability of the microalgae to adapt to high salinity. The maximum biomass concentration was suggestively higher in cultures supplemented with 1.0 g/L NaCl (3.827 g/L) compared to those with control (3.368 g/L) and 3.0–5.0 g/L supplementation varying from 3.431 to 3.521 g/L, which were not considerably different. Barghbani et al. [68] stated that adding 20.0 g/L NaCl to fresh water decreased the biomass of Chlorella vulgaris from 1.49 g/L (on a dry basis) to 0.55 g/L. Borowitzka [60] described that the growth of C. vulgaris enhanced in fresh water with no minimal salt concentration. 3.11. Anova Results For the above consequences the Anova table was made and the following F value is acquired for As(III) and As(V) (Table 5). ANOVA is a

Table 5 ANOVA table for growth of microalgae in presence of As(III) and As(V). Species As(III)

As(V)

Growth Error Total Growth Error Total

df

Sum of squares

Mean square

F value

Prob N F

1 10 11 1 10 11

2.44E − 05 9.66E − 06 3.41E − 05 2.56E − 05 9.74E − 06 3.54E − 05

2.44E − 05 9.66E − 07

25.28206

5.16E − 04

2.56E − 05 9.74E − 07

26.32927

4.43E − 04

Fig. 11. FT-IR spectra of C. pyrenoidosa control, As(III) ions exposed and As(V) ions exposed.

statistical technique that subdivides the total variation in a set of data into component parts associated with specific sources of ariation for the determination of testing hypothesis on the parameters of the model [69]. The statistical meaning of the ratio of the mean square variation because of the regression and mean square residual error is verified utilizing analysis of variance (ANOVA). According to the ANOVA (Table 5), the F statistic values for all regressions are higher. The higher value of F specifies that the most of the variation in the response can be clarified by the regression equation. The F table value for (1,10) degree of freedom is 4.9646 and F calculated value for As(III) is 25.28206 and for As(V) is 26.32927. In both the cases the F value estimated is more than the F table value. This indicates that the Null hypothesis which was previously assumed can be rejected and the alternative hypothesis can be accepted. So the consequence exhibits that there is a significant influence of arsenic addition on the specific growth rate of microalgae and this decreased specific growth rate is not by chance or by error. There is a 99.5% probability that this reduction is because of the addition of arsenic to phosphate. Hence ANOVA calculation indicates the addition of arsenic to the BG11 media containing phosphate for lower microalgal growth. The ANOVA table also displays a term for the residual error, which determines the amount of ariation in the response data left unclarified by the model. The form of the model selected for explaining the relationship between the factors and the response is exact and has very good agreement with the experimental data [70]. 3.12. Characterization of microalgae 3.12.1. FT-IR analysis The FT-IR spectra of the C. pyrenoidosa biomass with and without As(III) and As(V) ions loaded which were achieved to determine the possible functional groups, may have contributed to the phycoremediation of As(III) and As(V) ions, are presented in Fig. 11 and Table 6. The FT-IR spectra of the C. pyrenoidosa biomass without As(III) and As(V) ions loaded exhibited a number of absorption peaks, indicating the complex nature of the microalgal biomass. The spectra of unloaded and loaded with arsenic (As(III)/As(V)) ions are compared and observed the following shifting. The spectra of biomass exhibited an absorption band at 3394.155 cm−1 because of bonded \\OH and \\NH stretching vibration which was shifted to 3349.8 cm−1 (As(III)) and 3386.441 cm−1 (As(V)) which may be possibly because of the complexation of\\OH and\\NH groups with As(III) or As(V) ions [71,72]. Aliphatic C\\H stretching may be responsible for phycoremediation of As(III) and As(V) on the microalgal biomass as wavenumber shifted from 2923.602 cm−1 to 2925.53 cm−1

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Table 6 Wavenumber (cm−1) for the dominant peak from FT-IR for phycoremediation of As(III) and As(V). Functional groups

Native biomass

As(III) loaded biomass

As(V) loaded biomass

Surface O\ \H and N\ \H stretching Aliphatic C\ \H stretching Aldehyde C\ \H stretching \ \CH stretching vibration of alkyl chains Aliphatic acid C_O stretching Amide group (N\ \H stretching and C_O stretching vibration) Secondary amine group N\ \H group and carboxylate anion Carboxylate anion Symmetric bending of CH3 group \ \SO3 stretching C\ \N stretching vibrations of amino groups O\ \C\ \O scissoring vibration of polysaccharide As(III)\ \O As(V)\ \O

