Green microalgae for combined sewage and tannery effluent treatment: Performance and lipid accumulation potential

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Journal of Environmental Management 241 (2019) 167–178

Contents lists available at ScienceDirect

Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman

Research article

Green microalgae for combined sewage and tannery eﬄuent treatment: Performance and lipid accumulation potential

T

D. Saranya, S. Shanthakumar∗ Department of Environmental and Water Resources Engineering, School of Civil Engineering, Vellore Institute of Technology (VIT), Vellore, 632014, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Tannery eﬄuent Chromium Microalgae Phycoremediation Lipids

Microalgae have considerable interest owing to its phycoremediation potential and raw material for sustainable biofuel production. In this study, the performance of green algae Chlorella vulgaris (NRMCF0128) and Pseudochlorella pringsheimii (VIT_SDSS) was evaluated for the remediation of combined sewage and tannery efﬂuent under diﬀerent dilutions. Signiﬁcant reduction in pollutant concentration was observed in the efﬂuent: > 65% for NH3-N, 100% for PO4-P, > 63% for COD & > 80% for total chromium, at higher dilutions (up to 30%) of tannery eﬄuent (T) for both species. EDAX analysis conﬁrms the intracellular accumulation of heavy metal chromium and other elements such as aluminum, zinc, and iron by both microalgae. In addition, the maximum yield of biomass achieved was 3.51 g/L (for 30% Tannery eﬄuent) and 2.84 g/L (for 20% Tannery eﬄuent) for Chlorella vulgaris & Pseudochlorella pringsheimii, respectively. Between the two species, Pseudochlorella pringsheimii has shown high lipid accumulation potential of 25.4% compared to Chlorella vulgaris (9.3%) at 20% Tannery eﬄuent. Hence, it is evident that the green microalgae Pseudochlorella pringsheimii is promising for the sustainable treatment of combined sewage and tannery eﬄuent along with biofuel production.

1. Introduction Leather, one of the most extensively listed artefacts and has placed an outstanding position in the world economy. The revenue from leather industries is estimated to reach about US$ 91.2 billion by 2018, with an annual growth rate of 3.4% (Lucintel, 2013). Despite its ﬁnancial status, the tanning sector is considered as highly polluting among diﬀerent industrial sectors. It requires 30–40 m3 of water and 300 kg of various chemicals per tonne of hide processing (Camargo et al., 2003; Suthanthararajan et al., 2004; Durai and Rajasimman, 2011). Of which 90% of used water has been let out as toxic eﬄuent and is characterized by its high Total dissolved solids (TDS), Chemical oxygen demand (COD), Biological oxygen demand ((BOD5)/COD) ≤0.3, volatile organic compounds, sulﬁdes, ammonia, chromium etc. Intensiﬁed consumer demand for leather industry increases the pressure, concerning chemicals, water usage, and landﬁll issues. It requires ongoing research at production as well as eﬄuent treatment levels in order to ensure the stability of the leather industry. The selection of suitable treatment process depends, mainly on the characteristics of eﬄuent entering into the common eﬄuent treatment plants (CETP's) of tanning industries. Complex industrial outlets are preferably treated anaerobically due to its potential for handling high COD, power generation and reduced toxic sludge production. On the ∗

other hand, the anaerobic process suﬀers a lot due to low microbial growth rate, a low settling rate, process uncertainties, constraints of ammonium ion (NH4+) and hydrogen sulﬁde (HS−) release during post-treatment (Chan et al., 2009). Moreover, sulﬁde demands high oxygen for its removal (2-mol O2/mole sulﬁde) and this stipulation for O2 increases the total cost of eﬄuent treatment (McCarty et al., 2011; Chae and Kang, 2013). Hence, the tannery eﬄuent can be considered for aerobic/anaerobic treatment after adopting proper pre-treatment, or combined with other oxidation processes such as Ozonation, Photocatalysis, Fenton process, wet-air Oxidation etc. (Di-Iaconi et al., 2003; Szpyrkowicz et al., 2005; Oller et al., 2011; Mandal et al., 2010; Srinivasan et al., 2012; Lofrano et al., 2013). Nature uses its own sustainable routes to attain the goal of recycling without relying on external energy. One such way of remediating and capturing the organic and inorganic pollutant from the environment, without the requirement of aeration is phycoremediation (Cai et al., 2013; Rai et al., 2005; Hanumantha Rao et al., 2011). Many algal species, like Chlamydomonas, Chlorella and Scenedesmus, can assimilate organic matters to some extent under mixotrophic condition and organic acids under heterotrophic conditions (Kong et al., 2013; Evans et al., 2017). Algal-bacterial symbiosis has shown a signiﬁcant contribution towards wastewater treatment (Sun et al., 2018; Mujtaba and Lee, 2017; Tang et al., 2016; Su et al., 2011; Wang et al., 2010).

Corresponding author. E-mail address: [email protected] (S. Shanthakumar).

https://doi.org/10.1016/j.jenvman.2019.04.031 Received 27 October 2018; Received in revised form 27 March 2019; Accepted 9 April 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.

