Potential of Dictyosphaerium sp. LC172264 Concomitant Remediation of Cassava Wastewater and Accumulation of Lipids for Biodiesel Production

As a way of making algal feedstock feasible for biofuel production, simultaneous utilization of microalga Dictyosphaerium sp. LC172264 for cassava wastewater remediation and accumulation of lipids for biodiesel production was investigated. The algal biomass, lipid contents and composition were measured from the autotrophic, heterotrophic and mixotrophic cultured algal cells. Physicochemical parameters of the cassava wastewater and bioremediation potentials were measured. Biodiesel properties were deduced and compared with the standards. The results showed that mixotrophic culture was the best for both biomass accumulation (1.022 g/L) and lipid contents (24.53%). Irrespective of the culture condition, the predominant fatty acids were similar and included 11-Octadecenoic acid (vaccenic acid (C 19 H 36 O 2 ), oleic acid (C 18 H 34 O 2 ) and 14-methyl pentadecanoic acid (isopalmitic acid (C 17 H 34 O 2 ). The percentage reduction of total dissolved solids was remediation by Dictyosphaerium sp., mixotrophy showed superiority.


Introduction
The massive irreversible environmental degradation arising from the combustion of fossil fuels to meet the energy needs of man places a huge moral burden on society. The concomitant release of greenhouse gases into the atmosphere does almost incalculable damage to the protective ozone layer, consequently leading to global warming and other climate change issues with an undesirable negative impact on our wellbeing and environment [1]. If fossil fuel combustion is not stopped by 2100, the damage to the environment would be severe, pervasive and irreversible, causing long-lasting changes in all components of the climate system [2]. With the unrelenting increase in anthropogenic activities and the projected explosion of the human population by 2050, it is now no longer sustainable to continue to combust petroleum hydrocarbons at current rates. It has therefore never been more urgent to replace fossil fuels with greener, renewable and cheaper energy alternative sources.
Amongst several strategies and new methods for sustainable energy production from renewable carbon sources is the implementation of a "waste to wealth" strategy where potentially damaging wastes sources are converted to useful products and in the process eliminating the waste. This strategy also eliminates the potential competition for food sources that may affect global food security. The use of wastes for bioenergy production has been severally reported. These include municipal wastewater [3], industrial wastewater [4], agricultural wastewater [5], etc. The processing of cassava generates a lot of cassava wastewater (CWW) which is toxic, odoriferous and undesirable [5]. Cassava wastewater is a problematic acidic waste and a source of environmental concern because of its typically high hydrogen cyanide content (>400 mg·L −1 ) and biological oxygen demand (BOD) of 25,000 -50,000 mg·L −1 [6]. Nevertheless, it is a rich source of organic and inorganic nutrients capable of supporting the growth of organisms like microorganisms. Microalgae have the capability of using effluent as substrate to produce biomass from which oil can be extracted and subsequently used as precursor for products that substitute hydrocarbon fossil fuel.
Accordingly, Neves et al. [7] obtained total nitrogen (g/L) and a total phosphorous (g/L) of 0.25 ± 0.01 and 0.16 ± 0.01 respectively from cassava wastewater. They also obtained from the wastewater, a carbon to nitrogen ratio of 96.0 ± 4.24 and nitrogen to phosphorous ratio of 1.50 ± 0.071. These characteristics emissions. In addition, cassava starch hydrolysate has been used as culture medium for the production of high Ankistrodesmus sp. cell density and enhanced lipid content in a heterotrophic culture [8]. Nigeria, which is the highest producer of cassava in the world and with production accounting for about 20% of total world production [9], unfortunately, has no policy of handling and safely disposing of this hazardous wastewater, as the daily practices have been to recklessly and inappropriately discharge this effluent into the environment without caution into roadside ditches or fields and allow to flow freely, settling in shallow depressions, eventually percolating into the subsoil or flow into streams [5] [6].
Other strategies were bacterial-algal co-culture in wastewater for enhancing remediation and biofuel production. Leong et al. [10] in a quest to make algal biofuel production economically feasible and not compromising environmental prospects, co-cultured microalgae and bacteria alongside enhancing lipid-based biofuel production and municipal wastewater bioremediation.
Previous studies had recently demonstrated the use of cassava wastewater as substrate for microalgal biomass production and the harvesting of lipid for biodiesel production. Neves et al. [7] evaluated biodiesel production potentials of a microalga Phormidium autumnale using cassava wastewater as substrate under heterotrophic condition and obtained good quality biodiesel, high biomass density and high lipid contents. More recently, Ogbonna et al. [5] and Okpozu et al. [11] had demonstrated the use of Desmodesmus subspicatus LC172266 and Desmodesmus armatus respectively for the production of biomasses, lipids and biodiesel using cassava wastewater as substrate. They also demonstrated high quality bioremediation of the cassava wastewater by the algae. In a like manner, Kumar et al. [12] in a review demonstrated that there is a potential synergy of biofuel production with waste remediation along with value-added co-products recovery using membrane-based system for algal biorefinery. These are all in line with the recommendation of Chisti [13] that for algal biofuel production to be feasible in order to mitigate the high cultivation cost, incorporation of biofuel production with other co-product formation or processes is required. Dictyosphaerium sp. has been shown to be a high lipid producer and its applicability in biodiesel production has also been demonstrated [14]. Jiang et al. [15] used a high-lipid producing Dictyosphaerium ehrenbergianum grown in struvite precipitated liquid digestate for biodiesel production, with lipid and biomass productivities being higher than when cultured in the standard BG-11 medium. The microalga Dictyosphaerium ehrenbergianum Nageli has been reported to be useful in biodiesel production with oleic, palmitic and linoleic acids as the predominant fatty acids [16]. They also demonstrated that the qualities of the biodiesel produced with this organism as conforming to the standard qualities and therefore recommended Dictyosphaerium ehrenbergianum Nageli to be good feedstock for biodiesel production. In the present study, we evaluated the use of cassava wastewater as a medium for the cultivation of microalga Dictyosphaerium sp., as a means of bioremediating the wastewater and elaboration of lipids for biodiesel production. In addition to measuring the physicochemical parameters of the cassava wastewater and bioremediation potentials, algal biomass quantities, lipid contents and fatty acid profile were also measured from the alga cultivated under autotrophic, heterotrophic and mixotrophic culture conditions. Biodiesel properties were deduced and compared.

