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Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions
Journal Pre-proofs Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions Rifat Azam, Richa Kothari, Har Mohan Singh, Shamshad Ahmad, V. Ashok Kumar, V.V. Tyagi PII: DOI: Reference:
S0960-8524(20)30387-4 https://doi.org/10.1016/j.biortech.2020.123116 BITE 123116
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 January 2020 25 February 2020 1 March 2020
Please cite this article as: Azam, R., Kothari, R., Mohan Singh, H., Ahmad, S., Ashok Kumar, V., Tyagi, V.V., Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.123116
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Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions Rifat Azama, Richa Kotharia,b,*, Har Mohan Singhc, Shamshad Ahmada, V. Ashok Kumard, V. V. Tyagic aBioenergy
and Wastewater Treatment Laboratory, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow (U.P.), India
bDepartment cSchool
of Environmental Sciences, Central University of Jammu, Jammu, India
of Energy Management, Shri Mata Vaishno Devi University, Katra (J&K) India
dDepartment
of Chemical Technology, Chulaloangkorn University, Bangkok, Thailand
Abstract
Slaughterhouse produce large amount of wastewater, containing high pollutant load in terms of protein, fats and meat pieces, might lead to source of non-point contamination. Various concentrations (25%, 50%, 75%, and 100%) of slaughterhouse wastewater were used to increase the algal biomass production, pollutants removal and biochemical profile analysis under controlled conditions of C. pyrenoidosa. Results showed that the maximum biomass yield 430 mg L-1 was achieved at 50% concentration of wastewater to other concentration of wastewater. Direct relation was observed in between pollution load and nutrient load of SHWW with biochemical profile of C. pyrenoidosa. The COD/BOD ratio (1.9) was found to be significant on the scale of degradability by algal biomass. Sufficient nutrient removal efficiencies (23-42%, 1848%) and pollutant load efficiencies (17-31%, 7-29%) were observed. Findings showed that slaughterhouse wastewater is rich in nutrients, which can be utilized for algal biomass production and wastewater remediation for future endeavors. Keywords: Chlorella pyrenoidosa, Slaughterhouse wastewater, Nutrient removal, Biomass production * Corresponding Author (Dr. Richa Kothari) Email: - [email protected] 1
1. Introduction
The slaughter industry is one of the largest industries, which have economic importance for any country under the category of livestock sector. Slaughterhouses are the part of meat processing industries, but during the processing of meat a large amount of water is used mainly for cleaning and washing purposes. Among the various meat-producing countries, India is the fifth largest exporter of meat at international level (Kumar et al., 2018). So far, no reports are available on the use of discharged water estimation neither at global level nor at national level. It might be due to the non-point source at commercial level means very small to very large, based on quantity or animals (small/large) slaughtered for meat on daily basis. In Indian context very few data are available on the use of water foot-printing in slaughterhouses, but authorized information in the form of referred published data are not available. Although, physio-chemical characterization of slaughterhouse wastewater was reported by various researchers in peerreviewed articles. Research studies show that slaughter industry is the most significant contributors to water pollution, because it consists of high concentrations of contaminants like fats, suspended solids, chemical oxygen demand, chlorides and nitrates. Jais et al., (2015) has also reported that slaughterhouse wastewater is also rich in nitrogen and phosphorous. Besides, slaughterhouse wastewater pH was found in the range of 6.5 to 7.6 with high turbidity, red in color and offensive smell. These high concentrations of nutrients present in the slaughterhouse wastewater makes a favorable condition for algal growth and biofuel production (Kitrungloadjanaporn et al., 2017; Kothari et al., 2017). However, wastewater discharges without treatment to the surroundings largely contribute to the eutrophication. Earlier studies showed that microalgae have the capability of treating various types of wastewater, which includes dairy, domestic, municipal and textile wastewater and alternatively produce high biomass by utilizing their organic matter as a nutrient for growth (Ahmad et al., 2018; Maizatul et al., 2017; Kothari 2
et al., 2013). On the other hand, if same concept is developed with controlled/known algal strains to treat the slaughterhouse wastewater, it may provide new insight into the era of both water crisis and energy crisis. Since different types of phytoremediation approach have the capability to treat different types of wastewater more efficiently in comparison to other conventional methods like electro-coagulation, chemical coagulation, bed sequencing batch reactor, anaerobic treatments (UASB), etc. Nevertheless, all these methods are costly and need centralized systems for the treatment of wastewater at one place, and this could be the major challenge with these existing technologies. Therefore, to reduce the load of untreated wastewater from slaughterhouses in particular, decentralized type systems with a phycoremediation approach might be the best alternative and effective way for removal of nutrients as compared to other existing conventional methods (Emparen et al., 2018, Kothari et al., 2010).
Hence, the present study explores to assess the impact of variation in the concentration of slaughterhouse wastewater for the growth of C. pyrenoidosa for high biomass production. Pollutant removal through algal strain with an integrated approach of biomass production showed the benefits for the biomass growth using wastewater as a resource media (Ahmad et al., 2019; Salama et al., 2017). Different compositions of wastewater (dairy, textile, poultry, pond, municipal etc.) are treated with algal biomass by absorption of nutrient rich organic pollutants and their transformation into biomass which can be used to produce biodiesel by transesterification process as reported by the researchers with different microalgae species such as C. vulgaris, C. pyrenoidosa, C. sorokiniana, Chlamydomonas (Kothari et al., 2013; Ahmad et al., 2013; Venckus et al., 2017). C. vulgaris and Scenedesmus obliquus were utilized for biomass production and nutrients removal from wastewater in membrane photobioreactor (Gao et al.,
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2016). Capillary-driven photobioreactor also used for dual application of biomass production as well as nutrients removal of microalgae and this cultivation method shows higher light utilization efficiency (Xu et al., 2017). Liu et al., (2020) used for microalgae for wastewater treatment in addition with CO2 supply in vertical column photobioreactor.
To the best of our knowledge, so far no reports concerning the use of microalga C. pyrenoidosa for the treatment of slaughterhouse wastewater. Therefore, the objectives of the present study are focused on a novel approach for bioremediation because Chlorella species have a potential to remediate urban wastewater (Rasoul-Amini et al., 2014), poultry wastewater (Murugesan et al., 2010) and in our prior studies with dairy wastewater, textile wastewater (Pathak et al., 2014; Ahmad et al., 2018) for nitrate (57-79%), phosphate (84-87%), COD (43-79%), BOD (78-81%). That is the particular reason to investigate the potential of Chlorella pyrenoidosa in coupling with slaughterhouse wastewater for remediation of nutrient and pollution load as an objective in this study. Presently, the algal biomass production at commercial level using the existing freshwater resources which is not sustainable due to its demand for drinking and other purposes and available on high cost. It is also an alarming condition for environmental sustainable part and contributing as one the factor in capital production cost. Thus, the utilization of wastewater as an alternative source for freshwater and to reduce the cost in dual way i.e. as a medium for biomass and providing low-cost source nutrients from those instead of high-cost chemicals, decrease the production cost may break the limitations with its wide applications. 2. Materials and methods To study the objectives of this research study, this section is further divided into subsections such as: (i) Physio-chemical characterization of slaughterhouse wastewater: to know the real factual 4
composition of selected slaughterhouse wastewater; (ii) Algal (Chlorella pyrenoidosa) growth optimization with different concentration and analysis of biochemical profile of algae; (iii) Utilization of C. pyrenoidosa for removal of pollutant load and nutrient load in slaughterhouse wastewater. Correlation analysis investigation in between pollution load and biochemical profile of selected algae for possible outcomes in terms of value-added products also part of the objective. 2.1. Physical and chemical characterization of slaughterhouse wastewater Slaughtered wastewater was collected from the local market of Aminabad, Lucknow (UttarPradesh) India, which has the capacity to generate gallons of wastewater per day. Furthermore, approximate 30-35 cattle and 200-250 poultry (goat/sheep/buffalo/pig) cut per day, as per the information from local shopkeepers. The sample of slaughterhouse wastewater was collected and stored at 4 ˚C in sterilized water sampling bottles for analysis of quality. Four different concentrations of slaughterhouse wastewater were selected to study the possible impacts on algal growth, i.e. 25%, 50%, 75%, 100% and BG-11 media is taken as control for algal growth. In this study, distilled water media is used to maintain the concentration of wastewater as per our objectives in study of slaughterhouse wastewater. The physico-chemical parameters were assessed for study of color, odor, temperature, total dissolve solids (TDS), pH, BOD and COD by standard analytical methods of water and wastewater APHA, (2012). In slaughterhouse wastewater, the initial pH was analyzed by using a portable digital pH meter Model No -009 (1) A. The sample color was red which was seen by naked eyes. COD was analyzed by using titration method and BOD was determined after 5 days of incubation at 20 ºC by using incubator and then titrate the sample. The NO3- was estimated by (Cataldo et al., 1975) and PO43estimation by stannous chloride method (Sletten and Bach, 1961). All the parameters were 5
investigated in triplicate. The NO3- and PO43- was analyzed by using UV-Vis spectrophotometer (Systronics 2203). The physico-chemical parameters of slaughterhouse wastewater are shown in Table 1 with discharge limits prescribed by World Health Organization (WHO), European Union (EU), United States (US), Canadian and Australian standards. Prior to microalgal cell inoculation in slaughterhouse wastewater, pretreatment step is just followed by sieving after collection to remove hair and other fleshy materials etc. and autoclaving (for 20 minutes at 15 Psi and 121℃) before starting the experimental investigations. 2.2. Algal species For this study, microalgal organism Chlorella pyrenoidosa (NCIM 2738) collected from resource centre, National Collection of Industrial Microorganism (NCIM), Pune (Maharashtra), India. Chlorella sp. is selected to complete the objectives of the present research, with C. pyrenoidosa strain. BG-11 medium are used to maintain the culturing conditions with composition i.e. NaNO3,1.5 g L-1; K2HPO4,0.04 g L-1; MgSO4·7H2O, 0.075 g L-1; CaCl2·2H2O, 0.036 g L-1; citric acid, 0.006 g L-1; ferric ammonium citrate, 0.006 g L-1; EDTA (disodium salt), 0.001 g L-1; Na2CO3, 0.02 g L-1; 1 mL trace elements solution (in g L−1: H3BO3, 2.86; MnCl2·4H2O,1.81; ZnSO4·7H2O, 0.222; NaMoO4·2H2O 0.39; CuSO4·5H2O,0.079; Co (NO3)2·6H2O,0.0494) with pH 7.0 ± 2 as suggested by NCIM. The media was sterilized by autoclaving for 20 minutes at 15 Psi and 121℃. The glassware used for experiment, all clean and sterilized at 120℃ for 6-8 h before use. The medium was then inoculated with C. pyrenoidosa and incubated at 30±1℃ in a 12h light (10 Wm-2)/dark cycle.