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and A3 trace solution (g/L) components as 2.86 g H3BO3, 1.81 g MnCl2, 0.222 g ZnSO4.7H2O, 0.390 g Na2MoO4.2H2O, 0.079 g CuSO4.5H2O, 0.0494 g Co(NO3)2.6H2O. From the above stock 1 L media was prepared by adding 100 mL of A1 solution, 10 mL each from A2 constituents and 1 mL from A3 trace solution.

Metabolism of algae is precisely the reverse of heterotrophic bacteria: algae utilize carbon dioxide (CO2) (released by bacteria during organic mineralization), nutrients (Nitrogen, phosphates, and metal substances) from wastewater, and convert that into organic algal biomass and oxygen (Ramanan et al., 2016). In addition, the microalgal potential for photosynthetic oxygenation and lipid accumulation under stress condition has the ability to encounter oxygen and energy demand for conventional treatment, thereby reducing the overall cost of the treatment system (Xia et al., 2014). It has been assessed for the diﬀerent types of industrial wastewater and has shown favorable results (Abinandan et al., 2015; Abinandan and Shanthakumar 2015; Umamaheswari and Shanthakumar 2016; Nagabalaji et al., 2017). This approach shifts the treatment process from being an end-of-pipeline route to a means for energy and nutrient recovery. Untreated tannery eﬄuent does not support the growth of microalgae due to its turbidity, dissolved salts, toxic organic and inorganic load. Whereas pretreated or diluted eﬄuent is found to support the growth of microalgae (Dunn, 1977; Chandra et al., 2004). Rose et al. (1996) reported the growth of Cyanophyte Spirulina and chlorophyte Dunaliella sp. in the subsequent ponds of waste stabilization pond (WSP) due to mineralization, the complex interaction between algae and bacteria (aerobic & anaerobic), along with physicochemical and climatic inﬂuence. Based on the literature review, it has been perceived that phycoremediation of tannery eﬄuent was carried out in secondary treated eﬄuent (WSP) and diluted (distilled water/tap water) tannery eﬄuent for the removal of carbon load, nutrients (N&P) and heavy metals (Onyancha et al., 2008; Elumalai et al., 2014; Das et al., 2018). However, no research has been conducted with the view of algal lipid production under tannery eﬄuent stress. As the management of resources is depending mainly on economic and environmental aﬀordability of particular treatment technology, the use of distilled water or fresh water is not a sustainable way for reducing the toxicity of tannery eﬄuent. Currently, the sewage treatment plants are converted into wastewater treatment plants with resource recovery, as a part of sustainable and integrated wastewater management (McCarty et al., 2011; Mo and Zhang, 2013; Van Loosdrecht and Brdjanovic, 2014). With this view, an eﬀort has been made to assess the potential of green microalgae Chlorella vulgaris (NRMCF0128) and Pseudochlorella pringsheimii (VIT_SDSS) to treat combined sewage and tannery eﬄuent in diﬀerent dilutions and to evaluate its removal eﬃciency for Ammoniacal nitrogen, Total phosphorous, heavy metal (chromium) and COD under batch culture conditions. In addition, the biomass and lipid accumulation potential of microalgae in combined eﬄuent have been studied.

2.2. Microalgae identiﬁcation Morphology of the isolated microalgae species was perceived under a microscope and the genetic identiﬁcation was done by DNA extraction and PCR, according to the protocol described by Edwards et al., (1989). Sequencing reactions were performed using ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems). Later, the obtained sequence was compared with the National Centre for Biotechnology Information (NCBI) blast similarity search tool. The phylogeny analysis of query sequence with the closely related sequence of blast results was performed followed by multiple sequence alignment (Program MUSCLE 3.7). The program PhyML 3.0 aLRT was used for phylogeny analysis and HKY85 as Substitution model. 2.3. Sample collection and analysis Sewage sample was collected from sewage treatment plant located at VIT, Vellore, India and untreated composite tannery eﬄuent was collected from a CETP located in Vellore District of Tamil Nadu, India. The samples collected were preserved in a refrigerator at 4 °C and acidiﬁed for heavy metal analysis. The chemicals used in this work were obtained from Thomas Baker Ltd (Mumbai, India) and the reagents were prepared using double distilled water. For experimentation and analysis of various physicochemical parameters, pre-ﬁltered samples were used, to avoid interferences of solid particles in the subsequent phycoremediation process. Both the samples were analyzed for pH, TDS, BOD, COD, alkalinity, Sulphates, Sulphides, Chlorides, Sodium, Total Kjeldahl Nitrogen (TKN), Ammoniacal-Nitrogen (NH3-N), and phosphates as per the standard procedure prescribed in the American Public Health Association (APHA, 2012). Heavy metal-Total chromium was analyzed using Atomic absorption spectroscopy (AAS) (Agilent AA240). 2.4. Experimental methodology The exponential phase (13th day) inoculum of microalgae Chlorella vulgaris (3.8 × 105 cells/mL) and Pseudochlorella pringsheimii 5 (7.7 × 10 cells/mL) from BG11 medium were used in the study. All the experiments were carried out with 10% inoculum size in a 500 mL conical ﬂask with the working volume of 300 mL wastewater samples. One set for control and 2 experimental sets for both species were conducted in untreated tannery eﬄuent (100% T), sewage (100% S), and diluted tannery eﬄuent with sewage – 10% T (T:S-10:90), 20% T (T:S20:80), 30% T(T:S-30:70), 40% T(T:S-40:60), 50% T(T:S-50:50) under continuous illumination of light with 35 μmol m−2 s−1 intensity for the period of two weeks. In order to evaluate the removal of nutrients such as phosphates and ammoniacal nitrogen, the samples were monitored in an alternative day, COD was monitored once in a week and the cell growth was observed by cell count & chlorophyll a content in alternative days. Also, the pH of the experimental sets was monitored to observe the changes in wastewater and the ﬂasks were shaken every day thrice to prevent algal adhesion.