3394.155 2923.602 2858.033 2358.552 1735.647 1648.865 1542.798 1454.087 1394.303 1306.667 1251.595 1043.317 885.180 × ×

3349.800 2925.530 2867.675 2354.695 1728.372 1645.008 1540.869 1450.230 1398.160 1366.822 1257.674 1054.884 814.264 721.24 ×

3386.441 2929.387 2869.604 2327.696 1744.496 1657.054 1546.655 1417.445 1366.822 1317.829 1247.738 1039.460 896.744 × 819.845

and 2929.387 cm−1, respectively possibly because of the complexation with As(III) and As(V) ions [71]. Aldehyde C\\H stretching may be responsible for As(III) and As(V) phycoremediation on C. pyrenoidosa biomass as wavenumber shifted from 2858.033 cm−1 to 2867.675 cm−1 and also to 2869.604 cm−1, respectively [72]. The next absorption peaks at 2358.552 cm−1 was shifted at a lower frequency to 2354.695 cm−1 (As(III)) and 2327.696 cm−1 (As(V)), probably because of the complexation of\\CH stretching vibration of alkyl chains [73]. Table 6 also shows the responsibility of aliphatic acid C_O stretching for As(III) and As(V) phycoremediation by shifting the wavenumber from 1735.647 cm−1 to 1728.372 cm−1 and 1744.496 cm−1, respectively [72]. The next phycoremediation peaks at 1648.865 cm−1 shifted to 1645.008 cm−1 for As(III) and 1657.054 cm−1 for As(V), perhaps because of the complexation of amide group (N\\H stretching and C_O stretching vibration) with As(III) and As(V) ions [71,73]. Wavenumber shifted from 1542.798 cm−1 to 1540.869 cm−1 (As(III)) and 1546.655 cm−1 (As(V)), probably because of the complexation of secondary amine group with As(III) and As(V) ions [71]. Another shift was found from 1454.087 cm−1 to 1450.23 cm−1 (As(III)) and 1417.445 cm−1 (As(V)), possibly due to the complexation of nitrogen with As(III) and As(V) ions of the N\\H group [74] and also due to the complexation with carboxyl groups [71]. Wavenumber shifted from 1394.303 cm−1 to 1398.16 cm−1 (As(III)) and 1366.822 cm−1 (As(V)) assigned the reactivity of carboxylate anion C_O stretching for the phycoremediation process [75]. Wavenumber shifted from 1306.667 cm−1 to 1366.822 cm−1 (As(III)) and 1317.829 cm−1 (As(V)) assigned the symmetric bending of CH3 group [71]. Wavenumber 1251.595 cm−1 shifted to 1257.674 cm−1 (As(III)) and 1247.738 cm−1 (As(V)) assigned for \\SO3 stretching for the phycoremediation process [71]. The peaks at 1043.317 cm−1 may be attributed to the C\\N stretching vibrations of amino groups which was shifted and appeared at 1054.884 cm−1 and 1039.460 cm−1, respectively due to the interaction of nitrogen from the amino group with As(III) and As(V) ions [73]. The other weak phycoremediation peak shifted from 885.18 cm−1 to 814.264 cm−1 (As(III)) and 896.744 (As(V)), corresponding to the O\\C\\O scissoring vibration of polysaccharide [76]. Presence of As(III) and As(V) on the microalgal biomass can be assured from the bands appeared at 721.24 cm−1 and 819.845 cm−1, respectively [77,78]. It has to be cited here, that a clear band was very hard to be got in the case of both As(III) and As(V). This may be because of different mechanisms involved in As(III) and As(V) phycoremediation. It should be distinguished that the As\\O band after phycoremediation of arsenic was not clearly observed because of the broad overlapping peaks in this region [79]. 3.12.2. SEM analysis Fig. 12(a)–(c) shows the scanning electron microscopy (SEM) images of native microalgal biomass and As(III) and As(V) loaded