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2.2.1. Biochemical characterization The biochemical characterization of selected algal biomass for carbohydrate, protein, and chlorophyll were estimated by standard methods. The carbohydrate is estimated by Anthrone method (Dreywood, 1946) at an absorbance of 620 nm. The Lowry method is used for the estimation of protein at wavelength of 660 nm (Lowry et al., 1951). The chlorophyll is estimated by methanol method for the chlorophyll content in the cells (Mackinney, 1941) with absorbance at wavelengths, which consist of 750 nm, 666 nm, 475 nm and 653 nm by spectrophotometer. Biomass concentration was determined by using UV-Vis spectrophotometer (Model no. Systronics 2203) of the culture absorbance at a wavelength of 680 nm. 2.2.2. Optimization of algal growth on slaughterhouse wastewater The algal strain of C. pyrenoidosa was grown at different concentrations (25%, 50%, 75%, and 100%) using slaughterhouse wastewater. The experiment was started in 500 mL of the Erlenmeyer flask and incubated at 30±1ºC in a 12h light (10 Wm-2)/dark cycle. The algal strain was used for the inoculation (10 mL) of each flask. The exponential phase of algal species was determined with the help of optical density at 680 nm on alternate day by using double beam spectrophotometer (Model no. Systronics 2203). For the calibration curve observation, initial O.D. of 0.1 cells were dried at 60 °C for 24 hours and the regression equation obtained was y=298.23x-3.8 (R2=0.98), where y is the dry cell weight (mg L-1) and x is the absorbance at 680 nm. 2.3. Nutrient removal from slaughterhouse wastewater using Chlorella pyrenoidosa The nutrient removal investigation was carried out in 2 L conical flask, with working volume of 1 L of different concentrations (25%, 50%, 75%, and 100%) of using slaughterhouse wastewater. 7
The 10 mL C. pyrenoidosa suspension was inoculated in each flask with the above condition given in section 2.2. The quality of biomass was analyzed on 0th, 5th, 10th, and 15th day of inoculation for the reduction of pollutant load in slaughterhouse wastewater. Samples were centrifuged at 5000 rpm for 10 minutes at 4˚C and subsequently 0.45 mm filter paper used to filtrate. The filtrate was used to analyze BOD, COD, NO3-, and PO43- . The nutrient load was measured by using Eq. (1) after removal efficiency of pollutant load (APHA, 2012): Removal efficiency (%) = (Spo – Spt/Δt) ×100
(1)
Where, Spo (mg L-1) and Spt (mg L-1) are initial and final concentrations BOD, COD, NO3- and PO43- at time period of Δt. 2.4. Kinetics of algal growth For the measurement of microalgal growth kinetics, the specific growth rate was observed by using Eq. (2) as follow: µ= In (Po- Pt )/(t1-t0)
(2)
Where, µ is the specific growth rate (mg L-1 day-1) of C. pyrenoidosa. The graph Pad Prism Software v6 (student version) used to calculate the growth rate by applying the exponential Eq. (3) Pt=PO exp (r×t) is
(3)
Eqs. (2 and 3), Po and Pt are the size of the population at initial and final at time t and r is the growth rate of population (Van et al., 2015). 2.5. Correlation analysis Correlation analysis conducted between physico-chemical parameters of slaughterhouse wastewater and biochemical parameters of algal strain with help of Metabo Analyst, Statistical Analysis software (student version).
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3. Results and Discussions In this experimental study, slaughterhouse wastewater was treated using the microalga C. pyrenoidosa and produced algal biomass. The growth and algal biomass production was optimized with various concentrations of wastewater, and this experiment was conducted for a period of 15 days. During the growth period, the biochemical parameters were assessed at every 5 days interval, in terms of optical density (680 nm) with organic load (COD, BOD), nutrient load (nitrate and phosphate), reduction of pollution load, and nutrient load. Besides, the treated wastewater was analyzed thoroughly before it release to the surroundings. Co-relational analysis between physico-chemical and biochemical in slaughterhouse wastewater of algal biomass is also studied. 3.1. Slaughterhouse wastewater characteristics before treatment Slaughterhouse wastewater looks reddish color with alkaline pH (10.16), and offensive smell. The BOD and COD were found to be a higher range in comparison to discharge limits by Indian standards. Whereas, TDS range for discharge of slaughterhouse wastewater was not prescribed by anyone of the standard limits as given in the Table 1. Similarly, COD (3429 mg L-1) is also more than 10 times Indian standard (250 mg L-1), same trend was observed with BOD (1798 mg L-1) of slaughterhouse wastewater before treatment. The concentration of nitrate (331 mg L-1) was found to be six times greater than the approved limit (50 mg L-1). Similarly, the phosphate concentration was found to be again on higher side (34 mg L-1) in comparison to permissible range (5-9 mg L-1). According to literature, inorganic and organic forms of nitrogen present in the wastewater and it includes ammonia, nitrite and nitrate respectively. However natural decaying process of organic matter such as blood traces, fats, proteins etc. in case of slaughterhouse wastewater produced nitrates which is most stable form of nitrogen in water.
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Algal bloom is the harmful consequence of excessive nitrates level in the water bodies on the earth (Al-Gheeti et al., 2015). Yaakob et al., (2018) reported that most common form of orthophosphate (PO43-), which comes from use of soap and detergent products. Thus, it can be seen that high concentrations of nitrate and phosphate in wastewater are the main cause of eutrophication. Table 1 show the results obtained after treatment in favor of slaughterhouse wastewater discharge limits and compared with standards of permissible limits. The reason for high values of BOD and COD may also be due to presence of blood residues, fats, proteins, and fibers present in wastewater. The slaughter produces from the washing process of meat (Jaish et al., 2017). These types of wastewater have high level of nitrogen, phosphorus, COD and BOD (Maizatul et al., 2017). Most of the organic content of wastewater is available in the form of COD. So, the higher concentration of COD manifests the various chemical reactions among organic and inorganic substances of slaughterhouse wastewater. On the other side, BOD indicates the presence of biochemical oxidation of organic compounds available in complex forms of selected wastewater. Hence, the higher concentration of BOD which is directly proportional to higher microbial load in the slaughterhouse wastewater. As per the findings of analysis of selected wastewater without any treatment, exceeding the allowable level for all parameters. Similar results for slaughterhouse wastewater composition also found with successive experimental treatment by anaerobic baffle reactor based aerobic activated sludge reactor and UV/H2O2 photoreactor (Bustillo-lecompte et al., 2016). Therefore, COD/BOD ratio, indicator for degradable scale of wastewater also assessed by the initial parameters. According to review of literature, its value must be below 10 (Myra et al., 2015). While in the present study, it is 1.9, indicating that the compounds in the wastewater are relatively degradable and thus the algae can utilize the nutrient for their growth. Hence, the untreated wastewater discharge into the
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water bodies causes health problems and also pollutes the environment and causes eutrophication. Therefore, selecting the algal strain which have high potential for the treatment of slaughterhouse wastewater with optimization of concentration, observations and findings are discussed in detail in next section. 3.2. Algal growth and nutrient removal 3.2.1. Algal growth The growth condition was monitored using various concentrations of slaughterhouse wastewater, and it was assessed by optical density (680 nm). The growth of C. pyrenoidosa was optimized in the BG-11 growth medium (distilled water) as well as in slaughterhouse wastewater. During the period of cultivation, the exponential phase was observed between 5th – 9th days. Figure 1 shows that the algal growth increased with increasing concentration of wastewater from 25% to 75% and same trend observed with 100% concentration of slaughterhouse wastewater. In log and lag phase the trend of algal growth was found similar in all the four concentrations. Whereas, in stationary phase the algal growth was significantly found lower in the concentrations that are 25%, 75%, and 100% compared to 50% concentration. Although the survival rate of selected algal strain is observed good in all the concentrations of wastewater, however, 50% concentration found to be the best for the growth of algae (Figure 1). It may be due to existence of optimized values of nutrients (nitrate and phosphate) availability for algal biomass. There are two main limiting factors for microalgae growth which are nitrogen and phosphorus (Singh et al., 2019; Kothari et al., 2013; Kothari et al., 2012), as suggested by various researchers. In the absence of slaughterhouse wastewater i.e. in control 1 (only distilled water) the algae did not show any significant improvement with growth parameters.
The results indicate that the
algal growth without slaughterhouse wastewater did not support for the high biomass production
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is because of lack of nutrients. But in the controlled condition i.e. with BG-11 media (control 2), wastewater dependent growth of algae was also compared with BG-11 growth medium and delineated in Figure 1. The growth of algal biomass at 100% concentration was observed slow, and this may be due to higher level of contaminants. Thus, it inhibit the growth and presence of some nutrients in slaughterhouse wastewater, which are not used by C. pyrenoidosa efficiently. However, the growth of algae was stimulated and continued up to 12th day of treatment, very much noticed with 50% concentration of wastewater may be due to the availability of nutrients in optimized range required for growth, whereas, 25% concentration did not support the algal growth compared to control 2, that maybe due to low level of nutrients in wastewater. Therefore, this study found that 50% concentration of slaughterhouse wastewater could be the ideal condition for achieving highest biomass production using C. pyrenoidosa. 3.2.2. Nutrient removal Phyco-remediation is an advance stream of science using algal species for wastewater treatment and ultimately decreases the nutrient levels in the wastewater. After the optimization of algal growth with a 50% concentration of slaughterhouse wastewater, is selected for the study of pollutant removal rate. Microalgae prefer to orthophosphate (PO43-) for their food-material, which is easy to binds the ions for microalgae growth. The initial value of pH, TDS, COD, BOD, including nitrate and phosphate were observed and found to be in the higher amount than the allowable limit, as shown in Table 2. After inoculation of algal cells, the pollution load is measured in 0th day, 5th day, 10thday and 15th day of the treatment. Analysis of physicochemical parameters for 5th day of observation indicate increase in reduction level of TDS, COD, BOD, nitrate and phosphate concentration from 325 mg L-1 (18%), 1528 mg L-1 (17%), 839 mg L-1 (7%), 133 mg L-1 (22%), and 13.4 mg L-1 (17%) respectively as compared to the initial value
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obtained from inoculated wastewater (Figure 2). Furthermore, 10th day of experimental investigation for TDS, COD, BOD, nitrate and phosphate concentration were found in the ranges as 211 mg L-1 (35%), 1243 mg L-1 (18%), 778 mg L-1 (7%), 84 mg L-1 (36%), and 8.6 mg L-1 (35%) respectively. The algal cells absorbed the nutrients from wastewater for their growth, and increase the level of algal biomass, obtained result indicates that the nutrient load is decreasing in the selected concentration of wastewater. However, same trend was observed in the reduction of pollutant load up to 15th day of experiment. So, all the analysis shows that the algal-based wastewater treatment is a valuable alternative method for the remediation of pollutants from slaughterhouse wastewater. Because the algae feed, the nutrient from wastewater and result indicates that the level of pollution decreases. Although, many researchers find that microalgae can efficiently cultivate in nitrate and phosphate rich nutrient wastewater environment (Kothari et al., 2012; Wang et al., 2010) for various types of industrial effluents/influents, but the treatment of slaughterhouse wastewater with C. pyrenoidosa make this approach novel and ecofriendly to treat the wastewater with low-cost investment. Slaughterhouse wastewater discharges are harmful to the environment because nutrient level (nitrate and phosphate) is very high (Bustillo-Lecompte et al., 2016). Algal growth plays an important role in the treatment of such type of wastewater. During algal growth cycle, low-cost nutrients were used by the algal biomass which is reduced the pollution load in slaughterhouse wastewater. On the basis of nutrient, it has been reported that the slaughterhouse wastewater possesses a high amount of nitrogen and phosphorous (Jaish et al., 2015). From this study, it is evident that the use of algal biomass for treatment of slaughterhouse wastewater is viable alternative in the current water and energy crisis scenario because produced biomass has a potential for biofuel applications also as reported (Kumar et al., 2018; Kitrungloadjanaporn et al., 2017).