2. Materials and methods 2.1. Microalgae culture For this study, two microalgae species namely Chlorella vulgaris (NRMCF0128) and Pseudochlorella pringsheimii (VIT_SDSS) were utilized. Chlorella vulgaris (NRMCF0128) was obtained from National Repository for Microalgae and Cyanobacteria - Freshwater (NRMCF), Bharathidasan University, Tiruchirappalli, India as it is tolerable to heavy metal and saline stress (Mehta and Gaur, 1999; Shen et al., 2015) and was sub-cultured in BG11 medium. Pseudochlorella pringsheimii (VIT_SDSS) was an indigenous species isolated from sewage treatment plant located in the campus of VIT, Vellore, India. Isolation was carried out by following the standard procedure of serial dilution and spread plating in BG11 agar plates. And the axenic culture was maintained in BG11 broth under 35 μmol m−2 s−1 with 24 h illumination at 27 ± 1 °C. The media stock solution was prepared with the following composition of 100 mL each: A1 Solution of 1.5 g NaNO3, A2 constituents - 0.75 g MgSO4, 0.4 g K2HPO4, 0.36 g CaCl2, 0.2 g Na2CO3, 0.06 g citric acid, 0.06 g Ammonium ferric citrate, 0.01 g Na2. EDTA

2.4.1. Analytical methods used in the study The samples were centrifuged at 5000 rpm for 5 min for COD, Ammoniacal nitrogen and Total phosphates analysis as per methods prescribed in APHA (APHA, 2012). Cell growth was measured directly by cell count with a hemocytometer (ROHEM Silverlite) and chlorophyll a content. For chlorophyll analysis, the algal biomass obtained 168

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after centrifugation at 5000 rpm for 5 min was re-suspended in acetone and boiled until pellets become decolorized. Then the sample volume was brought to its original volume with acetone. The optical density O. D662 and O. D645 were measured with a spectrophotometer (Spectroquant@Pharo 300) using the solvent as blank. The chlorophyll a content was determined using equation (1) (Şükran et al., 1998):

Chlorophyll a (mg/L) = (11.75 × OD662) − (2.35 × OD645)

Table 1 Initial physicochemical characteristics of sewage and tannery eﬄuent.

(1)

2.4.2. Determination of biomass concentration For the determination of biomass concentration (on dry basis), algae cultures were harvested by ﬁltration through pre-weighed Whatman No.1 ﬁlter paper and dried at 60 °C for 24 h. The algal biomass weight (g/L) was obtained gravimetrically as dry cell weights (DCW) (Tang et al., 2011).

Parametersa

Sewage

Tannery eﬄuent

pH TDS BOD COD Total P as O-PO43TKN NH3-N Chlorides Sulfates Sulﬁdes Carbonates (as CaCO3) Bicarbonates (as CaCO3) Sodium Total chromium

6.9 690 84 448 3.8 124 71 805 76 8 154 290 165 Nil

7.2 27,320 1500 4800 1.7 760 532 9967 3296 203 Nil 1250 4535 20.9

a

2.4.3. Lipid extraction The algal biomass harvested by centrifugation at 5000 rpm for 5 min were disrupted through ultra-sonication for 10 min. Then the extraction of lipid was done by solvent extraction (2:1 ratio of chloroform: methanol) and phase separation method. The upper methanol-water layer was removed and the chloroform layer (containing lipid) was collected and weighed gravimetrically after evaporation for lipid content (% of dry biomass) (Chen et al., 2011). Lipid accumulation percentage was calculated using equation (2):

Lipid accumulation(%) = [(g of lipid)/(g of biomass)] × 100

3. Results and discussion 3.1. Identiﬁcation of isolated microalgae strain Morphology of the isolated microalgae revealed as green colored ellipsoidal cells without locomotor organelle and identiﬁed as Chlorella sp. The results obtained from 18s rDNA sequencing and phylogenetic analysis conﬁrmed that the isolate was belonging to the family of Chlorellaceae and member of the genus Pseudochlorella. The pairwise alignments gave 98% similarity with Pseudochlorella pringsheimii KY364701. Then the matched sequence was identiﬁed as Pseudochlorella pringsheimii strain VIT_SDSS (Gen Bank Accession No. MG889861).