Cassava Wastewater Collection and Analysis
The cassava wastewater used in this study was collected from local cassava milling plants in Benue State, Nigeria. The initial physicochemical parameters including the pH were measured. The cassava wastewater was prepared basically according to the method described by Ogbonna et al. [5]. Briefly, the wastewater was filtered through a typical sand filter as adapted using the improvised funnel, mesh, wire gauze and sand column [5]. A 50 mL quantity of one-molar tetra oxo sulphate (VI) acid was added to a ten-liter volume of the cassava wastewater filtrate. The mixture was heated with stirring until gelatinization occurred which eventually turned milky with further heating. The heated milky solution was mixed with 32.67 mL of 60% sodium hydroxide solution on a liter basis with further stirring to neutralize the mixture. According to Ogbonna et al. [5] and Okpozu et al. [11], to every millilitre volume of the cassava wastewater was added a 10 mL volume of the BBM was added. The pH of the CWW was adjusted to 7.0 using dilute HCl and 1% NaOH. This was thereafter sterilized by autoclaving at 121˚C for 10 min with subsequent addition of ampicillin and ketoconazole (250 and 200 mg respectively) upon cooling.
The microalga was subsequently maintained in Bold Basal Medium (BBM) [5]. 10 mL each of the sterile stock solutions 1 -6 were respectively added, followed by the addition of 1 mL each of stock solutions 7 -10. This was made up to a 1.0 L volume with sterile distilled water. The BBM was used for photoautotrophic cultivation whereas the heterotrophic and mixotrophic cultures were done using BBM medium supplemented with cassava wastewater (CWW) as described above. Each cultivation was done in a one-liter Erlenmeyer flask containing 500 mL of appropriate medium as adapted from Ogbonna et al. [5] and Okpozu et al. [11]. The inoculation of each set-up was done with 20% (v/v) of the isolate seed culture (approximately 10 6 CFU/mL of Dictyosphaerium sp. in exponential phase in BBM). The photoautotrophic cultures were exposed to approximately 12 hours of sunlight daily [average light intensity ~2000 LX (Digital light meter, model LX-1000, Japan)] at a temperature of 28˚C ± 2˚C. The set-up was agitated 2-times daily (vigorous hand-shaking for 2 min) during the hours of sunlight.
For heterotrophic and mixotrophic cultivations, the seed cultures were grown in darkness for 72 h for acclimatization before 20% v/v inoculation described previously. After inoculation as described above, the conical flasks for the heterotrophic cultures were wrapped in aluminum foil sheets and incubated in dark cupboards at a temperature of 28˚C ± 2˚C for the duration of its growth. For mixotrophic cultures, the acclimatized microalgal seed culture was inoculated into the 500 mL medium in two replicates. The set-up was exposed to sunlight for six hours daily from 7:00 am to 1:00 pm after which it was returned to stand in the dark. The experiments were done in two replicates for each growth mode.