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3.4. Biochemical profile of algae grown in slaughterhouse wastewater Biochemical profiles (chlorophyll, protein, and carbohydrate) of microalgal biomass are the source for the production of bio-based high quality products. Along with light and temperature, nutrients are the essential factors for biochemical compounds and biomass growth and development. Only 50%, concentration of wastewater was selected which used for the changes in chlorophyll, protein and carbohydrate along with growth dynamics monitored during the uptake of nutrients from wastewater. On the basis of growth, 50% concentration is best for the production of biomass (430 mg L-1 on dry wt. basis) in slaughterhouse wastewater. For the analysis of chlorophyll the concentration increases from 0th day to 15th days of the experiment are 0.31µg mL-1, 2.94µg mL-1, 9.50 µg mL-1 and 15.31µg mL-1 respectively. Therefore, it can be noted that the concentration of protein in slaughterhouse wastewater found to be increased from 0th to 15th days are 10 mg L-1 (2.32%), 32mg L-1 (7.44%), 42.4mg L-1 (9.86%), and 53.2 mg L-1 (12.37%). Thus, percentage of protein in dry microalgal biomass was analyzed to increase from 2.32% to 12.37% during the culture period with dry biomass (Figure 3). However, after inoculation, the carbohydrate content in slaughterhouse wastewater was measured to uninterruptedly increase up to 15th day of experiment 10.2 mg L-1 (2.37%), 22.3 mg L-1 (5.18%), 36.4 mg L-1 (8.46%) and 41.2 mg L-1 (9.58%). The present experimental investigation results clearly shown as the potential for algal biomass production with favorable biochemical profile with slaughterhouse wastewater at 50% concentration. Table 3 indicates the potential of Chlorella sp. with other wastewaters also but on comparison with slaughterhouse wastewater for biochemical profile of C. pyrenoidosa not as much high in meat wastewater (CUT), (Lu et al., 2015). It has been observed that the algae consume nitrate as nitrogen source and initial concentration of nitrogen pose a significant
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influence over bio-chemical profile of algal biomass (C. pyrenoidosa). Here, the initial concentration of nitrogen (as nitrate) in wastewater is high (172 mgL-1) and dilution of wastewater reduces its concentration. Therefore, higher concentration of nitrogen leads to produce higher amount of protein and increasing dilution level of wastewater reduces the concentration of nitrate which may be a reason of low accumulation of protein in present study. With 50 % concentration of slaughterhouse wastewater, only 42.8 % nitrate removed by the algae on 15th day, which responsible for accumulation of protein (12.3%) in the algal cell as observed in this study. Whereas, Lu et al., (2015) also reported that biomass yield of Chlorella sp. (UN5161) was very low with different processing steps in meat processing wastewater. It may be due to the low profile value of protein, which is important building block for growth of any algal cell, intake mechanism of growth nutrients also inhibited by the presence of toxic substances in the wastewater. Hence, growth rate and percentage of protein content in C. pyrenoidosa directly dependent on the initial concentration of nitrogen source in the medium/wastewater, this observation also supported by the Venckus et al., (2017) in his study with Chlorella vulgaris grown in municipal wastewater. As per present findings, dry weight of algal biomass is 430 mg L-1 (with 50%) concentration of slaughterhouse wastewater. Approximately, similar results were reported by Wang et al., (2010), Taskan, (2016) for Chlorella sp. and mixed consortium of algae (18 species), respectively, with high nutrient-rich wastewater. So, our findings are best on the ground of algal biomass productivity with half-percentage concentration of wastewater and use of single strain only. Similarly, Taskan, (2016) was taken initial cell concentration of 0.2 g L-l but in present study experiment proceed with 0.1 g L-l concentration of cell at initial level. Hence, biomass productivity and biochemical profile are significant with our studies in references to treatment of
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wastewater, which may be responsible for unhygienic and unhealthy conditions in surrounding, if discharged without any treatment. Use of photobioreator systems for treatment of wastewater by many researchers for algal growth in their lab studies but they are costly and energy-intensive. Further, lab-scale phycoremediation strategies for slaughterhouse wastewater with present approach also have similar applicability on the part of final findings but energy intensive in approaches. 4. Correlational analysis of slaughterhouse wastewater The correlational analysis of slaughterhouse wastewater was comprised of physicochemical and biological parameters of slaughterhouse wastewater. In the present study, the correlation between the different concentrations of slaughterhouse wastewater parameters, which consist of 25%, 50%, 75%, and 100%. In this section the co-relation between two variables indicates the high differences among them. The correlation coefficient analysis of parameters was presented in the given Figure 4. The highest positive correlation was observed in 25% and 50% concentration of slaughterhouse wastewater of TDS, BOD, COD, NO3- and PO43- between the chlorophyll, protein, and carbohydrate are highly correlated which is also present in the Figure 4. In 75% and 100% concentration, the correlation of TDS, BOD, COD,
NO3- and PO43- between the
chlorophyll, protein, and carbohydrate was not found highly correlated, because, due to the concentration of wastewater decrease. According to Lu et al., (2015) nutrient removal (NO3-, PO4-3 COD and BOD) efficiency positively correlated with biomass yield. In case of 75% the chlorophyll, protein, and carbohydrate are not positively correlated to BOD, COD, NO3- and PO43- except TDS. The same results were found in 100% concentration. Because slaughterhouse wastewater ratio of COD: BOD was 4:1, which is relatively high, direct affect the biomass yield and increase the time for degradation. High BOD and COD will lead to occurrence of
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eutrophication, which can be toxic to aquatic organisms, destruct organism nursery grounds, and extinction of certain species (Kundu et al., 2013; Yaakob et al., 2018). The best positive correlation was found with the 25% and 50% concentration of wastewater parameters for protein, carbohydrate and Chlorella pyrenoidosa. Similarly, a strong correlation reported with case of municipal wastewater between the nitrogen and phosphorous and in the context of protein, carbohydrates in the biomass (Venckus et al., 2017). Thus, the analysis and finding of the coefficient assist in rapid analysis in wastewater treatment and management. The significant correlation between the algal biomass and the nutrients indicates the importance of microalgal communities, and can be used as an important tool for the various phases of wastewater treatment. 5. Conclusion and recommendations Chlorella pyrenoidosa found to be strong algal strain for treatment of SHWW. Nutrient load present in wastewater helps in balancing bio-chemical profile with growth of algal cell. At optimization, 50% concentration was observed the best for biomass production (430 mgL-1) and removal of pollutant load (17-31%, 7-29%). This novel approach provides a sustainable solution for SHWW treatment because discharges of wastewater without treatment are always responsible for spread of pathogenic micro-organisms. Construction of on-site pits in combination with algal biomass near slaughterhouses able to solve the problem of untreated wastewater discharges for clean and commercial solution in favor of bio-economy. Acknowledgements We would like to thanks Department of Environmental Science, Babasaheb Bhimrao Ambedkar University to providing the instrumentation facilities for our research work. Har Mohan Singh is
17
grateful to MNRE (Govt. of India) for providing the Senior Research Fellowship in the programme of NRE Fellowship at SoEM, Shri Mata Vaishno Devi University, Katra (J &K). References 1. Ahmad, F., Khan, A. U., Yasar, A. 2013. The potential of Chlorella vulgaris for wastewater treatment and biodiesel production. Pak. J. Bot. 1, 461-465. 2. Ahmad, S., Kothari, R., Pathania, D. and Tyagi, V.V., 2019. Optimization of nutrients from wastewater using RSM for augmentation of Chlorella pyrenoidosa with enhanced lipid productivity, FAME content, and its quality assessment using fuel quality index. Biomass Conversion and Biorefinery, 1-18. 3. Ahmad, S., Pathak, V.V., Kothari, R., Kumar, A. and Krishna, S.B.N., 2018. Optimization of nutrient stress using C. pyrenoidosa for lipid and biodiesel production in integration with remediation in dairy industry wastewater using response surface methodology. 3 Biotech, 8(8), 326. 4. APHA, 20112 Standard Methods for the Examination of Water and Wastewater, twentysecond ed., American Public Health Association, New York. 5. Bustillo-Lecompte, C., Mehrvar, M. 2017. Slaughterhouse wastewater: treatment, management and resource recovery. Robina Farooq, Zaki Ahmad, (Eds.). Physico-chemical wastewater treatment and resource recovery. 153-174 6. Bustillo-Lecompte, C., Mehrvar, M., Quinones-Bolanos, E. 2016. Slaughterhouse wastewater characterization and treatment: an economic and public health necessity of the meat processing industry in Ontario, Canada. J. Geosci. Environ Protect. 04, 175. 7. Cataldo, D.A., Maroon, M., Schrader, L.E., Young, V.L. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. soil sci. plant anal. 1, 71-80. 8. Chew, K.W., Yap, J.Y., Show, P.L., Suan, N.H., Juan, J.C., Ling, T.C., Lee, D.J., Chang, J.S., 2017. Microalgae biorefinery: high value products perspectives. Bioresour. Technol. 229, 53–62. 9. Choi, H. J., 2016. Dairy wastewater treatment using microalgae for potential biodiesel application. Environ. Eng. Res. 4, 393-400 10. Dreywood, R. 1946. Qualitative test for carbohydrate material. Ind. Eng. Chem. 8, 499-499. 18
11. Emparan, Q., Harun, R., Danquah, M.K., 2019. Role of phycoremediation for nutrient removal from wastewaters: Appl. Ecol. Environ. Res.1, 889-915. 12. Gao, F., Li, C., Yang, Z.H., Zeng, G.M., Feng, L.J., Liu, J.Z., Liu, M. and Cai, H.W., 2016. Continuous
microalgae
cultivation
in
aquaculture
wastewater
by
a
membrane