(2)

2.4.4. Heavy metal - chromium analysis At the end of incubation time, the dry biomass obtained were investigated for morphological changes through a Scanning electron microscope (SEM) and chromium remediation through Energy-dispersive X-ray spectroscopy (EDAX) (Zeiss EVO18). After biomass separation, the supernatant was analyzed for total chromium concentration using AAS.

3.2. Physicochemical characteristics of sewage and tannery eﬄuent The initial characteristics of sewage and tannery eﬄuent after removal of suspended solids are furnished in Table 1. The results showed that the tannery eﬄuent was characterized by high TDS, Organic load, Total chromium, Total Kjeldahl and ammoniacal nitrogen content, high concentrations of chlorides sulfates and sulﬁdes. Except pH, other parameters have made the tannery eﬄuent more toxic, compared to the sewage. Hence, in this study, the tannery eﬄuent was diluted with sewage to support the phycoremediation.

2.4.5. Removal eﬃciency The removal or reduction eﬃciencies (%) of various parameters were calculated using equation (3).

R= [(C0 − Cf )/C0] × 100

Except pH, all the values are in (mg/L).

(3)

where, 3.3. Assessment of microalgae growth under diﬀerent culture conditions

R – Removal or reduction eﬃciency (%) C0 – Initial concentration of a given parameter (mg/L) Cf – Remaining concentration of a given parameter on a given sampling time (mg/L)

The growth responses of microalgal strain Chlorella vulgaris and Pseudochlorella pringsheimii in untreated tannery eﬄuent (100% T), sewage (100% S) and diluted tannery eﬄuents (10% T, 20% T, 30% T, 40% T, and 50% T) were analyzed based on cell count and chlorophyll a content. The results have shown enhanced microalgal growth when mixed with tannery eﬄuent at low concentration (10% T, 20% T, 30% T) as compared to higher tannery eﬄuent concentration (40% T, 50% T) and sewage (100%S). The maximum cell density of 6.07 × 106 cells/ mL for Chlorella vulgaris in 30% T and 9.1 × 106 cells/mL for Pseudochlorella pringsheimii in 20% T was observed at the end of incubation period (Fig. 1(a) and (b)). These results indorsed that the toxicity reduction as well as the nutrient (C, N, P) utilization within sewage and tannery eﬄuent, would enhance the microalgal cell division and metabolic process in diluted eﬄuent. The microalgal growth responses obtained in this study were similar to the trend obtained in ammonium enriched synthetic media (Tam and Wong, 1996) and salinity increased media (Heredia-Arroyo et al., 2011). Both the microalgal species started their exponential phase in 3 days in 100% S, 10% T, 20% T& 30% T eﬄuent. However, microalgal tendency to reach its stationary phase in the diﬀerent dilutions was diﬀered signiﬁcantly

2.5. Statistical analysis All the statistical analysis were carried out using SPSS Statistics for Windows, version 23 (IBM Corp., Armonk, N.Y., USA). The growth of microalgae species was correlated with the reduction of various pollutants such as COD, NH3-N & PO4-P concentrations in the eﬄuent during the experimental period using bivariate analysis. The results are speciﬁed as the Pearson correlation coeﬃcient of which values are statistically signiﬁcant at p < 0.01. Univariate analysis of variance (ANOVA) was done to check the signiﬁcant eﬀects (at p < 0.05) of various factors such as diﬀerent tannery eﬄuent dilutions, treatment with two diﬀerent microalgae species, and their interaction eﬀects (dilution & species) on pollutants (COD, NH3-N & PO4-P) removal efﬁciency. Subsequently, pair-wise comparisons were performed between microalgae species and control for pollutant removal eﬃciency using post hoc Bonferroni tests at signiﬁcant p value < 0.05. 169

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Fig. 1. Growth curves for (a) C. vulgaris & (b) P. pringsheimii grown in 100% S, 100% T & diluted tannery eﬄuents. Chlorophyll a synthesis trends for (c) C. vulgaris & (d) P. pringsheimii in 100% S, 100% T & diluted tannery eﬄuents. (T - Tannery eﬄuent; S –sewage; [10%T, 20%T, 30%T, 40%T, 50%T] - concentration of tannery eﬄuent in diﬀerent dilutions made with sewage).

was very less initially (< 30%), due to the presence of persistent organic pollutant compared to sewage (Fig. 2) (Table S4). However, COD reduction has increased at the end of incubation time, showed > 69% removal with Chlorella vulgaris and > 63% with Pseudochlorella pringsheimii up to 30% T dilution (Table 2). It endorsed the carbon mineralization and utilization by the acclimatized algal-bacterial community, which in turn supported the microalgae growth in diluted tannery efﬂuent (Gupta et al., 2016). Whereas, the COD reduction results obtained for control sets were similar to microalgae inoculated at higher tannery eﬄuent concentration (40%T, 50%T and 100%T), which was due to the stress posed by eﬄuent constituents on microbial adaptation. The signiﬁcance of the carbon source for wastewater remediation was apparent from carbon enrichment study. Evans et al. (2017) reported the signiﬁcant increase in cell density (5 times the initial cell density and reduction in COD) in 3 days for Chlorella vulgaris supplemented with organic carbon (glucose, glycerol), compared to unenriched wastewater.