Determination of Growth, Lipid Content and Composition
Determination of growth rates for each culture mode was done using spectrophotometer at Optical Density 680 nm (HACH, Loveland, Colorado, USA). A growth curve was generated based on the Optical Density (OD 680 ) readings and a standard curve was used to convert to cell dry weight. The dried cell (DC) (0.5 g) was used for the lipid extraction. Lipid was extracted from the DC by the method of Bligh and Dyer [19] in chloroform: methanol: water solvent system. The

Determination of Fatty Acid Methyl Ester (Biodiesel) Property
Fatty acid methyl ester property was determined by means of a Biodiesel Analyzer software [20].

Measurement of Electrical Conductivity (EC)
Using Ademoroti [21] recommended protocol, the electrical conductivity (EC) was measured by using a conductivity probe (Hanna combo pH/EC meter HI 98129). The electrical conductance (micro second per centimeter (μS/cm)) of the CWW was determined by inserting the probe into the wastewater and taking reading after 3 min.

Measurement of Total Dissolved Solids (TDS) of the CWW
The total dissolved solid (TDS) of the CWW was measured using filtration method of American Standard Test Methods [22]. In brief, a 50 mL CWW sample added into an evaporating dish was completely dried by heating at 180˚C. Upon dryness to constant weight, the TDS was calculated using the equation below in Equation (1).

Measurement of Dissolved Oxygen and Biochemical Oxygen Demand
The Dissolved Oxygen (DO) was measured using a modification of Winkler-Azide protocol while the Biochemical Oxygen Demand (BOD) was measured as the difference between DO of samples immediately after collection and DO of samples after incubation at 20˚C for five days [22] as adapted from Okpozu et al. [11].

Measurement of Chemical Oxygen Demand (COD)
The COD was measured using a method adapted from Ademoroti [23] by a titrimetric method with Ferroin indicator as presented recently by Okpozu et al. [11].

Measurement of pH of CWW Solution
The CWW sample pH was measured by a means of a calibrated pH meter (Hanna Instrument C-99-USA). The calibration was done using buffers at pH 4, 7 and 10 [21].

Measurement of Hydrogen Cyanide (HCN) Content of the Solution
Hydrogen cyanide content of the CWW was measured using the method adapted from Ezeh et al. [24] and Sawyerr et al. [25] with modification. This was determined by spectrometric method using alkaline picarate solution. The sand-filtered CWW was further filtered using Whatman No. 1  was prepared by dissolving 1 g of picric acid and 2 g of anhydrous sodium carbonate (Na 2 CO 3 ) in a small volume of warm water and making up to a 100 mL volume. This was stored in an amber-colored bottle in a refrigerator (4˚C) until used. In the measurement of HCN content, 4 mL of alkaline picarate was added to a 5 mL quantity of the filtered CWW. This was warmed for 5 min in a water bath (55˚C) for brown color development and was allowed to cool and read against a blank at 490 nm (UV-Spectrophotometer). The blank was prepared by using distilled water in the place of CWW. The HCN content was expressed as mg/L and was extrapolated from a standard curve.