microalgal biomass, respectively. In both images, two types of structure have been seen. The spherical shape represents C. pyrenoidosa. Comparing Fig. 12(a), (b) and (c), it is found that in native microalgal biomass the surface is smooth while after arsenic (either As(III) or As(V)) treatment the surface becomes rough in both the microalgal structures. Such roughness of the surface may be because of the phycoremediation of arsenic (either As(III) or As(V)) over the surface that made the surface coarser than its original form [80]. It was also seen that there was very little or no change in the fraction of spherical shape of C. pyrenoidosa before and after arsenic (either As(III) or As(V)) removal. It recommends that the presence of arsenic (either As(III) or As(V)) does not make the medium selective toward any of the strains and the biological nature of the consortium remains fairly constant. Densities of the nodules also seemed to be unaffected by the presence of arsenic (either As(III) or As(V)) signifying that the growth kinetics of the consortium remains unaffected in the presence of arsenic (either As(III) or As(V)) in simulated wastewater. But, the nodules were not clearly visible in the SEM. The cells seemed to be glued to each other. It was because of more EPS production, which is one of the well-known responses against stress. The corresponding EDX spectra of the unloaded and loaded microalgae was collected and given in Fig. 12(a)–(c). The presence of arsenic on the loaded microalgae surface was exposed evidently. This outcome again established the occurrence of phycoremediation of arsenic by the microalgae. 4. Conclusion Fuel shortage in the near future poses a serious challenge; hence, a renewable energy resource having less environmental effect is necessary. Application of microalgae for such a purpose is an effective step. In the present study, an efficient microalgae Chlorella pyrenoidosa was found to be suitable for the removal of both As(III) and As(V) from wastewater. The health status of the culture was checked by continuous dissolved oxygen (DO) and pH readings. A kinetic study has been performed in pure media as well as in simulated wastewater. The microalgae C. pyrenoidosa removes 81.74% and 85.08% of As(III) and As(V), respectively from synthetic wastewater after 168 h of treatment with initial arsenic concentration of 50 mg/L at pH 9.0 and inoculum size of 10% (v/v). As(V) was more poisonous than As(III), particularly at the near neutral pH 7.0, but at higher pH ( pH 9.0) As(III) was much more poisonous than As(V). higher than with As(III), signifying more As(V) than As(III) availability and uptake. The presence of arsenic (either As(III) or As(V)) ions in the growth media inhibited the growth of the microalgae irreversibly and this influence of inhibition reduced with the increase in nitrate ions concentrations. The Monod model has been employed for depicting the growth kinetics of the microalgae C. pyrenoidosa in pure media at various concentrations of nitrate ions.

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Fig. 12. Scanning electron micrographs (SEM) (1500×) and EDX of (a) native C. pyrenoidosa, (b) As(III) loaded biomass and (c) As(V) loaded biomass.

Maximum specific growth rate and saturation constant of microalgae were 0.146 d−1 and 8.29E − 04 g g/L, respectively in pure media containing different concentration of nitrate ions. An increase in concentration of phosphate in growth medium increased the growth of microalgae. Increasing the concentrations of NaHCO3 and NaCl resulted in corresponding reductions in the growth of C. pyrenoidosa. Media with 1.0 g/L of NaCl indicated the highest algal growth compared to the control. This could be probably because of the adaptation of the microalgae to the lower salinity (1.0 to 2.0 g/L). However while the salinity level was enhanced, the specific growth rate reduces quickly. From the above results it can be decided that the microalga C. pyrenoidosa can competently use both CO2 gas and

bicarbonate salt as carbon source in culture media. Yield of biomass production was highest with 1.0 g/L NaHCO3. Thus it can be stated that the microalgae C. pyrenoidosa can be employed as an effective biomaterial for remediation as well as renewable energy source for biofuel production. However this is a preliminary work involving a simulated solution of either As(III) and As(V) and a comprehensive study with real industrial wastewater encompassing a detailed parametric study in a continuous reactor system is needed. Nomenclature C0 initial concentration of arsenic in the solution (mg/L)

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Ce w1 w2 CA Ks K0 K1 K2 OD

equilibrium concentration of arsenic in the solution (mg/L) the dry biomass concentration when cultured in wastewater (g/L) the dry biomass concentration when cultured in pure media (g/L) the limiting substrate concentration at time t (g/L) the substrate saturation constant (g/L) reaction coefficient reaction coefficient reaction coefficient optical density

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