Abstract: Microalgae are considered one of the viable feedstocks having potential to replace agricultural products for bioethanol production. The culture costs of microalgae remain the major hurdle in using them to produce bioethanol. This study focuses upon cultivation of a freshwater branched microalga Stigeoclonium sp., Kütz. BUM11007 in domestic wastewater and use the biomass for bioethanol production by Saccharomyces cerevisiae. Initially the microalga Stigeoclonium sp., was tested for their efficiency to remove the nutrients from different wastewater. When the organism was allowed to grow in wastewater they almost use all the available nutrients from the medium within 14 day. Among the various culture setups established, maximum growth (2.38±0.34 g L–1 dry cell weight) was observed from the setup composed of a wastewater (WW1) with 50% strength of Chu10 medium. The carbohydrates and starch contents of the cell were almost similar among the different culture setups. The algal biomass was harvested and tested for their usefulness in bioethanol production. The algal biomass was acid treated to dissolve the complex sugars. The sugar extract was used as the fermentation medium for bioethanol production with Saccharomyces cerevisiae. After 24 h of anaerobic fermentation, the yield was quantified as 0.195 g ethanol/g of microalgal biomass. The result proves the possibility of developing integrated process for bioethanol production with microalgal biomass cultivated from wastewater.
INTRODUCTION
Over the past decades, rising population has drastically increased mechanized transportation, consequently augmented fuel demand. In the present scenario transportation is mainly relying on fossil fuels, thereof release in carbon dioxide to the atmosphere remains obvious resulting in global warming. The fossil fuel reserves are projected to be exhausted within next 50 years. Considering their deleterious effects on the atmosphere and insecurity about the supplies, views of promising researchers have turned towards finding alternatives to the fossil fuels (Chisti, 2008). Biofuel are renewable, sustainable, nontoxic, biodegradable, eco-friendly and considered potential source to replace fossil fuels (Praveenkumar et al., 2012a). To date, bio-ethanol is one of the largely produced and widely used biofuels. Brazil, Canada and the USA are the leading producers of bio-ethanol around the world (Chiaramonti, 2007). Bio-ethanol is renewable, biodegradable and easily blended with gasoline. It emits lower amount of carbon monoxide, hydrocarbons, carbon dioxide and NOx gases (Wyman, 1996). All the desirable characteristics makes bioethanol much attractive than petrochemical products (Sanchez and Cardona, 2008). In spite of such remarkable properties bioethanol still remains a partial substitute to fossil fuels. Till date bioethanol is produced from edible crops (sugarcane and corn) which raised socio-economic and political issues on food supply. Over a decade strong opposition has been shown on utilization of food crops for bioethanol production. Considering the negative impact United Nation Environmental Protection agency (UNEP) waived regulation to use limited amount of ethanol for vehicle fuel (Auld, 2012). At this point selection of a suitable feedstock addressing the issues of both economic viability and food security remains decisive. For the past years several alternative feedstocks; sucrose based, starch based and lignocellulosics have been tested for bio-ethanol production. Microalgae are phototrophic organisms that have ability to grow rapidly by fixing atmospheric carbon dioxide and accumulate carbohydrates mainly in the form of starch, Moreover micro-algae do not warrant arable land for their growth thereby do not compete food crops, thereby micro-algae provide greater advantages sustainably (Matsumoto et al., 2003). Holding all the desirable characteristics, micro-algae are considered interesting and eligible feedstock for bio-ethanol (Gao et al., 2010). The drawback associated with microalgal feedstocks is their cost of cultivation and requirement of huge water resource (Uri et al., 2010). One possible and attractive solution to overcome this issue is to cultivate the micro-algae in wastewater. Some studies have shown that microalgae efficiently use wastewater nutrients NH4+, NO3–, PO43– for its growth (Kim and Jeune, 2009; Craggs et al., 1997). Thus by using the wastewater we can bring down the culturing cost of micro-algae and water demand. To date, few studies have reported cultivating microalgae in wastewater for biodiesel production (Sydney et al., 2011; Zhou et al., 2011) but there are no such reports dealing with utilization of wastewater nutrients to cultivate micro algae for bio ethanol production. In this background the present study aimed at finding the possibility of cultivating an indigenous micro-alga Stigeoclonium sp., Kütz. BUM11007 in domestic wastewater and evaluating their efficiency as feedstock for bio-ethanol. The fresh water branched micro-alga Stigeoclonium sp., was grown in domestic wastewater. The time course of nutrient removal by the alga was monitored. Sugars from the cultivated algal biomass was extracted and used for ethanol fermentation by Saccharomyces cerevisiae.