photobioreactor for biomass production and nutrients removal. Ecolo. Engine. 92, 55-61. 13. Hena, S., Fatimah, S., Tabassum, S., 2015. Cultivation of algae consortium in a dairy farm wastewater for biodiesel production. Water. Resour. Indus. 10, 1-14. 14. Jais, N.M., Mohamed, R.M.S.R., Al-Gheethi, A.A., Hashim, M.A., 2017. The dual roles of phycoremediation of wet market wastewater for nutrients and heavy metals removal and microalgae biomass production. Clean. Technol. Environ. 1, 37-52. 15. Jais, N.M., Mohammed, R.M.S.R., Apandi, W.A.W.M, Peralta H.M.M. 2015. Removal of nutrients Removal of Nutrients and Selected Heavy Metals in Wet Market Wastewater by Using Microalgae Scenedesmus sp. Mech. Mater 773, 1210-1214. 16. Kitrungloadjanaporn, P., Sripongpun, G., Triampo, W., 2017. Nutrient removal from the effluent of swine slaughterhouse wastewater by Chlorella vulgaris TISTR 8580. Int. J. Adv. Agric. Environ. Eng. 4, 28-32. 17. Kothari, R., Pandey, A., Ahmad, S., Kumar, A., Pathak, V.V. and Tyagi, V.V., 2017. Microalgal cultivation for value-added products: a critical enviro-economical assessment. 3 Biotech, 7(4), 243. 18. Kothari, R., Pathak, V.V., Kumar, V., Singh, D.P., 2012. Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy waste water: an integrated approach for treatment and biofuel production. Bioresour. Technol. 116, 466-470. 19. Kothari, R., Prasad, R., Kumar, V., Singh, D.P. 2013. Production of biodiesel from microalgae
Chlamydomonas
polypyrenoideum
grown
on
dairy
industry
wastewater. Bioresour. Technol. 144, 499-503. 20. Kumar, V., Nanda, M., Joshi, H.C. Singh, A., Sharma, S., Verma, M., 2018. Production of biodiesel and bioethanol using algal biomass harvested from fresh water river. Renew. Energ. 116, 606-612. 21. Kundu, P., Debsarkar, A., & Mukherjee, S. 2013. Treatment of slaughter house wastewater in a sequencing batch reactor: performance evaluation and biodegradation kinetics. BioMed research international, 2013. 19
22. Lee, J., Lee, J., Shukla, S. K., Park, J., Lee, T. K. 2016. Effect of algal inoculation on COD and nitrogen removal, and indigenous bacterial dynamics in municipal wastewater. J. Microbiol. Biotechnol. 5, 900-8. 23. Liu, Z., Joo, J. C., Choi, S. H., Jang, N. H. J., Hur, J. W., 2018. Assessment of Surface Water Quality in Geum River Basin, Korea using Multivariate Statistical Techniques. Int. J. Appl. Eng. Res. 9, 6723-6732. 24. Liu, X., Chen, G., Tao, Y. and Wang, J., 2020. Application of effluent from WWTP in cultivation of four microalgae for nutrients removal and lipid production under the supply of CO2. Rene. Ener. 149, 708-715. 25. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 26. Lu, Q., Zhou, W., Min, M., Ma, X., Chandra, C., Doan, Y. T.& Chen, P 2015. Growing Chlorella sp. on meat processing wastewater for nutrient removal and biomass production. Bioresour. Technol, 198, 189-197. 27. Lu, Q., Zhou, W., Min, M., Ma, X., Chandra, C., Doan, Y.T., Ma, Y., Zheng, H., Cheng, S., Griffith, R. and Chen, P., 2015. Growing Chlorella sp. on meat processing wastewater for nutrient removal and biomass production. Bioresour. Technol. 198, 189-197. 28. Mackinney, G. 1941. Absorption of light by chlorophyll solutions. J. biol. Chem. 2, 315-322. 29. Maizatul, A. Y., Mohamed, R. M. S. R., Al-Gheethi, A. A., & Hashim, M. A. 2017 An overview of the utilisation of microalgae biomass derived from nutrient recycling of wet market wastewater and slaughterhouse wastewater. Int Aq Res 9(3), 177-193. 30. Murugesan, S., Venkatesh, P., Dhamotharan, R., 2010. Phycoremediation of poultry wastewater by micro alga. Biosci. Biotech. Res. Comm. 2, 14. 31. Myra, T., David, H., Judith, T., Marina, Y., Ricky, B.J., Reynaldo, E. 2015. Biological treatment of meat processing wastewater using anaerobic sequencing batch reactor (ASBR). Int. Res. J. Biol. Sci. 3, 66-75. 32. Pathak, V.V., Kothari, R., Chopra, A.K. and Singh, D.P., 2015. Experimental and kinetic studies for phycoremediation and dye removal by Chlorella pyrenoidosa from textile wastewater. J. of Environ Manag, 163,270-277. 33. Pathak, V.V., Singh, D.P., Kothari, R. and Chopra, A.K., 2014. Phycoremediation of textile wastewater by unicellular microalga Chlorella pyrenoidosa. Cell Mol Biol, 60(5)35-40. 20
34. Rasoul-Amini, S., Montazeri-Najafabady, N., Shaker, S., Safari, A., Kazemi, A., Mousavi, P., Ghasemi, Y., 2014. Removal of nitrogen and phosphorus from wastewater using microalgae free cells in bath culture system. Biocatal. Agric. Biotechnol. 2, 126-131. 35. Rath, B., 2012. Microalgal bioremediation: current practices and perspectives. J. Biochem. Technol. 3, 299-304. 36. Safafar, H., Uldall Norregaard, P., Ljubic, A., Moller, P., Løvstad Holdt, S. and Jacobsen, C., 2016. Enhancement of protein and pigment content in two Chlorella species cultivated on industrial process water. J. Mar. Sci Eng. 4, 84. 37. Salama, E.S., Kurade, M.B., Abou-Shanab, R.A., El-Dalatony, M.M., Yang, I.S., Min, B., Jeon, B.H., 2017. Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew. Sustain. Energ. Rev.79, 1189-1211. 38. Singh, H. M., Pathak, A. K., Chopra, K., Tyagi, V. V., Anand, S., & Kothari, R. 2019 Microbial fuel cells: a sustainable solution for bioelectricity generation and wastewater treatment. Biofuels, 10(1), 11-31. 39. Singh, H.M., Kothari, R., Gupta, R. and Tyagi, V.V., 2019. Bio-fixation of flue gas from thermal power plants with algal biomass: Overview and research perspectives. J Environ Manage, 245, 519-539 40. Sletten, O., Bach, C.M., 1961. Modified stannous chloride reagent for orthophosphate determination. JAM. Water. Works. Ass. 8, 1031-1033. 41. Tan X.B., Zhao X.C., Yang L.B., 2019. Strategies for enhanced biomass and lipid production by Chlorella pyrenoidosa culture in starch processing wastewater. J. Clean. Prod. 236, 117671 42. Taşkan E., 2016. Performance of mixed algae for treatment of slaughterhouse wastewater and microbial community analysis. Environ Sci Pollut Res 23:20474–20482 43. Venckus, P., Kostkeviciene, J., Bendikienė, V., 2017. Green algae Chlorella vulgaris cultivation in municipal wastewater and biomass composition. J. Environ. Eng. Landsc Manag. 1, 56-63. 44. Viegas, C.V., Hachemi, I., Maki-Arvela, P., Smeds, A., Aho, A., Freitas, S.P., Kumar, N., 2015. Algal products beyond lipids: Comprehensive characterization of different products in direct saponification of green alga Chlorella sp. Algal Res. 11, 156-164.
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45. Wang, L., Min, M., Li, Y., 2010. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Appl. Biochem. Biotechnol. 4, 1174-86. 46. Xu, X.Q., Wang, J.H., Zhang, T.Y., Dao, G.H., Wu, G.X. and Hu, H.Y., 2017. Attached microalgae cultivation and nutrients removal in a novel capillary-driven photo-biofilm reactor. Alga. Rese. 27, 198-205. 47. Yaakob, M. A., Mohamed, R. M. S. R., Al-Gheethi, A. A. S., & Kassim, A. H. M 2018 Characteristics of chicken slaughterhouse wastewater. Chem Engg Transac, 63, 637-642. 48. Zhao, X.C., Tan, X.B., Yang, L.B., Liao, J.Y. and Li, X.Y., 2019. Cultivation of Chlorella pyrenoidosa in anaerobic wastewater: The coupled effects of ammonium, temperature and pH conditions on lipids compositions. Bioresour. Technol. 284, 90-97.
To, The Editor-in-Chief, Bioresource Technology. Subject: Credit Author Statement I have submitted that this final manuscript is sharing with all authors and all authors provides its suggestions to improve this manuscript. As I, Dr. Richa Kothari (corresponding Author) is responsible for ensuring that the descriptions are accurate and agreed by all authors. Conflict of Interest: None Thanks Dr. Richa Kothari 22
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Rifat Azam Richa Kothari Har Mohan Singh Shamshad Ahmad V. Ashok Kumar V. V. Tyagi
Graphical Abstract
23
HIGHLIGHTS
Treatment of slaughterhouse wastewater (SHWW) with Chlorella pyrenoidosa
Maximized growth observed with 50% concentration in 430 mgL-1 of biomass (dry wt.)
COD, NO3- and PO4-3 reduction ≈17-31%, 23-42%, 18-48% with 50% concentration of SHWW
Correlation analysis investigation in between parameters of SHWW and algal strain
SHWW treatment with Chlorella supports pollutant reduction and biochemical profiles
24
List of Figure Figure 1: Growth optimization of algae on slaughterhouse wastewater Figure 2: Percent reduction in pollutant load of slaughterhouse wastewater with selected concentrations. Figure 3: Biochemical profile of slaughterhouse wastewater with 50% concentration Figure 4: Correlation analysis for physico-chemical parameters of SHWW and biochemical profile of C. pyrenoidosa with selected concentrations.
25
Biomass (mg L⁻¹)
500 450 400 350 300 250 200 150 100 50 0
25%.
0
2
4
50%.
6
75%.
8 10 Days
100%.
12
14
16
control
18
Figure 1: Growth optimization of algae on slaughterhouse wastewater
26
20
Percentage Reduction (%)
120 100 80
25%
60
50%
40
75% 100%
20 0 BOD
COD Nitrate Parameters
Phosphate
Figure 2: Percent reduction in pollutant load of slaughterhouse wastewater with selected concentrations.
27
Concentration (mg L-1)
Chlorophyll
Protein
Carbohydrate
70 60 50 40 30 20 10 0 0
5
10
15
Days Figure 3: Biochemical profile of slaughterhouse wastewater with 50% concentration
28
Figure 4: Correlation analysis for physico-chemical parameters of SHWW and biochemical profile of C. pyrenoidosa with selected concentrations
List of Table Table 1.Worldwide standards limits for the discharges of slaughterhouse wastewater (Bustillo-Lecompte and Mehrvar, 2017) Table 2. Pollutant reduction at 0th day to 15th day by using Chlorella pyrenoidosa with 50% concentration of wastewater Table 3. Comparative analysis of biochemical profile of algae with various types of wastewater with current study
29
Table 1: Worldwide standards limits for the discharges of slaughterhouse wastewater (Bustillo-Lecompte and Mehrvar, 2017) S.NO Parameters Present study World EU US Canadian Australian Indian Standards Standards Standards Standards Standards Bank Standards 1
pH
10.16±0.32
6-9
NA
6-9
6-9
5-9
5-9
2
Color
Dark red
-
-
-
-
-
-
3
Odor
Offensive smell
-
-
-
-
-
-
4
TDS
774.66±70.39
-
-
-
-
-
-
5
COD
3429.33±61.04
125
125
NA
NA
3×BOD
250
6
BOD
1798.33±14.01
30
25
26
5-30
10-15
100
30
7
NO3-
331.66±7.50
10
10
8
1
0.1-15
10-50
8
PO4-3
34.13±1.32
6-9
NA
6-9
6-9
5-9
5-9
Table 2. Pollutant reduction at 0th day to 15th day by using Chlorella pyrenoidosa with 50% concentration of wastewater Time period
Parameters Color
0th day
Reddish
pH
10.2
Odor
Offensive meat smell
TDS (mg L-1)
Nitrate
Phosphate
BOD
COD
(mg L-1)
(mg L-1)
(mg L-1)
(mg L-1)
398
172.3
16.3
900
1846.6
31
Percent reduction (%)
-
-
-
-
-
-
-
5th day
9.9
Offensive meat smell
325
133.3
13.4
839
1528.3
Percent reduction (%)
-
-
18.3
22.6
17.7
6.7
17.2
10th day
9.6
Offensive meat smell
211
84
8.6
778
1243.3
35.1
36.9
35.8
7.3
18.7
148
48
4.4
547.6
855.6
29.9
42.8
48.8
29.6
31.2
Light reddish green
Light reddish green
Percent reduction (%)
-
15th day
9.3
Green
Percent reduction (%)
-
Offensive meat smell
32
Table 3. Comparative analysis of biochemical profile of algae with various types of wastewater with current study Wastewater
Algal Strain
Biochemical Parameters Carbohydrate
Protein
(%)
(%)
27
51.58
Meat wastewater (CUT)
Chlorella sp.