(F = 11.319, p < 0.05) (Table S3). In 100% S, the microalgae reach its stationary phase, within 5–7 days compared to 10% T (reached on the 10th day). Wherein, the microalgae from 20% T to 30% T has taken even longer time to reach their stationary phase (≥15 days) and the prolonged lag phase was observed for microalgae at 40%T. This would be due to algal acclimatization and adaptation in a subsequent higher concentration of tannery eﬄuent rather than the availability of nutrients (Erdmann and Hagemann, 2001; Borowitzka, 2018), and the same trend was cross-checked with chlorophyll a content (Fig. 1(c) and (d)). Further, the growth inhibition observed in 50%T & 100% T efﬂuent advocated the microalgal stress, due to the high TDS, chromium, sulﬁdes, ammonium concentration, and organic load compared to diluted eﬄuent (González-Camejo et al., 2017; Collos and Harrison, 2014). Generally, the microbial species found in the tannery eﬄuent demands more oxygen for sulﬁdes and organic matter reduction (Chan et al., 2009). In this study, the oxygen supplement and pollutant removal were done by algal-bacterial symbiosis. Reduction in organic load was monitored (as COD) in control and experimental sets. Compared to control, algae inoculated eﬄuents has shown increased COD reduction. In sewage, initially the COD removal was highly rapid (> 77%) for both species, but at the end of incubation time (after reaching stationary phase), there was an increase in COD concentration due to the release of extracellular products by microalgae (Myklestad, 1995). In tannery and its diluted eﬄuents, the COD removal eﬃciency

3.4. Biomass concentration and lipid accumulation Generally, the indigenous microorganisms present in sewage will compete for microalgae for organic and inorganic nutrients and hence be limiting the algal growth (Pittman et al., 2011). But no such limitations were taking place in this study. Moreover, the addition of sewage to the tannery eﬄuent has shown an induced growth eﬀect (at 170

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Fig. 2. COD reduction proﬁle of raw and diluted eﬄuents treated with (a) C. vulgaris (b) P. pringsheimii (c) Control (Eﬄuents without microalgae) (The treatment acronyms 100% T, 100%S, 10%T- 50%T are same as given in Fig. 1.).

The eﬀect of tannery eﬄuent dilution on biomass concentration & lipid accumulation of Chlorella vulgaris & Pseudochlorella pringsheimii were evaluated at the end of the incubation period. The maximum yield of biomass was 3.51 g/L for Chlorella vulgaris in 30% T and 2.84 g/L for Pseudochlorella pringsheimii in 20%T, which was comparatively higher than that of microalgae from sewage (100% S) (Fig. 3a). The biomass obtained was subjected to lipid analysis and the outcomes were presented as percentage lipid accumulation (Fig. 3b). The

dilutions up to 30%T) on Chlorella vulgaris and Pseudochlorella pringsheimii compared to sewage alone. Further, a noteworthy change was observed after 5 days of inoculation in 40%T due to acclimatization and it was conﬁrmed through cell count and chlorophyll a analysis. Initially, at 40%T the chlorophyll a content for both species was less than 1 g/L till 5th day and started to increase gradually afterward (which includes dead matters) (Fig. 1 (c) & (d)). No such appreciative changes were observed at 50% T, & 100% T eﬄuent during the incubation period.

Table 2 The overall percentage removal eﬃciency of COD, Ammoniacal nitrogen and phosphates in sewage, tannery and its diluted eﬄuents treated with C.vulgaris; P. pringsheimii and Control (Eﬄuents without microalgae). Eﬄuent dilutiona

COD Initial (mg/L)

100%S 10%T 20%T 30%T 40%T 50%T 100%T

448 883.2 1318 1753 2188.8 2624 4800

% Removal Control

C.vulgaris

P. pringsheimii

91.07 58.67 62.82 68.05 57.05 54.26 43.75

75.89 68.29 63.65 69.88 61.89 59.67 48.95

70.3 63.76 63.58 69.59 61.94 57.31 45.83

Ammoniacal Nitrogen

PO4-P

Initial (mg/L)

Initial (mg/ L)

70.92 117.07 163.23 209.39 255.5 301.6 532.4

% Removal Control

C.vulgaris

P. pringsheimii

68.97 47 46.61 48.6 38.94 32.36 29

100 100 95.62 65.37 46.77 34.68 33.63

100 100 89.70 62.74 47.16 33.68 32.75

3.78 3.57 3.37 3.16 2.96 2.75 1.73

% Removal Control

C.vulgaris

P. pringsheimii

50.5 29.97 25.81 17.7 39.81 34.54 47.9

100 100 100 100 100 78.5 66.4

100 100 100 100 100 78.1 51.4

a (T - Tannery eﬄuent; S –sewage; 100% T- Tannery eﬄuent without dilution, 100% S- Sewage (without tannery eﬄuent mix), [10%T, 20%T, 30%T, 40%T, and 50% T] - concentration of tannery eﬄuent in diﬀerent dilutions made with sewage).