MATERIALS AND METHODS
Wastewater sample: The wastewater samples used in this study were collected from the Department of Plant Science (WW1) and Department of Biomedical Science (WW2), Bharathidasan University, Tiruchirappalli, India. The samples were allowed to settle for 10 days to remove the sludge and filtered through 0.45 μm cellulose acetate membrane prior to use. The samples were characterized before each experiment using several specific analyses, determining pH, salinity and nutrient contents such as nitrate, phosphate, ammonia and sulphate.
Culture and growth: The microalgae Stigeoclonium sp., Kütz. BUM011007 was obtained from Microalgal Repository of Division of Microbial Biodiversity and Bioenergy (DMBB). The alga was grown in Chu10 medium. The composition of Chu10 medium in 1000 mL is as follows: Calcium nitrate-0.232 g, dipotassium hydrogen phosphate -0.01 g, magnesium sulphate -0.025 g, sodium carbonate -0.02 g, sodium silicate-0.044 g, ferric citrate-3.5 mg, citric acid-3.5 mg, boric acid-2.4 mg, manganese chloride-1.4 g, zinc chloride-0.4 mg, cobalt (II) chloride-20 μg and copper (II) chloride -0.1 μg. For experimentation the alga was grown in five different setups: A-Chu10 medium, B- WW1, C-WW2, D-WW1 with 50% Chu10 medium and E-WW2 with 50% Chu10 medium. The pH of the culture setups were maintained at 7 and incubated at 24°C under a light intensity of 50 μmol m‾2 sec–1 with 14/10 light/dark cycles.
Analysis of growth, nutrient removal and biochemical compositions: Culture supernatant was collected every 2 days for 14 days for the following analyses. Growth rate of the alga under different culture conditions were monitored following the values given by Praveenkumar et al. (2012b). The initial pH and salinity was measured. The removal nutrients such as nitrate, ammonia, phosphate and sulphate from the wastewater were estimated spectrophotometrically (APHA, AWWA and WPCF., 2005). The total carbohydrates (Gerhardt et al., 1994) and starch (Rose et al., 1991) contents of alga were measured spectrophotometrically.
Extraction of sugars from microalgal biomass: After 14 days of culture, the biomass was harvested and lyophilized. About 5% (w/v) of dried cell powder was treated in 3% (v/v) sulphuric acid at 110°C for 30 min and centrifuged (Nguyen et al., 2009). The extracted sugars were obtained from the supernatant. The sugar filtrate was neutralized with sodium hydroxide, sterilized and stored at 4°C until further use.
Ethanol fermentation by Saccharomyces cerevisiae: Saccharomyces cerevisiae cell were activated by dissolving 5 g of ordinary baker buts yeast in 50 mL of warm water and left for 15 min with proper agitation. Saccharomyces cerevisiae inoculum was prepared by inoculating 3% (v/v) of yeast in sterilized LB broth. The culture was incubated at 30°C for 24 h at 200 rpm. Ten percentage of overnight grown S. cerevisiae inoculum was transferred to the pre-sterilized sugar filtrate under aseptic condition. The flask was left for fermentation under anaerobic condition at 30°C for 24 h. The fermentation mixture was filter sterilized through 0.2 μm membrane filter and subjected to GC analysis.
Gas chromatographic analysis: The fermented samples were analyzed by gas chromatography (Shimadzu, GC2014, Japan) with Flame Ionization Detector (FID). One micro liter of the sample was injected in the Rtx-5- Amines column (Restek, USA) (5% diphenyl/95% dimethyl polysiloxane). Temperature program was as follows: Initial 60°C with 1 min hold; ramp 4°C/min to 70°C with a 1 min hold. Column flow was set at 22.2 mL min–1. Instrument condition was as follows: Carrier gas nitrogen; FID set at 260°C, total run time for a single sample was 4.5 min. Identification was made by comparison with standard. A standard calibration curve for ethanol was generated with different concentrations (1-5% v/v) to detect the actual ethanol concentration in the sample.