Industrial process water
Chlorella pyrenoidosa
NA
65.2
Industrial process water
Chlorella vulgaris
NA
Artificial anaerobic effluents
Chlorella pyrenoidosa
Starch processing wastewater
Chlorella
Slaughter house wastewater
Chlorella
Chlorophyll
Culture
References
Period (Days) 9
Lu et al., 2015
5.41 mg g-1 DW
16
Safafar et al., 2016
55.1
32.44 mg g-1 DW
16
Safafar et al., 2016
25.7
63.7
3.7 %
15
Zhao et al., 2019
25.4
62.3
3.9 %
12
Tan et al., 2019
9.58%
12.37
15.31 µg mL-1
15
Present study
(including nucleic acid)
pyrenoidosa
pyrenoidosa
33
S0960-8524(20)30387-4 https://doi.org/10.1016/j.biortech.2020.123116 BITE 123116
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
7 January 2020 25 February 2020 1 March 2020
Please cite this article as: Azam, R., Kothari, R., Mohan Singh, H., Ahmad, S., Ashok Kumar, V., Tyagi, V.V., Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.123116
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Production of algal biomass for its biochemical profile using slaughterhouse wastewater for treatment under axenic conditions Rifat Azama, Richa Kotharia,b,*, Har Mohan Singhc, Shamshad Ahmada, V. Ashok Kumard, V. V. Tyagic aBioenergy
and Wastewater Treatment Laboratory, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow (U.P.), India
bDepartment cSchool
of Environmental Sciences, Central University of Jammu, Jammu, India
of Energy Management, Shri Mata Vaishno Devi University, Katra (J&K) India
dDepartment
of Chemical Technology, Chulaloangkorn University, Bangkok, Thailand
Abstract
Slaughterhouse produce large amount of wastewater, containing high pollutant load in terms of protein, fats and meat pieces, might lead to source of non-point contamination. Various concentrations (25%, 50%, 75%, and 100%) of slaughterhouse wastewater were used to increase the algal biomass production, pollutants removal and biochemical profile analysis under controlled conditions of C. pyrenoidosa. Results showed that the maximum biomass yield 430 mg L-1 was achieved at 50% concentration of wastewater to other concentration of wastewater. Direct relation was observed in between pollution load and nutrient load of SHWW with biochemical profile of C. pyrenoidosa. The COD/BOD ratio (1.9) was found to be significant on the scale of degradability by algal biomass. Sufficient nutrient removal efficiencies (23-42%, 1848%) and pollutant load efficiencies (17-31%, 7-29%) were observed. Findings showed that slaughterhouse wastewater is rich in nutrients, which can be utilized for algal biomass production and wastewater remediation for future endeavors. Keywords: Chlorella pyrenoidosa, Slaughterhouse wastewater, Nutrient removal, Biomass production * Corresponding Author (Dr. Richa Kothari) Email: - [email protected] 1
1. Introduction
The slaughter industry is one of the largest industries, which have economic importance for any country under the category of livestock sector. Slaughterhouses are the part of meat processing industries, but during the processing of meat a large amount of water is used mainly for cleaning and washing purposes. Among the various meat-producing countries, India is the fifth largest exporter of meat at international level (Kumar et al., 2018). So far, no reports are available on the use of discharged water estimation neither at global level nor at national level. It might be due to the non-point source at commercial level means very small to very large, based on quantity or animals (small/large) slaughtered for meat on daily basis. In Indian context very few data are available on the use of water foot-printing in slaughterhouses, but authorized information in the form of referred published data are not available. Although, physio-chemical characterization of slaughterhouse wastewater was reported by various researchers in peerreviewed articles. Research studies show that slaughter industry is the most significant contributors to water pollution, because it consists of high concentrations of contaminants like fats, suspended solids, chemical oxygen demand, chlorides and nitrates. Jais et al., (2015) has also reported that slaughterhouse wastewater is also rich in nitrogen and phosphorous. Besides, slaughterhouse wastewater pH was found in the range of 6.5 to 7.6 with high turbidity, red in color and offensive smell. These high concentrations of nutrients present in the slaughterhouse wastewater makes a favorable condition for algal growth and biofuel production (Kitrungloadjanaporn et al., 2017; Kothari et al., 2017). However, wastewater discharges without treatment to the surroundings largely contribute to the eutrophication. Earlier studies showed that microalgae have the capability of treating various types of wastewater, which includes dairy, domestic, municipal and textile wastewater and alternatively produce high biomass by utilizing their organic matter as a nutrient for growth (Ahmad et al., 2018; Maizatul et al., 2017; Kothari 2
et al., 2013). On the other hand, if same concept is developed with controlled/known algal strains to treat the slaughterhouse wastewater, it may provide new insight into the era of both water crisis and energy crisis. Since different types of phytoremediation approach have the capability to treat different types of wastewater more efficiently in comparison to other conventional methods like electro-coagulation, chemical coagulation, bed sequencing batch reactor, anaerobic treatments (UASB), etc. Nevertheless, all these methods are costly and need centralized systems for the treatment of wastewater at one place, and this could be the major challenge with these existing technologies. Therefore, to reduce the load of untreated wastewater from slaughterhouses in particular, decentralized type systems with a phycoremediation approach might be the best alternative and effective way for removal of nutrients as compared to other existing conventional methods (Emparen et al., 2018, Kothari et al., 2010).
Hence, the present study explores to assess the impact of variation in the concentration of slaughterhouse wastewater for the growth of C. pyrenoidosa for high biomass production. Pollutant removal through algal strain with an integrated approach of biomass production showed the benefits for the biomass growth using wastewater as a resource media (Ahmad et al., 2019; Salama et al., 2017). Different compositions of wastewater (dairy, textile, poultry, pond, municipal etc.) are treated with algal biomass by absorption of nutrient rich organic pollutants and their transformation into biomass which can be used to produce biodiesel by transesterification process as reported by the researchers with different microalgae species such as C. vulgaris, C. pyrenoidosa, C. sorokiniana, Chlamydomonas (Kothari et al., 2013; Ahmad et al., 2013; Venckus et al., 2017). C. vulgaris and Scenedesmus obliquus were utilized for biomass production and nutrients removal from wastewater in membrane photobioreactor (Gao et al.,
3
2016). Capillary-driven photobioreactor also used for dual application of biomass production as well as nutrients removal of microalgae and this cultivation method shows higher light utilization efficiency (Xu et al., 2017). Liu et al., (2020) used for microalgae for wastewater treatment in addition with CO2 supply in vertical column photobioreactor.
To the best of our knowledge, so far no reports concerning the use of microalga C. pyrenoidosa for the treatment of slaughterhouse wastewater. Therefore, the objectives of the present study are focused on a novel approach for bioremediation because Chlorella species have a potential to remediate urban wastewater (Rasoul-Amini et al., 2014), poultry wastewater (Murugesan et al., 2010) and in our prior studies with dairy wastewater, textile wastewater (Pathak et al., 2014; Ahmad et al., 2018) for nitrate (57-79%), phosphate (84-87%), COD (43-79%), BOD (78-81%). That is the particular reason to investigate the potential of Chlorella pyrenoidosa in coupling with slaughterhouse wastewater for remediation of nutrient and pollution load as an objective in this study. Presently, the algal biomass production at commercial level using the existing freshwater resources which is not sustainable due to its demand for drinking and other purposes and available on high cost. It is also an alarming condition for environmental sustainable part and contributing as one the factor in capital production cost. Thus, the utilization of wastewater as an alternative source for freshwater and to reduce the cost in dual way i.e. as a medium for biomass and providing low-cost source nutrients from those instead of high-cost chemicals, decrease the production cost may break the limitations with its wide applications. 2. Materials and methods To study the objectives of this research study, this section is further divided into subsections such as: (i) Physio-chemical characterization of slaughterhouse wastewater: to know the real factual 4
composition of selected slaughterhouse wastewater; (ii) Algal (Chlorella pyrenoidosa) growth optimization with different concentration and analysis of biochemical profile of algae; (iii) Utilization of C. pyrenoidosa for removal of pollutant load and nutrient load in slaughterhouse wastewater. Correlation analysis investigation in between pollution load and biochemical profile of selected algae for possible outcomes in terms of value-added products also part of the objective. 2.1. Physical and chemical characterization of slaughterhouse wastewater Slaughtered wastewater was collected from the local market of Aminabad, Lucknow (UttarPradesh) India, which has the capacity to generate gallons of wastewater per day. Furthermore, approximate 30-35 cattle and 200-250 poultry (goat/sheep/buffalo/pig) cut per day, as per the information from local shopkeepers. The sample of slaughterhouse wastewater was collected and stored at 4 ˚C in sterilized water sampling bottles for analysis of quality. Four different concentrations of slaughterhouse wastewater were selected to study the possible impacts on algal growth, i.e. 25%, 50%, 75%, 100% and BG-11 media is taken as control for algal growth. In this study, distilled water media is used to maintain the concentration of wastewater as per our objectives in study of slaughterhouse wastewater. The physico-chemical parameters were assessed for study of color, odor, temperature, total dissolve solids (TDS), pH, BOD and COD by standard analytical methods of water and wastewater APHA, (2012). In slaughterhouse wastewater, the initial pH was analyzed by using a portable digital pH meter Model No -009 (1) A. The sample color was red which was seen by naked eyes. COD was analyzed by using titration method and BOD was determined after 5 days of incubation at 20 ºC by using incubator and then titrate the sample. The NO3- was estimated by (Cataldo et al., 1975) and PO43estimation by stannous chloride method (Sletten and Bach, 1961). All the parameters were 5
investigated in triplicate. The NO3- and PO43- was analyzed by using UV-Vis spectrophotometer (Systronics 2203). The physico-chemical parameters of slaughterhouse wastewater are shown in Table 1 with discharge limits prescribed by World Health Organization (WHO), European Union (EU), United States (US), Canadian and Australian standards. Prior to microalgal cell inoculation in slaughterhouse wastewater, pretreatment step is just followed by sieving after collection to remove hair and other fleshy materials etc. and autoclaving (for 20 minutes at 15 Psi and 121℃) before starting the experimental investigations. 2.2. Algal species For this study, microalgal organism Chlorella pyrenoidosa (NCIM 2738) collected from resource centre, National Collection of Industrial Microorganism (NCIM), Pune (Maharashtra), India. Chlorella sp. is selected to complete the objectives of the present research, with C. pyrenoidosa strain. BG-11 medium are used to maintain the culturing conditions with composition i.e. NaNO3,1.5 g L-1; K2HPO4,0.04 g L-1; MgSO4·7H2O, 0.075 g L-1; CaCl2·2H2O, 0.036 g L-1; citric acid, 0.006 g L-1; ferric ammonium citrate, 0.006 g L-1; EDTA (disodium salt), 0.001 g L-1; Na2CO3, 0.02 g L-1; 1 mL trace elements solution (in g L−1: H3BO3, 2.86; MnCl2·4H2O,1.81; ZnSO4·7H2O, 0.222; NaMoO4·2H2O 0.39; CuSO4·5H2O,0.079; Co (NO3)2·6H2O,0.0494) with pH 7.0 ± 2 as suggested by NCIM. The media was sterilized by autoclaving for 20 minutes at 15 Psi and 121℃. The glassware used for experiment, all clean and sterilized at 120℃ for 6-8 h before use. The medium was then inoculated with C. pyrenoidosa and incubated at 30±1℃ in a 12h light (10 Wm-2)/dark cycle.