171

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Fig. 3. (a) Biomass Concentration (dry weight basis) & (b) Lipid accumulation (%) of microalgae collected from sewage & diluted tannery eﬄuents (Media- BG11 media grown algae; Treatment acronyms 100% T, 100%S, 10%T- 50%T are same as given in Fig. 1.).

Harrison, 2014). The rate of microalgae metabolism depends mainly on the concentration of dissolved salts in the growing medium. High concentrations of dissolved salts (> 10 g/L) have a greater inhibitory eﬀect on cell division and biomass accumulation in freshwater algae (Yeh and Chang, 2012).

results ascribed that Pseudochlorella pringsheimii has high lipid accumulation potential of 25.4% compared to 9.3% of Chlorella vulgaris in 20% T eﬄuent. Further, it can be noted that at 20% T dilution, the lipid accumulation potential of Pseudochlorella pringsheimii was 5 times and 1.5 times more than that of sewage (100% S) and BG11 grown microalgae, respectively. It has been reported that diﬀerent factors induce the lipid productivity and biomass productivity in algae, includes diﬀerent mode of cultivation, salinity stress (pH) or osmotic stress, temperature stress, N-starvation, inorganic carbon supplements etc. (Yeh and Chang, 2012; Chiu et al., 2009; Hirooka et al., 2014). The maximum saline stress varied between 0 and 50 g/L reported inducing the biomass productivity and lipid accumulation according to nutrient availability (Shen et al., 2015). However, in this study, the biomass obtained and lipid accumulation at dilutions ≥50% T were absolutely zero for both species, irrespective of dissolved salt stress levied by the eﬄuent. This was due to the presence of growth inhibiting parameters of tannery eﬄuent such as chromium, sulﬁdes, ammonia, and TDS at higher tannery eﬄuent concentration. Chromium has a lethal eﬀect when Cr6+ approximately was 2 mg/L for C. pyrenoidosa (Hörcsik et al., 2006). The average tolerable concentration of free ammonia (at pH > 9) for microalgae belongs to the Chlorophyceae family was 7600 μM (Collos and

3.5. Phycoremediation of eﬄuent Nitrogen and phosphorous are the essential nutrients which support the growth of microalgae in wastewater. To understand the assimilation and uptake of nutrients under the toxic eﬄuent condition, pH, COD, NH3-N, and PO4-P were monitored over the experimental period in diﬀerent eﬄuents (sewage, tannery, and its diluted eﬄuents) and the overall percentage removal was presented in Table 2. Variations in pH were monitored in both experimental and control sets. Microalgal carbon ﬁxation through RuBisCO in the Calvin cycle counterbalances OH− and H+ ions, resulting in ﬂuctuations of eﬄuent pH (Chi et al., 2011). Besides, the rise in pH has an inﬂuence on NH3-N reduction from the eﬄuent. Though ammonium (NH4+) is the favored inorganic nitrogen source for microalgae, a pH rise above 9.4 leads to the formation of free ammonia (NH3), which is more toxic to aquatic 172

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Fig. 4. pH changes in eﬄuents treated for a period of 15 days with (a) C. vulgaris (b) P. pringsheimii and (c) Control (Eﬄuents without microalgae) (Treatment acronyms 100% T, 100%S, 10%T- 50%T are same as given in Fig. 1.).

was initially more rapid (79% for Chlorella vulgaris and 53% for Pseudochlorella pringsheimii on day 5) and reached 100% on the 10th day. Whereas, in diluted Tannery eﬄuent (10% T, 20% T, 30%T) NH3-N removal increased gradually and reached a maximum of 100%, ≥90%, > 60% respectively for both species (Fig. 5). These results ascribed that the assimilation of inorganic nitrogen-ammonium into amino acids in microalgae is strongly reliant on available carbon substrates and its correlation was conﬁrmed through bivariate analysis (Pearson Correlation r = 0.865 & 0.757 for C. vulgaris and P. pringsheimii respectively, signiﬁcant at p < 0.01) (Tables S1 and S2). It is evident from the previous study, that the signiﬁcant reduction in NH3-N (84% removal in 3 days) needs carbon enrichment (Evans et al., 2017). Further, the ammonium incorporation has been shown to increase the demand for that tricarboxylic acid cycle (TCA) intermediates in microalgae (Wett and Rauch, 2003). Therefore, the higher NH3-N removal eﬃciency observed in 100% S and diluted tannery eﬄuent (up to 30% T) in the present study would be due to dilution and assimilation of bioavailable carbon and nitrogen by microalgae.