Statistical Analysis
The data obtained from this study were analyzed using Statistical Package for Social Science (SPSS version 20.0).

Pre-Treatment of Cassava Wastewater
This study focused on the evaluation of Dictyosphaerium sp. LC172264 for dual cassava wastewater remediation and concomitant accumulation of lipids for biodiesel production. Before the wastewater was used, it was pre-treated via sand filtration, acid hydrolysis, alkali neutralization and subsequently autoclave-sterilized. The sand filtration was done to simulate natural water filtration taking place in the soil and this resulted in the removal of large particulate matter in the wastewater with the CWW appearing ash to colorless. Pre-treatment processes have been customary in wastewater treatment by microalgae. Hence, Rui et al. [26] used anaerobic digestion of the wastewater by microorganisms before the cultivation of microalgae on the wastewater. The present sand filtration has been recently employed in the bioremediation of cassava wastewater by other species of microalgae in previous studies [5] [11]. Cassava processing wastewater was recently demonstrated to be an ideal platform for third generation biodiesel production [7] because of the nutrients it contains.

Growth, Lipid Content and Composition Dictyosphaerium sp. LC172264
The growth pattern of Dictyosphaerium species under autotrophic, heterotrophic and mixotrophic cultivation modes are presented in Figure 1. The mixotrophic culture had the highest biomass content with a maximum cell dry weight value of 1.022 g/L equivalent to optical density value of 2.842 ± 0.12 on the day 8 of incubation. Heterotrophic culture accumulated its highest biomass concentration of 0.63 g/L (OD value equivalent to 1.981 ± 0.70, on day 10 of incubation) while autotrophic growth was the lowest of the three with its highest biomass concentration of 0.238 g/L (OD value equivalent to 1.477 ± 0.51, day 10 of incubation). Cells presented in g/L were extrapolated from the standard curve of the plot of cell dry weight (g/L) against the optical density readings (OD 680 ) with R 2 = 0.8982. The three growth curves did not show any pronounced lag period and  whereas under heterotrophic cultivation, Dictyosphaerium sp. LC172264 accumulated approximately half the biomass obtained under mixotrophic condition, photoautotrophic cultured cells were only able to get 1/6 th approximated equivalence. This therefore implies that the organism prefers mixotrophic metabolism needing both light energy and an organic carbon substrate. Evidence supporting superiority of mixotrophic cultivation of some microalgae in organic carbon substrates had been recorded [27] [28]. In a previous study with the microalga, Ogbonna and Ogbonna [14] had demonstrated the heterotrophic and mixotrophic growth ability of the isolate using glucose and glycerol as substrates. However, using glucose and glycerol as substrates, higher biomass content was recorded than the biomass of the present study.
The percentage lipid content of Dictyosphaerium sp. and final biomass concentration under the different cultivation conditions is presented in Figure 2.
The Dictyosphaerium sp. grown in mixotrophic condition had more biomass concentration and accumulated more lipids than the other cultivation modes. The highest lipid content (%) for autotrophic, heterotrophic and mixotrophic culture modes were 15.32, 16.54 and 21.16 respectively whereas the biomass contents were 0.238, 0.63 and 1.022 g/L respectively. Using CWW as carbon and additional nitrogen source gave lower percentage lipid content than what was previously reported (42.3% ± 1.33%) where glucose and glycerol were the carbon sources [14]. The reason for the disparity could be the differences in medium compositions since glucose as a carbon source is more easily assimilated than hydrolyzed cassava starch.
The predominant fatty acid obtained with Dictyosphaerium sp. grown on glucose and glycerol carbon substrates [14] were similar to the present study implying that irrespective of the growth condition, the fatty acid composition of microalgae may be species dependent.