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2.2.1. Biochemical characterization The biochemical characterization of selected algal biomass for carbohydrate, protein, and chlorophyll were estimated by standard methods. The carbohydrate is estimated by Anthrone method (Dreywood, 1946) at an absorbance of 620 nm. The Lowry method is used for the estimation of protein at wavelength of 660 nm (Lowry et al., 1951). The chlorophyll is estimated by methanol method for the chlorophyll content in the cells (Mackinney, 1941) with absorbance at wavelengths, which consist of 750 nm, 666 nm, 475 nm and 653 nm by spectrophotometer. Biomass concentration was determined by using UV-Vis spectrophotometer (Model no. Systronics 2203) of the culture absorbance at a wavelength of 680 nm. 2.2.2. Optimization of algal growth on slaughterhouse wastewater The algal strain of C. pyrenoidosa was grown at different concentrations (25%, 50%, 75%, and 100%) using slaughterhouse wastewater. The experiment was started in 500 mL of the Erlenmeyer flask and incubated at 30±1ºC in a 12h light (10 Wm-2)/dark cycle. The algal strain was used for the inoculation (10 mL) of each flask. The exponential phase of algal species was determined with the help of optical density at 680 nm on alternate day by using double beam spectrophotometer (Model no. Systronics 2203). For the calibration curve observation, initial O.D. of 0.1 cells were dried at 60 °C for 24 hours and the regression equation obtained was y=298.23x-3.8 (R2=0.98), where y is the dry cell weight (mg L-1) and x is the absorbance at 680 nm. 2.3. Nutrient removal from slaughterhouse wastewater using Chlorella pyrenoidosa The nutrient removal investigation was carried out in 2 L conical flask, with working volume of 1 L of different concentrations (25%, 50%, 75%, and 100%) of using slaughterhouse wastewater. 7
The 10 mL C. pyrenoidosa suspension was inoculated in each flask with the above condition given in section 2.2. The quality of biomass was analyzed on 0th, 5th, 10th, and 15th day of inoculation for the reduction of pollutant load in slaughterhouse wastewater. Samples were centrifuged at 5000 rpm for 10 minutes at 4˚C and subsequently 0.45 mm filter paper used to filtrate. The filtrate was used to analyze BOD, COD, NO3-, and PO43- . The nutrient load was measured by using Eq. (1) after removal efficiency of pollutant load (APHA, 2012): Removal efficiency (%) = (Spo – Spt/Δt) ×100
(1)
Where, Spo (mg L-1) and Spt (mg L-1) are initial and final concentrations BOD, COD, NO3- and PO43- at time period of Δt. 2.4. Kinetics of algal growth For the measurement of microalgal growth kinetics, the specific growth rate was observed by using Eq. (2) as follow: µ= In (Po- Pt )/(t1-t0)
(2)
Where, µ is the specific growth rate (mg L-1 day-1) of C. pyrenoidosa. The graph Pad Prism Software v6 (student version) used to calculate the growth rate by applying the exponential Eq. (3) Pt=PO exp (r×t) is
(3)
Eqs. (2 and 3), Po and Pt are the size of the population at initial and final at time t and r is the growth rate of population (Van et al., 2015). 2.5. Correlation analysis Correlation analysis conducted between physico-chemical parameters of slaughterhouse wastewater and biochemical parameters of algal strain with help of Metabo Analyst, Statistical Analysis software (student version).
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3. Results and Discussions In this experimental study, slaughterhouse wastewater was treated using the microalga C. pyrenoidosa and produced algal biomass. The growth and algal biomass production was optimized with various concentrations of wastewater, and this experiment was conducted for a period of 15 days. During the growth period, the biochemical parameters were assessed at every 5 days interval, in terms of optical density (680 nm) with organic load (COD, BOD), nutrient load (nitrate and phosphate), reduction of pollution load, and nutrient load. Besides, the treated wastewater was analyzed thoroughly before it release to the surroundings. Co-relational analysis between physico-chemical and biochemical in slaughterhouse wastewater of algal biomass is also studied. 3.1. Slaughterhouse wastewater characteristics before treatment Slaughterhouse wastewater looks reddish color with alkaline pH (10.16), and offensive smell. The BOD and COD were found to be a higher range in comparison to discharge limits by Indian standards. Whereas, TDS range for discharge of slaughterhouse wastewater was not prescribed by anyone of the standard limits as given in the Table 1. Similarly, COD (3429 mg L-1) is also more than 10 times Indian standard (250 mg L-1), same trend was observed with BOD (1798 mg L-1) of slaughterhouse wastewater before treatment. The concentration of nitrate (331 mg L-1) was found to be six times greater than the approved limit (50 mg L-1). Similarly, the phosphate concentration was found to be again on higher side (34 mg L-1) in comparison to permissible range (5-9 mg L-1). According to literature, inorganic and organic forms of nitrogen present in the wastewater and it includes ammonia, nitrite and nitrate respectively. However natural decaying process of organic matter such as blood traces, fats, proteins etc. in case of slaughterhouse wastewater produced nitrates which is most stable form of nitrogen in water.
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Algal bloom is the harmful consequence of excessive nitrates level in the water bodies on the earth (Al-Gheeti et al., 2015). Yaakob et al., (2018) reported that most common form of orthophosphate (PO43-), which comes from use of soap and detergent products. Thus, it can be seen that high concentrations of nitrate and phosphate in wastewater are the main cause of eutrophication. Table 1 show the results obtained after treatment in favor of slaughterhouse wastewater discharge limits and compared with standards of permissible limits. The reason for high values of BOD and COD may also be due to presence of blood residues, fats, proteins, and fibers present in wastewater. The slaughter produces from the washing process of meat (Jaish et al., 2017). These types of wastewater have high level of nitrogen, phosphorus, COD and BOD (Maizatul et al., 2017). Most of the organic content of wastewater is available in the form of COD. So, the higher concentration of COD manifests the various chemical reactions among organic and inorganic substances of slaughterhouse wastewater. On the other side, BOD indicates the presence of biochemical oxidation of organic compounds available in complex forms of selected wastewater. Hence, the higher concentration of BOD which is directly proportional to higher microbial load in the slaughterhouse wastewater. As per the findings of analysis of selected wastewater without any treatment, exceeding the allowable level for all parameters. Similar results for slaughterhouse wastewater composition also found with successive experimental treatment by anaerobic baffle reactor based aerobic activated sludge reactor and UV/H2O2 photoreactor (Bustillo-lecompte et al., 2016). Therefore, COD/BOD ratio, indicator for degradable scale of wastewater also assessed by the initial parameters. According to review of literature, its value must be below 10 (Myra et al., 2015). While in the present study, it is 1.9, indicating that the compounds in the wastewater are relatively degradable and thus the algae can utilize the nutrient for their growth. Hence, the untreated wastewater discharge into the
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water bodies causes health problems and also pollutes the environment and causes eutrophication. Therefore, selecting the algal strain which have high potential for the treatment of slaughterhouse wastewater with optimization of concentration, observations and findings are discussed in detail in next section. 3.2. Algal growth and nutrient removal 3.2.1. Algal growth The growth condition was monitored using various concentrations of slaughterhouse wastewater, and it was assessed by optical density (680 nm). The growth of C. pyrenoidosa was optimized in the BG-11 growth medium (distilled water) as well as in slaughterhouse wastewater. During the period of cultivation, the exponential phase was observed between 5th – 9th days. Figure 1 shows that the algal growth increased with increasing concentration of wastewater from 25% to 75% and same trend observed with 100% concentration of slaughterhouse wastewater. In log and lag phase the trend of algal growth was found similar in all the four concentrations. Whereas, in stationary phase the algal growth was significantly found lower in the concentrations that are 25%, 75%, and 100% compared to 50% concentration. Although the survival rate of selected algal strain is observed good in all the concentrations of wastewater, however, 50% concentration found to be the best for the growth of algae (Figure 1). It may be due to existence of optimized values of nutrients (nitrate and phosphate) availability for algal biomass. There are two main limiting factors for microalgae growth which are nitrogen and phosphorus (Singh et al., 2019; Kothari et al., 2013; Kothari et al., 2012), as suggested by various researchers. In the absence of slaughterhouse wastewater i.e. in control 1 (only distilled water) the algae did not show any significant improvement with growth parameters.
The results indicate that the
algal growth without slaughterhouse wastewater did not support for the high biomass production
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is because of lack of nutrients. But in the controlled condition i.e. with BG-11 media (control 2), wastewater dependent growth of algae was also compared with BG-11 growth medium and delineated in Figure 1. The growth of algal biomass at 100% concentration was observed slow, and this may be due to higher level of contaminants. Thus, it inhibit the growth and presence of some nutrients in slaughterhouse wastewater, which are not used by C. pyrenoidosa efficiently. However, the growth of algae was stimulated and continued up to 12th day of treatment, very much noticed with 50% concentration of wastewater may be due to the availability of nutrients in optimized range required for growth, whereas, 25% concentration did not support the algal growth compared to control 2, that maybe due to low level of nutrients in wastewater. Therefore, this study found that 50% concentration of slaughterhouse wastewater could be the ideal condition for achieving highest biomass production using C. pyrenoidosa. 3.2.2. Nutrient removal Phyco-remediation is an advance stream of science using algal species for wastewater treatment and ultimately decreases the nutrient levels in the wastewater. After the optimization of algal growth with a 50% concentration of slaughterhouse wastewater, is selected for the study of pollutant removal rate. Microalgae prefer to orthophosphate (PO43-) for their food-material, which is easy to binds the ions for microalgae growth. The initial value of pH, TDS, COD, BOD, including nitrate and phosphate were observed and found to be in the higher amount than the allowable limit, as shown in Table 2. After inoculation of algal cells, the pollution load is measured in 0th day, 5th day, 10thday and 15th day of the treatment. Analysis of physicochemical parameters for 5th day of observation indicate increase in reduction level of TDS, COD, BOD, nitrate and phosphate concentration from 325 mg L-1 (18%), 1528 mg L-1 (17%), 839 mg L-1 (7%), 133 mg L-1 (22%), and 13.4 mg L-1 (17%) respectively as compared to the initial value
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obtained from inoculated wastewater (Figure 2). Furthermore, 10th day of experimental investigation for TDS, COD, BOD, nitrate and phosphate concentration were found in the ranges as 211 mg L-1 (35%), 1243 mg L-1 (18%), 778 mg L-1 (7%), 84 mg L-1 (36%), and 8.6 mg L-1 (35%) respectively. The algal cells absorbed the nutrients from wastewater for their growth, and increase the level of algal biomass, obtained result indicates that the nutrient load is decreasing in the selected concentration of wastewater. However, same trend was observed in the reduction of pollutant load up to 15th day of experiment. So, all the analysis shows that the algal-based wastewater treatment is a valuable alternative method for the remediation of pollutants from slaughterhouse wastewater. Because the algae feed, the nutrient from wastewater and result indicates that the level of pollution decreases. Although, many researchers find that microalgae can efficiently cultivate in nitrate and phosphate rich nutrient wastewater environment (Kothari et al., 2012; Wang et al., 2010) for various types of industrial effluents/influents, but the treatment of slaughterhouse wastewater with C. pyrenoidosa make this approach novel and ecofriendly to treat the wastewater with low-cost investment. Slaughterhouse wastewater discharges are harmful to the environment because nutrient level (nitrate and phosphate) is very high (Bustillo-Lecompte et al., 2016). Algal growth plays an important role in the treatment of such type of wastewater. During algal growth cycle, low-cost nutrients were used by the algal biomass which is reduced the pollution load in slaughterhouse wastewater. On the basis of nutrient, it has been reported that the slaughterhouse wastewater possesses a high amount of nitrogen and phosphorous (Jaish et al., 2015). From this study, it is evident that the use of algal biomass for treatment of slaughterhouse wastewater is viable alternative in the current water and energy crisis scenario because produced biomass has a potential for biofuel applications also as reported (Kumar et al., 2018; Kitrungloadjanaporn et al., 2017).