life (Collos and Harrison, 2014). The pH of 100% S, 100% T & diluted tannery eﬄuent (up to 30% T) were increased from 7 to 9.3 over an 8day duration and subsequent peak changes were observed thereon (Fig. 4). This alkalization indorsed the limitations and reduction in inorganic carbon due to its buﬀering actions in the growing environment. Hence, the removal of NH3-N and PO4-P from eﬄuents inoculated with microalgae had inﬂuenced the overall shift in extracellular H+ concentration through carbon assimilation (Evans et al., 2017). Whereas in control sets no such prominent peak changes were observed except during initial period owing to its onset of bacterial action. 3.5.1. Inorganic nutrient NH3-N removal Generally, biological NH3-N reduction ensues through its conversion to NO2, NO3, and N2 by nitrifying and denitrifying microorganisms. However, pH increases and carbon limitations during phycoremediation limits the formation of NO2-N (Wett and Rauch, 2003). Hence, in this study, the amplest form of nitrogen: Ammoniacal nitrogen was monitored in sewage (100% S) as well as in tannery eﬄuent for its removal by microalgae. The statistical results have shown that the dilution has imposed a signiﬁcant eﬀect on NH3-N removal eﬃciency at p value < 0.05 (F = 11.945; p = 0.000) (Table S5). The removal percentage was decreased with increase in tannery eﬄuent concentrations and there were no signiﬁcant diﬀerences observed in removal eﬃciency between species (post hoc test, p = 0.977) at p < 0.05 (Table S7). In sewage alone (100% S), the removal eﬃciency

3.5.2. Inorganic nutrient PO4-P removal In sewage and diluted tannery eﬄuents (up to 40% T), 100% PO4-P removal eﬃciency was achieved at the end of the incubation period and there was no signiﬁcant diﬀerence observed between dilutions and between species (Tables S6 and S8). Initially, PO4-P was drastically reduced from 3.57 mg/L to 0.126 mg/L on the 5th day for Chlorella vulgaris and 0.064 mg/L on the 7th day for Pseudochlorella pringsheimii 173

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Fig. 5. NH3-N reduction in sewage, tannery, and its diluted eﬄuents for (a) C. vulgaris (b) P. pringsheimii (c) Control (Eﬄuents without microalgae) Treatment acronyms 100% T, 100%S, 10%T- 50%T are same as given in Fig. 1.

biomass separation endorsed > 80% chromium reduction for both species in 10% T, 20%T, 30%T eﬄuent and ∼50% in lower dilutions (40%T, 50%T, and 100% T) (Table 3). It conﬁrmed that the chromium removal from tannery eﬄuent (untreated & diluted) was done, not only by microalgae, also due to the reduction of oxidation states of chromium in the presence of electron donors such as organic matter or reduced inorganic matter, as bacterial reaction proceeds over the course of time (Mwinyihija, 2010). Lethal eﬀect of chromium mainly depends on its oxidation state: Cr6+ is more toxic than Cr3+ form, due to its strong oxidative potential as well as free diﬀusion across cell membrane transporter. The reported lethal chromium concentration (Cr6+) was approximately 20 mg/L and EC50 was 2 mg/L for C. pyrenoidosa (Hörcsik et al., 2006). The surface morphology of both microalgae biomass (grown in 10%T, 20%T, 30%T, and BG11 medium) was observed under 3000× magniﬁcation using scanning electron microscopy (SEM). The corresponding micrographs, showing the surface variation due to contaminants adsorption with respect to growing media (Fig. 7). Elemental analysis of microalgal biomass was carried out with EDAX. Its spectrum endorses not only chromium remediation by microalgae and also other elements such as calcium, magnesium, sodium, potassium, aluminum, zinc, iron, in diluted tannery eﬄuent (Fig. 8). Further, Hörcsik et al. (2006) reported that increased chromium on the cell wall would accumulate more calcium over the cell wall through ion exchange. Whereas iron and magnesium have an inverse relation with chromium

in 10%T eﬄuent with a removal eﬃciency of > 96% and remained below detectable level afterward, till the end of incubation period. Whereas for 100% S, the removal eﬃciency of > 96% was achieved on the 10th day and remained below detectable level thereafter (Fig. 6). Also, the correlation analysis results have shown the positive correlation between N & P removal within two species (Pearson Correlation signiﬁcant at p < 0.01; r = 0.708 & 0.793 for C. vulgaris and P. pringsheimii grown eﬄuent respectively) (Tables S1 and S2). This describes the rate at which P removal was mainly depended on available nitrogen source (Ruiz et al., 2011). Further, the phosphates removed at a higher eﬄuent concentration (50%T & 100%T), and control sets would describe the possibility of phosphates precipitation as calcium phosphate when pH rises above 8.5 during bacterial action along with the algal assimilation and calcium precipitation (Ferguson et al., 1973).

