Abstract

Microalgae and Cyanobacteria are potentially diverse, dominant photosynthetic organisms in nature. Initially, eight strains belonging to Marine cyanobacteria and Microalgae, based on growth, were selected. C. vulgaris BDU G91771 was used to study the possibility of utilizing ossein effluents. The organism was inoculated in two different effluents (HTDS and LTDS) with seawater and nutrient dilutions, namely (effluent, effluent with Seawater with N:P. The maximum lipid productivity was observed in 100%LTDS + Nutrients (Urea and superphosphate) with 16.8%. Lipid composition in fatty acids appeared to be suitable for biodiesel production. The results showed that the marine microalgae C. vulgaris BDU G91771 could be used to treat ossein effluents, and at the same time, to produce biodiesel sustainably.

1. Introduction

Biofuel production coupled with carbon dioxide sequestration through photosynthetic microorganisms has been a good process since the last century. ¹ Microalgae and cyanobacteria, photosynthetic organisms, uptake nutrients and convert carbon dioxide into carbon-rich lipids ². Algae govern the consumption of CO_2, and they can remove contaminants and uptake the nutrients in industrial wastewater effluent; It also has been proven that nutrients in most types of wastewater, such as municipal wastewater, agricultural wastewater, food industry wastewater, etc., could be effectively utilized by microalgae ³. The use of microalgae for effluent treatment has been employed for decades, as a low-cost process, especially in areas with increased ambient temperature and adequate sunshine throughout the year. ⁴ Ossein (decalcified bone) is the chief organic substance of the animal bone tissue obtained as a residue in the clarification process of the gelatin production system. High total dissolved solids (HTDS)-released on heat treatment of the decalcified bones and low total dissolved solids (LTDS) effluent-discharged after final washing and settlement of Ossein. ⁵ Microalgae-based advanced nutrient removal treatment has outstanding advantages, including cost-effective and environmentally friendly as no additional chemicals are required, while oxygen generation, carbon dioxide mitigation, and metal ion reduction can be realized at the same time; and potential utilization of the harvested microalgal biomass for production of food, feed, fuel, fertilizers, and fine chemicals. ^{6, 7} Microalgae biomass contains products with high commercial importance like proteins, lipids, carbohydrates. ⁸ Nutrient availability significantly impacts the growth and propagation of microalgae and overall effects on their lipid and fatty acid composition, especially Lipids are one of the main components of microalgae. The interest in microalgae for oil production is due to some species' high lipid content and the fact that lipid synthesis, especially of the non-polar TAGs, which are the best substrate to produce biodiesel, can be modulated by varying growth conditions. ⁹ Microalgae contain around 2-60 % lipids of the total cell dry weight depending on the species and growth conditions. Lipids derived from microalgae have been the focus of considerable interest because these oils contain fatty acid and triglyceride compounds. ¹⁰ To study the growth characteristics of eight strains based on the lipid selected, the best strain for the ossein effluent growth study, C. vulgaris used to remove nutrients from the effluent. The use of both LTDS and HTDS effluent wastewater for microalgae biomass production is a plausible approach as a renewable and sustainable process removing nutrients and producing by-products economically. The study aims to demonstrate the performance of C. vulgaris in treating ossein effluent and estimating biomass and lipid simultaneously under different combinations under Laboratory conditions. Moreover, according to the experimental results to describe, growth, nutrients consumption and promote the strains further investigated in the Large scale cultivation.

2. Materials and Methods

A total of eight strains were obtained from the repository of the National Facility for Marine Cyanobacteria (Sponsored by DBT, Govt of India) for the study. Based on the screening of the chosen eight strains collected from various coastal areas.