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3.4. Biochemical profile of algae grown in slaughterhouse wastewater Biochemical profiles (chlorophyll, protein, and carbohydrate) of microalgal biomass are the source for the production of bio-based high quality products. Along with light and temperature, nutrients are the essential factors for biochemical compounds and biomass growth and development. Only 50%, concentration of wastewater was selected which used for the changes in chlorophyll, protein and carbohydrate along with growth dynamics monitored during the uptake of nutrients from wastewater. On the basis of growth, 50% concentration is best for the production of biomass (430 mg L-1 on dry wt. basis) in slaughterhouse wastewater. For the analysis of chlorophyll the concentration increases from 0th day to 15th days of the experiment are 0.31µg mL-1, 2.94µg mL-1, 9.50 µg mL-1 and 15.31µg mL-1 respectively. Therefore, it can be noted that the concentration of protein in slaughterhouse wastewater found to be increased from 0th to 15th days are 10 mg L-1 (2.32%), 32mg L-1 (7.44%), 42.4mg L-1 (9.86%), and 53.2 mg L-1 (12.37%). Thus, percentage of protein in dry microalgal biomass was analyzed to increase from 2.32% to 12.37% during the culture period with dry biomass (Figure 3). However, after inoculation, the carbohydrate content in slaughterhouse wastewater was measured to uninterruptedly increase up to 15th day of experiment 10.2 mg L-1 (2.37%), 22.3 mg L-1 (5.18%), 36.4 mg L-1 (8.46%) and 41.2 mg L-1 (9.58%). The present experimental investigation results clearly shown as the potential for algal biomass production with favorable biochemical profile with slaughterhouse wastewater at 50% concentration. Table 3 indicates the potential of Chlorella sp. with other wastewaters also but on comparison with slaughterhouse wastewater for biochemical profile of C. pyrenoidosa not as much high in meat wastewater (CUT), (Lu et al., 2015). It has been observed that the algae consume nitrate as nitrogen source and initial concentration of nitrogen pose a significant
14
influence over bio-chemical profile of algal biomass (C. pyrenoidosa). Here, the initial concentration of nitrogen (as nitrate) in wastewater is high (172 mgL-1) and dilution of wastewater reduces its concentration. Therefore, higher concentration of nitrogen leads to produce higher amount of protein and increasing dilution level of wastewater reduces the concentration of nitrate which may be a reason of low accumulation of protein in present study. With 50 % concentration of slaughterhouse wastewater, only 42.8 % nitrate removed by the algae on 15th day, which responsible for accumulation of protein (12.3%) in the algal cell as observed in this study. Whereas, Lu et al., (2015) also reported that biomass yield of Chlorella sp. (UN5161) was very low with different processing steps in meat processing wastewater. It may be due to the low profile value of protein, which is important building block for growth of any algal cell, intake mechanism of growth nutrients also inhibited by the presence of toxic substances in the wastewater. Hence, growth rate and percentage of protein content in C. pyrenoidosa directly dependent on the initial concentration of nitrogen source in the medium/wastewater, this observation also supported by the Venckus et al., (2017) in his study with Chlorella vulgaris grown in municipal wastewater. As per present findings, dry weight of algal biomass is 430 mg L-1 (with 50%) concentration of slaughterhouse wastewater. Approximately, similar results were reported by Wang et al., (2010), Taskan, (2016) for Chlorella sp. and mixed consortium of algae (18 species), respectively, with high nutrient-rich wastewater. So, our findings are best on the ground of algal biomass productivity with half-percentage concentration of wastewater and use of single strain only. Similarly, Taskan, (2016) was taken initial cell concentration of 0.2 g L-l but in present study experiment proceed with 0.1 g L-l concentration of cell at initial level. Hence, biomass productivity and biochemical profile are significant with our studies in references to treatment of
15
wastewater, which may be responsible for unhygienic and unhealthy conditions in surrounding, if discharged without any treatment. Use of photobioreator systems for treatment of wastewater by many researchers for algal growth in their lab studies but they are costly and energy-intensive. Further, lab-scale phycoremediation strategies for slaughterhouse wastewater with present approach also have similar applicability on the part of final findings but energy intensive in approaches. 4. Correlational analysis of slaughterhouse wastewater The correlational analysis of slaughterhouse wastewater was comprised of physicochemical and biological parameters of slaughterhouse wastewater. In the present study, the correlation between the different concentrations of slaughterhouse wastewater parameters, which consist of 25%, 50%, 75%, and 100%. In this section the co-relation between two variables indicates the high differences among them. The correlation coefficient analysis of parameters was presented in the given Figure 4. The highest positive correlation was observed in 25% and 50% concentration of slaughterhouse wastewater of TDS, BOD, COD, NO3- and PO43- between the chlorophyll, protein, and carbohydrate are highly correlated which is also present in the Figure 4. In 75% and 100% concentration, the correlation of TDS, BOD, COD,
NO3- and PO43- between the
chlorophyll, protein, and carbohydrate was not found highly correlated, because, due to the concentration of wastewater decrease. According to Lu et al., (2015) nutrient removal (NO3-, PO4-3 COD and BOD) efficiency positively correlated with biomass yield. In case of 75% the chlorophyll, protein, and carbohydrate are not positively correlated to BOD, COD, NO3- and PO43- except TDS. The same results were found in 100% concentration. Because slaughterhouse wastewater ratio of COD: BOD was 4:1, which is relatively high, direct affect the biomass yield and increase the time for degradation. High BOD and COD will lead to occurrence of
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eutrophication, which can be toxic to aquatic organisms, destruct organism nursery grounds, and extinction of certain species (Kundu et al., 2013; Yaakob et al., 2018). The best positive correlation was found with the 25% and 50% concentration of wastewater parameters for protein, carbohydrate and Chlorella pyrenoidosa. Similarly, a strong correlation reported with case of municipal wastewater between the nitrogen and phosphorous and in the context of protein, carbohydrates in the biomass (Venckus et al., 2017). Thus, the analysis and finding of the coefficient assist in rapid analysis in wastewater treatment and management. The significant correlation between the algal biomass and the nutrients indicates the importance of microalgal communities, and can be used as an important tool for the various phases of wastewater treatment. 5. Conclusion and recommendations Chlorella pyrenoidosa found to be strong algal strain for treatment of SHWW. Nutrient load present in wastewater helps in balancing bio-chemical profile with growth of algal cell. At optimization, 50% concentration was observed the best for biomass production (430 mgL-1) and removal of pollutant load (17-31%, 7-29%). This novel approach provides a sustainable solution for SHWW treatment because discharges of wastewater without treatment are always responsible for spread of pathogenic micro-organisms. Construction of on-site pits in combination with algal biomass near slaughterhouses able to solve the problem of untreated wastewater discharges for clean and commercial solution in favor of bio-economy. Acknowledgements We would like to thanks Department of Environmental Science, Babasaheb Bhimrao Ambedkar University to providing the instrumentation facilities for our research work. Har Mohan Singh is
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grateful to MNRE (Govt. of India) for providing the Senior Research Fellowship in the programme of NRE Fellowship at SoEM, Shri Mata Vaishno Devi University, Katra (J &K). References 1. Ahmad, F., Khan, A. U., Yasar, A. 2013. The potential of Chlorella vulgaris for wastewater treatment and biodiesel production. Pak. J. Bot. 1, 461-465. 2. Ahmad, S., Kothari, R., Pathania, D. and Tyagi, V.V., 2019. Optimization of nutrients from wastewater using RSM for augmentation of Chlorella pyrenoidosa with enhanced lipid productivity, FAME content, and its quality assessment using fuel quality index. Biomass Conversion and Biorefinery, 1-18. 3. Ahmad, S., Pathak, V.V., Kothari, R., Kumar, A. and Krishna, S.B.N., 2018. Optimization of nutrient stress using C. pyrenoidosa for lipid and biodiesel production in integration with remediation in dairy industry wastewater using response surface methodology. 3 Biotech, 8(8), 326. 4. APHA, 20112 Standard Methods for the Examination of Water and Wastewater, twentysecond ed., American Public Health Association, New York. 5. Bustillo-Lecompte, C., Mehrvar, M. 2017. Slaughterhouse wastewater: treatment, management and resource recovery. Robina Farooq, Zaki Ahmad, (Eds.). Physico-chemical wastewater treatment and resource recovery. 153-174 6. Bustillo-Lecompte, C., Mehrvar, M., Quinones-Bolanos, E. 2016. Slaughterhouse wastewater characterization and treatment: an economic and public health necessity of the meat processing industry in Ontario, Canada. J. Geosci. Environ Protect. 04, 175. 7. Cataldo, D.A., Maroon, M., Schrader, L.E., Young, V.L. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. soil sci. plant anal. 1, 71-80. 8. Chew, K.W., Yap, J.Y., Show, P.L., Suan, N.H., Juan, J.C., Ling, T.C., Lee, D.J., Chang, J.S., 2017. Microalgae biorefinery: high value products perspectives. Bioresour. Technol. 229, 53–62. 9. Choi, H. J., 2016. Dairy wastewater treatment using microalgae for potential biodiesel application. Environ. Eng. Res. 4, 393-400 10. Dreywood, R. 1946. Qualitative test for carbohydrate material. Ind. Eng. Chem. 8, 499-499. 18
11. Emparan, Q., Harun, R., Danquah, M.K., 2019. Role of phycoremediation for nutrient removal from wastewaters: Appl. Ecol. Environ. Res.1, 889-915. 12. Gao, F., Li, C., Yang, Z.H., Zeng, G.M., Feng, L.J., Liu, J.Z., Liu, M. and Cai, H.W., 2016. Continuous
microalgae
cultivation
in
aquaculture
wastewater
by
a
membrane