Properties of the Fatty Acid Methyl Esters (Biodiesel)
To ascertain the qualities of these fatty acids obtained under autotrophic, heterotrophic and mixotrophic cultivation modes for use as biodiesel feedstock, biodiesel property analyses (BiodieselAnalyzer software, [20] were carried out and whose values were presented in Table 4. The iodine value (gI 2 /100g), the cetane number, and kinematic viscosity were within the recommended limits (EN 14214 and ASTM D6751) for biodiesel obtained from Dictyosphaerium sp.
grown under the three cultivation conditions. This could signify the suitability of use of Dictyosphaerium sp. as a feedstock for biodiesel production using CWW as a substrate. The density of biodiesel produced via heterotrophic metabolism was 0.816 g/cm 2 and was therefore lower than the EN 14214 set standard. However, biodiesel from Dictyosphaerium sp. cultured under autotrophic and mixotrophic  conditions had densities within recommended standards. As a general rule, the qualities of the fatty acid methyl esters from the different cultivation modes were similar even though the quantities slightly varied. This is probably because the same substrate and microalga were used, thereby implying that the cultivation conditions (autotrophic, heterotrophic and mixotrophic) had no remarkable influence in biodiesel quality. It is also remarkable to note that most of the parameters assessed were within the ASTM D6751 and EN 14214 recommended values. Compared to the values reported for Desmondesmus subspitacus and Desmondesmus armatus, and plant oil methyl esters, the results of the present study had slight variations with saponification values of Dictyosphaerium sp. methyl ester (present study) being higher than Desmodesmus subspitcatus oil methyl ester and Desmodesmus armatus oil methyl ester (Table 4).

Bioremediation Potential of Microalga Dictyosphaerium Species Grown on Cassava Wastewater
What could make the production of biofuel from CWW attractive is the ability to combine the fuel production with bioremediation. Cassava wastewater is an odoriferous, high insect (mosquito) breeding liquid pollutant. Concomitant utilization of microalgae for biomass production for sustainable biofuels and phytoremediation had been demonstrated [5] [7] [11] [31]. In the very recent times, several attempts have also been made to bioremediate different categories of wastewater with a concomitant production of algal based biofuel. For instance, whereas Nguyen et al. [32] investigated the relationship between bacteria growth and lipid production by cultivation of microalgae Chlorella vulgaris in seafood wastewater, Leong et al. [33] compared the performances of microalgal-bacterial co-cultivation to bioremediate synthetic and municipal wastewaters whilst producing biodiesel sustainably. Rosli et al. [34]    Characteristically, CWW has been described to be high in hydrogen cyanide content (>400 mg·L −1 ) and biological oxygen demand (BOD) (25,000 -50,000 mg·L −1 ) [6]. High quantities of these two environmental parameters (hydrogen cyanide and BOD) in water indicate high pollution. In remediating CWW, Neves et al. [7] obtained a total nitrogen (g/L) and a total phosphorous (g/L) of 0.25 ± 0.01 and 0.16 ± 0.01 respectively from cassava wastewater. They also obtained from the wastewater, a carbon to nitrogen ratio of 96.0 ± 4.24 and nitrogen to phosphorous ratio of 1.50 ± 0.071. Whereas Leong et al. [10] and Leong et al. [35] focused on nitrogen removal efficiency, the present work reports cyanide removal from the culture medium. Cyanide is however a nitrogen-rich compound and its measurement alongside biofuel production and bioremediation could stand as a novel approach and a major contribution. The cyanide removal of the present study was efficient.

Conclusion
Dictyosphaerium species LC172264 under the two cultivation modes showed good remediation potentials; however, the mixotrophic cultures had more biomass and lipid contents than the heterotrophic and autotrophic counterparts.
Overall, the Dictyosphaerium species of the present study is ideal for concomitant cassava wastewater remediation and biodiesel production under mixotrophic condition.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper. Percentage reduction (%)