An inverted light microscope observed eight strains' cell size and morphological characteristics (unialgal, axenic cyanobacterial) (Leica DMI 3000B). A measuring ocular calibrated to the different magnification (10X, 40X, and 100X) was used to calculate cell sizes. At least 100 microalgal cells if each species were measured as its stationary period. ¹¹

To select the suitable organisms for sustainable bioenergy, eight organisms were screened based on growth and lipid analyses. 30 ml of the culture with high cell density were inoculated in triplicate into 250ml Erlenmeyer flasks containing 150 ml of ASN Medium prepared in sterilized conditions. Cultures were maintained at 27 ± 2°C, with a photoperiod of 16:8 h light / dark cycles under a light intensity of 250 µmol photons m⁻² s⁻¹ provided by cool white fluorescent tubes and culture for Seven days. The cell count was measured every alternate day, and the growth rate of all eight strains was determined in triplicates for seven days following. ¹²

Calcium-rich ossein effluent was collected at three different clarification stages from the gelatin manufacturing industry, Pioneer Jellice Industries, Cuddalore, Tamil Nadu, India. Two effluents, namely, high total dissolved solids (HTDS) low total dissolved solids (LTDS), have been used for the present study. The effluent differed in amounts of the total dissolved solids (TDS), hence named as high TDS (HTDS) and low TDS (LTDS). The collected effluents are stored in black plastic cans at 4°C to avoid microbial growth till use. Autoclaved effluent was used for the experiments.

Sterilized Erlenmeyer flasks (250 ml) were used for the experiments. The effluent's initial physicochemical analysis was estimated following the Standard Methods (APHA, 1975). The following treatments were employed to study the interaction of C.vulgaris with the Industrial Ossein effluent. Effluent uninoculated was taken as a control for physicochemical analysis (C). ASN medium inoculated with C.vulgaris BDUG91771 for the estimation of growth. Inoculation was made by adding a 5.0 ml uniform suspension of C.vulgaris BDU G91771 (0.5 OD). The experiment was conducted under controlled conditions (Temperature 25 ± 2^oC with light intensities of 1200lux provided from overhead cool with fluorescent tubes). Effluents were diluted at different combinations Table 1 The growth of C.vulgaris BDU G91771 was studied in the rich cultures harvested on the 7^th day. The filtered effluents (inoculated and control) were used for physicochemical analysis.

The un-inoculated effluent served as control. The clear supernatant was used for the analysis after centrifugation of the effluent-grown microalgal culture. The physicochemical parameters of the two types of effluents were analyzed on Initial and Final days for the experimental period. The total solids (TS), total suspended solids (TSS), and total dissolved solids (TDS) were evaluated according to the standard methods ¹³ by drying the sample at 100°C using pre-weighed crucibles. The nutrient removal by strains from the effluents was evaluated by determining the amount of dissolved oxygen (DO), total Kjeldahl nitrogen (TKN), nitrate, nitrite, ammonia, total phosphorus, inorganic phosphorus, calcium, and magnesium by following the standard methods for the examination of water and wastewater.

Chloroform: methanol (2:1) extraction method was followed to ensure complete lipid extraction. Lipid extraction from algal cells requires cell membrane disruption physically or chemically. As it is a temperature tolerance study, the direct solvent extraction method has been used. The biomass and solvent mixture (chloroform: methanol (2:1)) containing RB flask equipped with a condenser was refluxed for about two hours. In the extracted lipid, distilled water was added to remove water-soluble impurities. The lower chloroform layer containing lipid was carefully collected, and the upper layer was discarded. (for hexane and petroleum ether facilitated extraction, the top layer was collected) A moisture content of collected lipid was eliminated by passing it to the sodium sulfate bed. The lipid was transferred to the preweighed glass container and dried in a Rotary evaporator (Evator II), and the lipid weight was measured gravimetrically.

Where,

Wt – Total weight of dried lipid container

Wp – Pre weight of the container

D – Dry cell weight of algal biomass

In the transesterification process, the purified lipid was taken into Round Bottom (RB) flask and was preheated in a water bath to get rid of any water. The acid catalyst (3% sulfuric acid) was thoroughly dissolved in methanol, and the resulted mixture was poured into the preheated lipid at room temperature. The reaction setup was equipped with a reflux condenser, magnetic stirrer, thermometer, and reactions for 2 h at 65±10°C. After completion of the reaction, the mixture was carefully transferred to a separating funnel and allowed to stand for 30 min, a Lower layer containing glycerol, unreacted methanol, and catalysts was drawn off, and the upper layer comprising methyl ester was collected, Methyl ester thus obtained was purified by repeated water wash until the lower water layer reached pH 7.0, The resulting purified methyl ester was dried at 70°C in a Rotary evaporator (Evator II) and used for gas chromatographic analysis.