photobioreactor for biomass production and nutrients removal. Ecolo. Engine. 92, 55-61. 13. Hena, S., Fatimah, S., Tabassum, S., 2015. Cultivation of algae consortium in a dairy farm wastewater for biodiesel production. Water. Resour. Indus. 10, 1-14. 14. Jais, N.M., Mohamed, R.M.S.R., Al-Gheethi, A.A., Hashim, M.A., 2017. The dual roles of phycoremediation of wet market wastewater for nutrients and heavy metals removal and microalgae biomass production. Clean. Technol. Environ. 1, 37-52. 15. Jais, N.M., Mohammed, R.M.S.R., Apandi, W.A.W.M, Peralta H.M.M. 2015. Removal of nutrients Removal of Nutrients and Selected Heavy Metals in Wet Market Wastewater by Using Microalgae Scenedesmus sp. Mech. Mater 773, 1210-1214. 16. Kitrungloadjanaporn, P., Sripongpun, G., Triampo, W., 2017. Nutrient removal from the effluent of swine slaughterhouse wastewater by Chlorella vulgaris TISTR 8580. Int. J. Adv. Agric. Environ. Eng. 4, 28-32. 17. Kothari, R., Pandey, A., Ahmad, S., Kumar, A., Pathak, V.V. and Tyagi, V.V., 2017. Microalgal cultivation for value-added products: a critical enviro-economical assessment. 3 Biotech, 7(4), 243. 18. Kothari, R., Pathak, V.V., Kumar, V., Singh, D.P., 2012. Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy waste water: an integrated approach for treatment and biofuel production. Bioresour. Technol. 116, 466-470. 19. Kothari, R., Prasad, R., Kumar, V., Singh, D.P. 2013. Production of biodiesel from microalgae
Chlamydomonas
polypyrenoideum
grown
on
dairy
industry
wastewater. Bioresour. Technol. 144, 499-503. 20. Kumar, V., Nanda, M., Joshi, H.C. Singh, A., Sharma, S., Verma, M., 2018. Production of biodiesel and bioethanol using algal biomass harvested from fresh water river. Renew. Energ. 116, 606-612. 21. Kundu, P., Debsarkar, A., & Mukherjee, S. 2013. Treatment of slaughter house wastewater in a sequencing batch reactor: performance evaluation and biodegradation kinetics. BioMed research international, 2013. 19
22. Lee, J., Lee, J., Shukla, S. K., Park, J., Lee, T. K. 2016. Effect of algal inoculation on COD and nitrogen removal, and indigenous bacterial dynamics in municipal wastewater. J. Microbiol. Biotechnol. 5, 900-8. 23. Liu, Z., Joo, J. C., Choi, S. H., Jang, N. H. J., Hur, J. W., 2018. Assessment of Surface Water Quality in Geum River Basin, Korea using Multivariate Statistical Techniques. Int. J. Appl. Eng. Res. 9, 6723-6732. 24. Liu, X., Chen, G., Tao, Y. and Wang, J., 2020. Application of effluent from WWTP in cultivation of four microalgae for nutrients removal and lipid production under the supply of CO2. Rene. Ener. 149, 708-715. 25. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 26. Lu, Q., Zhou, W., Min, M., Ma, X., Chandra, C., Doan, Y. T.& Chen, P 2015. Growing Chlorella sp. on meat processing wastewater for nutrient removal and biomass production. Bioresour. Technol, 198, 189-197. 27. Lu, Q., Zhou, W., Min, M., Ma, X., Chandra, C., Doan, Y.T., Ma, Y., Zheng, H., Cheng, S., Griffith, R. and Chen, P., 2015. Growing Chlorella sp. on meat processing wastewater for nutrient removal and biomass production. Bioresour. Technol. 198, 189-197. 28. Mackinney, G. 1941. Absorption of light by chlorophyll solutions. J. biol. Chem. 2, 315-322. 29. Maizatul, A. Y., Mohamed, R. M. S. R., Al-Gheethi, A. A., & Hashim, M. A. 2017 An overview of the utilisation of microalgae biomass derived from nutrient recycling of wet market wastewater and slaughterhouse wastewater. Int Aq Res 9(3), 177-193. 30. Murugesan, S., Venkatesh, P., Dhamotharan, R., 2010. Phycoremediation of poultry wastewater by micro alga. Biosci. Biotech. Res. Comm. 2, 14. 31. Myra, T., David, H., Judith, T., Marina, Y., Ricky, B.J., Reynaldo, E. 2015. Biological treatment of meat processing wastewater using anaerobic sequencing batch reactor (ASBR). Int. Res. J. Biol. Sci. 3, 66-75. 32. Pathak, V.V., Kothari, R., Chopra, A.K. and Singh, D.P., 2015. Experimental and kinetic studies for phycoremediation and dye removal by Chlorella pyrenoidosa from textile wastewater. J. of Environ Manag, 163,270-277. 33. Pathak, V.V., Singh, D.P., Kothari, R. and Chopra, A.K., 2014. Phycoremediation of textile wastewater by unicellular microalga Chlorella pyrenoidosa. Cell Mol Biol, 60(5)35-40. 20
34. Rasoul-Amini, S., Montazeri-Najafabady, N., Shaker, S., Safari, A., Kazemi, A., Mousavi, P., Ghasemi, Y., 2014. Removal of nitrogen and phosphorus from wastewater using microalgae free cells in bath culture system. Biocatal. Agric. Biotechnol. 2, 126-131. 35. Rath, B., 2012. Microalgal bioremediation: current practices and perspectives. J. Biochem. Technol. 3, 299-304. 36. Safafar, H., Uldall Norregaard, P., Ljubic, A., Moller, P., Løvstad Holdt, S. and Jacobsen, C., 2016. Enhancement of protein and pigment content in two Chlorella species cultivated on industrial process water. J. Mar. Sci Eng. 4, 84. 37. Salama, E.S., Kurade, M.B., Abou-Shanab, R.A., El-Dalatony, M.M., Yang, I.S., Min, B., Jeon, B.H., 2017. Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew. Sustain. Energ. Rev.79, 1189-1211. 38. Singh, H. M., Pathak, A. K., Chopra, K., Tyagi, V. V., Anand, S., & Kothari, R. 2019 Microbial fuel cells: a sustainable solution for bioelectricity generation and wastewater treatment. Biofuels, 10(1), 11-31. 39. Singh, H.M., Kothari, R., Gupta, R. and Tyagi, V.V., 2019. Bio-fixation of flue gas from thermal power plants with algal biomass: Overview and research perspectives. J Environ Manage, 245, 519-539 40. Sletten, O., Bach, C.M., 1961. Modified stannous chloride reagent for orthophosphate determination. JAM. Water. Works. Ass. 8, 1031-1033. 41. Tan X.B., Zhao X.C., Yang L.B., 2019. Strategies for enhanced biomass and lipid production by Chlorella pyrenoidosa culture in starch processing wastewater. J. Clean. Prod. 236, 117671 42. Taşkan E., 2016. Performance of mixed algae for treatment of slaughterhouse wastewater and microbial community analysis. Environ Sci Pollut Res 23:20474–20482 43. Venckus, P., Kostkeviciene, J., Bendikienė, V., 2017. Green algae Chlorella vulgaris cultivation in municipal wastewater and biomass composition. J. Environ. Eng. Landsc Manag. 1, 56-63. 44. Viegas, C.V., Hachemi, I., Maki-Arvela, P., Smeds, A., Aho, A., Freitas, S.P., Kumar, N., 2015. Algal products beyond lipids: Comprehensive characterization of different products in direct saponification of green alga Chlorella sp. Algal Res. 11, 156-164.
21
45. Wang, L., Min, M., Li, Y., 2010. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Appl. Biochem. Biotechnol. 4, 1174-86. 46. Xu, X.Q., Wang, J.H., Zhang, T.Y., Dao, G.H., Wu, G.X. and Hu, H.Y., 2017. Attached microalgae cultivation and nutrients removal in a novel capillary-driven photo-biofilm reactor. Alga. Rese. 27, 198-205. 47. Yaakob, M. A., Mohamed, R. M. S. R., Al-Gheethi, A. A. S., & Kassim, A. H. M 2018 Characteristics of chicken slaughterhouse wastewater. Chem Engg Transac, 63, 637-642. 48. Zhao, X.C., Tan, X.B., Yang, L.B., Liao, J.Y. and Li, X.Y., 2019. Cultivation of Chlorella pyrenoidosa in anaerobic wastewater: The coupled effects of ammonium, temperature and pH conditions on lipids compositions. Bioresour. Technol. 284, 90-97.
To, The Editor-in-Chief, Bioresource Technology. Subject: Credit Author Statement I have submitted that this final manuscript is sharing with all authors and all authors provides its suggestions to improve this manuscript. As I, Dr. Richa Kothari (corresponding Author) is responsible for ensuring that the descriptions are accurate and agreed by all authors. Conflict of Interest: None Thanks Dr. Richa Kothari 22
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Rifat Azam Richa Kothari Har Mohan Singh Shamshad Ahmad V. Ashok Kumar V. V. Tyagi
Graphical Abstract
23
HIGHLIGHTS
Treatment of slaughterhouse wastewater (SHWW) with Chlorella pyrenoidosa
Maximized growth observed with 50% concentration in 430 mgL-1 of biomass (dry wt.)
COD, NO3- and PO4-3 reduction ≈17-31%, 23-42%, 18-48% with 50% concentration of SHWW
Correlation analysis investigation in between parameters of SHWW and algal strain
SHWW treatment with Chlorella supports pollutant reduction and biochemical profiles
24
List of Figure Figure 1: Growth optimization of algae on slaughterhouse wastewater Figure 2: Percent reduction in pollutant load of slaughterhouse wastewater with selected concentrations. Figure 3: Biochemical profile of slaughterhouse wastewater with 50% concentration Figure 4: Correlation analysis for physico-chemical parameters of SHWW and biochemical profile of C. pyrenoidosa with selected concentrations.
25
Biomass (mg L⁻¹)
500 450 400 350 300 250 200 150 100 50 0
25%.
0
2
4
50%.
6
75%.
8 10 Days
100%.
12
14
16
control
18
Figure 1: Growth optimization of algae on slaughterhouse wastewater
26
20
Percentage Reduction (%)
120 100 80
25%
60
50%
40
75% 100%
20 0 BOD
COD Nitrate Parameters
Phosphate
Figure 2: Percent reduction in pollutant load of slaughterhouse wastewater with selected concentrations.
27
Concentration (mg L-1)
Chlorophyll
Protein
Carbohydrate
70 60 50 40 30 20 10 0 0
5
10
15
Days Figure 3: Biochemical profile of slaughterhouse wastewater with 50% concentration
28
Figure 4: Correlation analysis for physico-chemical parameters of SHWW and biochemical profile of C. pyrenoidosa with selected concentrations
List of Table Table 1.Worldwide standards limits for the discharges of slaughterhouse wastewater (Bustillo-Lecompte and Mehrvar, 2017) Table 2. Pollutant reduction at 0th day to 15th day by using Chlorella pyrenoidosa with 50% concentration of wastewater Table 3. Comparative analysis of biochemical profile of algae with various types of wastewater with current study
29
Table 1: Worldwide standards limits for the discharges of slaughterhouse wastewater (Bustillo-Lecompte and Mehrvar, 2017) S.NO Parameters Present study World EU US Canadian Australian Indian Standards Standards Standards Standards Standards Bank Standards 1
pH
10.16±0.32
6-9
NA
6-9
6-9
5-9
5-9
2
Color
Dark red
-
-
-
-
-
-
3
Odor
Offensive smell
-
-
-
-
-
-
4
TDS
774.66±70.39
-
-
-
-
-
-
5
COD
3429.33±61.04
125
125
NA
NA
3×BOD
250
6
BOD
1798.33±14.01
30
25
26
5-30
10-15
100
30
7
NO3-
331.66±7.50
10
10
8
1
0.1-15
10-50
8
PO4-3
34.13±1.32
6-9
NA
6-9
6-9
5-9
5-9
Table 2. Pollutant reduction at 0th day to 15th day by using Chlorella pyrenoidosa with 50% concentration of wastewater Time period
Parameters Color
0th day
Reddish
pH
10.2
Odor
Offensive meat smell
TDS (mg L-1)
Nitrate
Phosphate
BOD
COD
(mg L-1)
(mg L-1)
(mg L-1)
(mg L-1)
398
172.3
16.3
900
1846.6
31
Percent reduction (%)
-
-
-
-
-
-
-
5th day
9.9
Offensive meat smell
325
133.3
13.4
839
1528.3
Percent reduction (%)
-
-
18.3
22.6
17.7
6.7
17.2
10th day
9.6
Offensive meat smell
211
84
8.6
778
1243.3
35.1
36.9
35.8
7.3
18.7
148
48
4.4
547.6
855.6
29.9
42.8
48.8
29.6
31.2
Light reddish green
Light reddish green
Percent reduction (%)
-
15th day
9.3
Green
Percent reduction (%)
-
Offensive meat smell
32
Table 3. Comparative analysis of biochemical profile of algae with various types of wastewater with current study Wastewater
Algal Strain
Biochemical Parameters Carbohydrate
Protein
(%)
(%)
27
51.58
Meat wastewater (CUT)
Chlorella sp.
Industrial process water
Chlorella pyrenoidosa
NA
65.2
Industrial process water
Chlorella vulgaris
NA
Artificial anaerobic effluents
Chlorella pyrenoidosa
Starch processing wastewater
Chlorella
Slaughter house wastewater
Chlorella
Chlorophyll
Culture
References
Period (Days) 9
Lu et al., 2015
5.41 mg g-1 DW
16
Safafar et al., 2016
55.1
32.44 mg g-1 DW
16
Safafar et al., 2016
25.7
63.7
3.7 %
15
Zhao et al., 2019
25.4
62.3
3.9 %
12
Tan et al., 2019
9.58%
12.37
15.31 µg mL-1
15
Present study
(including nucleic acid)
pyrenoidosa
pyrenoidosa
33