3.5.3. Heavy metal - chromium removal Microalgal potential to adsorb or accumulate heavy metals through biosorption has a signiﬁcant contribution to heavy metal remediation treatment (Singh and Thakur, 2015). Its defense system functions against heavy metal through negative functional group and metal-ion transporters found on its cell wall (Pacheco et al., 2015; Kumar et al., 2015). In this present study, the chromium removal eﬃciency of Chlorella vulgaris and Pseudochlorella pringsheimii inoculated in untreated and diluted tannery eﬄuent was evaluated at the end of the experimental period using AAS and EDAX. Analysis of supernatant after 174

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Fig. 6. PO4-P removal from sewage, tannery, and its diluted eﬄuents for (a) C. vulgaris (b) P. pringsheimii (c) Control (Eﬄuents without microalgae) (Treatment acronyms 100% T, 100%S, 10%T- 50%T are same as given in Fig. 1.).

Table 3 Overall Chromium removal eﬃciency from Tannery and its diluted eﬄuents treated with C.vulgaris and P.pringsheimii. Eﬄuenta dilution

10%T 20% T 30 %T 40% T 50% T 100% T

Initial Concentration (mg/L)

2.08 4.17 6.25 8.34 10.42 20.85

C.vulgaris

P.pringsheimii

Final Concentration (mg/L)

% Removal

Final Concentration (mg/L)

% Removal

BDL 0.103 1.249 2.822 5.728 11.91

100 97.52 80.01 66.16 45.02 42.87

0.032 0.276 1.287 2.597 5.478 11.12

98.46 93.38 79.4 68.86 47.42 46.6

a (T - Tannery eﬄuent, 100% T- Tannery eﬄuent without dilution, [10%T, 20%T, 30%T, 40%T, 50%T] - concentration of tannery eﬄuent in diﬀerent dilutions made with sewage).

(r = 0.892 & 0.737) & PO4-P (r = 0.800 & 0.725 respectively) at signiﬁcant p value < 0.01. Further, the signiﬁcant eﬀects of diﬀerent dilutions, treatment with two diﬀerent microalgae species, interaction eﬀects of dilution & microbial growth on pollutants (COD, NH3-N & PO4-P) removal eﬃciencies were analyzed and presented in Supplementary Tables S3–S6. In addition, a pair-wise comparison between microalgae species and control for pollutant removal eﬃciency were analyzed and presented in Supplementary Tables S7 and S8.

concentrations on cell wall binding. The weight percentage of chromium observed were 16.96%, 19.23%, 19.47% for Chlorella vulgaris and 21.47%, 15.64%, 9.45% for Pseudochlorella pringsheimii in 10% T, 20%T, 30% T eﬄuent respectively. 3.6. Statistical analysis The correlation between the study parameters such as the growth of microalgae (cell count), change in pH, eﬄuent dilution, percentage removal of various pollutants such as COD, NH3-N & PO4-P in the efﬂuent during the experimental period was analyzed and presented in Supplementary Tables S1 and S2. From the table it can be noted that the microalgae growth for C. vulgaris & P. pringsheimii is highly correlated with percentage removal of COD (r = 0.745 & 0.661), NH3-N

4. Conclusion The present study highlights the eﬃciency of Chlorella vulgaris NRMCF0128 and Pseudochlorella pringsheimii (strainVIT_SDSS) for 175

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Fig. 7. SEM image of biomass (3000× magniﬁcation) collected at the end of incubation period - C. vulgaris grown in (a) BG11 Medium (b) 10%T (c) 20%T (d) 30%T & P. pringsheimii grown in (e) BG11 Medium (f) 10%T (g) 20% T (h) 30%T.

eﬄuent concentrations (dilutions > 40%T), inorganics (N, P, and heavy metal) was not reduced to the levels as those achieved when their initial concentrations were lower. Hence, suggesting the potential of microalgae in treating the tannery eﬄuent with sewage may be limited by the adaptation of microalgae to initial eﬄuent constituents: chromium, TDS, available carbon, and other inorganic elements. Overall, the dilution of tannery eﬄuent with sewage suggested the reliability of

treating the tannery eﬄuent when combined with sewage in diﬀerent dilution under batch cultivation conditions. The results showed that both species are apparent to treat tannery eﬄuent when the dilutions are 10% T, 20% T and 30% T, with the substantial removal of COD, NH3-N, PO4-P & heavy metal chromium. In addition, Pseudochlorella pringsheimii has higher lipid accumulation potential than Chlorella vulgaris irrespective of the saline stress. However, at the higher tannery 176

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Fig. 8. EDAX peaks for biomass collected at the end of incubation period - C. vulgaris grown in (a) 10%T (b) 20% T (c) 30%T & P. pringsheimii grown in (d) 10%T (e) 20% T (f) 30%T.

eﬀective industrial eﬄuent treatment through phycoremediation along with the production of lipids in microalgae biomass.

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Acknowledgment The authors would like to thank the Vellore Institute of Technology (VIT), Vellore for the necessary facilities and support provided to carry out the research work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2019.04.031. Declaration of interest None. References Abinandan, S., Bhattacharya, R., Shanthakumar, S., 2015. Eﬃcacy of Chlorella pyrenoidosa and Scenedesmus abundans for nutrient removal in rice mill eﬄuent (paddy

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