For the separation of FAMEs, one µl of transesterified samples dissolved in hexane used as a solvent was injected onto a gas chromatograph (Perkin Elmer Clarus 500) equipped with SP 2560 capillary column (100 m length) and Flame Ionization Detector. The chromatography conditions were; carrier gas flow rate -1 mL min-1 (1ml –nitrogen, 10ml – Hydrogen, 100ml -Zeroair), oven temperature 140-240°C; injection port temperature 260°C; detector temperature 260°C and the total run time was 45 min. Supelco, 37 component FAME mix, was used as a standard to identify the analytes. The response of each fatty acid separated on the chromatograph was determined as the peak area.

3. Results and Discussion

Eight strains were obtained from the repository of the National Facility for Marine Cyanobacteria (Sponsored by DBT, Govt of India) for the study. The chosen strains are listed in Table 2.

All the procured eight strains from the repository were further authenticated for their morphological characters under an inverted light microscope (Leica DMI 3000B) for morphological and unialgal conformity, and their microscopical images were illustrated below in Plate 1.

To identify a high lipid yielding marine candidate, the chosen eight strains on the 7^th day in ASN III medium were estimated for their maximum lipid using 2:1 chloroform: methanol solvent mixture and expressed gravimetrically. Among the eight strains, Chlorella sp. BDUG 91771 possessed high lipid content of approximately 22%, which is of substantial value from a biodiesel perspective. Both marine microalgae and cyanobacteria equally competed for lipid content, of which cyanobacteria showed approximately 16%, which was comparatively lower than the lipid produced by microalgae.

Microalgae and cyanobacteria could serve as feedstock for biodiesel production if they meet all the above requirements. They have high growth and provide a high lipid fraction to produce biodiesel. ¹⁴ Marine cyanobacteria exhibited polyvalence in lipid content across genera among the eight selected strains. The previous study reported that Phormidium might accumulate lipids up to 0.001 to 14, while Phormidium angustissimum is the lowest lipid producer of the genus. Likewise, The previous research showed Phormidium sp. FW01, Phormidium sp. FW02, Oscillatoria sp. FW01, and Oscillatoria sp. FW02 showed 6.7, 8.2, 10.2, and 9.4% lipids, respectively. In our present study, P.valderianum BDU142231 showed 14%, and O.laatevirens BDU100891 showed 16.2 % lipid. This was comparatively the same in the Phormidium and above in the oscillatoria. ¹⁵ The previous study reported that S. platensis resulted in the highest lipid production of 4.7%, grown in the medium of Zarrouk’s synthetic medium; compare these results ¹⁶. Plectonema sp. BDU 51111 showed 9% of lipid produced in the ASN medium in our results. In this study, 4 are Chlorella sp., namely BDUG 91771, BDUG 22101, BDUG 10101, BDUG 20601 Plectonema sp BDU 51111 and L. confervodies BDU 142001 were obtained. When comparing the previous study in Phormidium, maximum lipids accounted for approximately 15% of total biomass mass. ¹⁷ (Table 3) Moreover, the previous study reported using five input cultivation variables (i.e., illumination flux, CO₂, NaNO₃ ) the maximum lipids was found 1.99 g L^-1in stichococcus ¹⁸. In the present study, L. confervodies BDU142001 showed 7% of lipid for the economical production of biofuels from microalgae, biomass, and lipid content significant role. In a previous study, Chlorella strains, among which C. emersonii, C. minutissima, and C. vulgaris increased lipid content of 63%, 56%, and 40% biomass by dry weight, respectively ¹⁹. Our previous research reported that C. vulgaris BDUG 91771 was a potent strain to yield 22.2% lipid ²⁰. This research also showed 22% lipid in C.vulgaris BDUG 91771. The screening results manifestly confirmed that, of the nominated eight strains, Due to the high lipid content coupled with moderate growth, further optimization studies due to the biodiesel perspective were restricted to C.vulgaris BDUG 91771.

Lipid content for C.vulgaris BDU91771 grown in different effluent and seawater combinations were tested and expressed gravimetrically as percentage lipid. The maximum lipid productivity was observed in both 100%LTDS + Nutrients (Urea and superphosphate) and LTDS with seawater and nutrients containing 16.8% on equal. The minimum lipid was observed in the LTDS + seawater with nutrient (1:1:1) combination. In the tested HTDS effluent condition, the maximum lipid production was about 14%. This indicates that lipid productivity was high in LTDS than HTDS effluent condition. (Figure 1).

The chosen high lipid yielding strain C.vulgaris BDU G91771 was further characterized for their fatty acid profile is defined ASN medium (control), low-cost seawater based medium and both the effluent (LTDS and HTDS) and the maximum lipid yielding condition, LTDS with seawater and nutrients (I:1).

In a previous study, Calcium-rich ossein effluent from a gelatin-producing industry was treated by O. willei BDU130791 and P. valderianum BDU20041. Results show calcification which appears to be a plausible alternative to removing calcium in effluents and sequestration of CO₂ from the point source ⁵. Our current study also uses the same ossein effluent for C.vulgaris BDU G91771. Previous research studied the growth rate and bulk, macro, and micronutrient removal capability of C.vulgaris in the three different wastewaters (primary wastewater, PWW, secondary wastewater, SWW, and petroleum effluent, PE). Calcium, sulfur, magnesium, potassium was high in this waste effluent. It was removed 66% to 80% ²¹. Various studies reported that using bioremediation options by microalgae is viable for treating wastewater some previous studies said phosphorus and nitrogen removal for wastewater injected CO₂ by C. vulgaris, respectively ²². The lipid content increased to 34.1% ²³. In our current study, the maximum lipid content was 16% compared to the control of ASN medium, with 22% of lipid produced. The previous results in the study of C. vulgaris microalgae using wastewater had palmitoleic acid (C16: 1) of 30.54%, higher when compared with 10.2% photobioreactor and 10.4% open pond. Stearic acid (C18: 4) was 13.65% lower when compared with photobioreactor of 21.6% and open pond by 21.4%. There is no linolenic acid (C18: 3) when linolenic acid [18:3] at photobioreactor 14.9% and open pond 14.3%. ²⁴ In the current study, The fatty acid profile varied inevitably in the different conditions tested. The selected strain showed the predominance of middle and long-chain fatty acid, namely palmitic (C16), palmitoleic (C16:1), cis 10- heptadecenoic acid (C17:1), stearic (C18:0), oleic (C18:1n9c), linoleic acid ((C18:3n3), in LTDS with seawater and nutrients (I:1) condition.

The total fraction of the fatty acid methyl esters (FAME) extracted and the specific composition of the individual detected FAME are shown in Table 5. The concentration of palmitic and linoleic acid was found to be high in the tested LTDS effluent, which is a significant observation.

4. Conclusion

C.vulgaris BDU G91771grown well in 100% LTDS + Nutrients (Urea and superphosphate). It utilizes the nutrients in the effluent and converts them into biomass and high lipid content. C.vulgaris BDU G91771 contains essential fatty acids that are relatively abundant. In brief, C.vulgaris BDU G91771 is a better solution for utilizing the nutrients treated the Ossein effluent efficiently and meanwhile producing valuable feedstock with a relatively cheap and environmental friendly approach.

Acknowledgments

The authors thank the Department of Biotechnology, Govt. of India (BT/IN/Indo-UK/SUBB/28/LU/dt: 29.10.2013) for the Indo-UK project financial support and Pioneer Jellice industries, Cuddalore, TamilNadu, India for gifting the oseein effluent. Also, authors sincerely thank Department of Biotechnology for funding the facility, National Facility for Marine Cyanobacteria, Bharathidasan University, Trichy, TamilNadu, India.

References