Statistical Analysis: Data was statistically analyzed using SPSS Statistics 17.0 software (Statistical Program for Social Sciences 17.0) and the results were expressed as Means±Standard Deviation of three independent replicates. The significance level used for all conclusions was p<0.001 and was derived from an analysis of variance (ANOVA) followed by Duncan test.
RESULTS AND DISCUSSION
Removal of nutrients from the medium: The initial pH of both wastewater WW1 and WW2 was 7 and salinity was zero. Stigeoclonium sp., was tested for their ability to utilize the nutrients from wastewater. Sufficient amount of phosphate and sulphate was found in WW1 and WW2 next to nitrates (Table 1). Phosphates and sulphates are well known for their ability to stimulate algal growth. Apart from the available phosphates nitrates and sulphates, the wastewater may also be rich in organic carbon. Comparing the nutrient profile of all five culture setups, D significantly (p<0.001) stands alone with almost all the nutrients falling either under upper or near upper classification (Duncan). The alga grown in setup A readily starts depleting the nutrients from the first day onwards. Where as in other setups say B, C, D and E the alga takes a lag phase of 2-4 days and once gets adapted, utilizes the nutrients faster than the later (Fig. 1).
| Table 1: | Nutrient analysis report of different culture setups |
A: Chu10 medium, B: WW1, C: WW2, D: 50% Chu10 with WW1, E: 50% Chu10 with WW2, Data represent the Mean ±Standard Deviation of three separate trials, Within the same row, significant differences are indicated by different superscripts (p<0.001) | |
| Fig. 1(a-e): | Time course of nutrient removal from different medium by Stigeoclonium sp. Data represents the Mean±Standard Deviation (w/v) of three separate trials. (a) A: Chu10 medium, (b) B: WW1, (c) C: WW2, (d) D: 50% Chu10 with WW1 and (e) E: 50% Chu10 with WW2 |
| Fig. 2: | Dynamics of Stigeoclonium sp., Growth in different medium. Data represents the Mean±Standard Deviation (w/v) of three separate trials, A: Chu10 medium, B: WW1, C: WW2, D: 50% Chu10 with WW1 and E: -50% Chu10 with WW2 |
At the 14th days of culture the nutrients in the medium were almost depleted by the alga. At the end of cultivation nitrate/phosphate/ammonia/sulphate removal percentage in Chu10 medium was about 90/50/10/40, whereas in the setup D it was 95/45/45/60. The alga was well adapted to setup D and exhibited maximum removal of the available nutrients. Earlier report suggests that the maximum removal rate of nitrogen and phosphorous from domestic wastewater with Chlorella vulgaris was 53.8 and 49.8% (Singh and Dhar, 2007). Another report states that at 50% concentration of wastewater Spirulina platensis and Scenedesmus quadricauda removes lower amount of nitrogen and phosphate (Gantar et al., 1991). Comparatively the present results show much higher rate of nutrient removal. This proves the efficiency of the test algae in wastewater treatment.
Growth and biochemical compositions of alga: The growth rate and biochemical composition of Stigeoclonium sp., was tested separately in Chu10 medium, wastewater and combination with 50% Chu10 medium. In Chu10 medium the organism exhibited a steady growth reaching the maximum biomass concentration of 1.57±0.09 g L–1 at 14th day of culture. Notably the mixture of WW1 and Chu10 medium (D) supported maximum growth of the alga (2.38±0.12 g L–1) than in any other setups (Fig. 2). The maximum biomass concentration under setup D is attributed to its congenial nutrient profile. The increased growth rate in setup D may be due to the availability of certain micro nutrients supplemented through 50% of Chu10 medium. This finding is consistent with other report, demonstrating that wastewater can be used as an efficient growth substrate for microalgae in addition with some growth nutrient (Mutanda et al., 2011). Thus the above results indicate that the use of WW1 with 50% of Chu10 medium can promote high rate of nutrient removal and maximum biomass yield and thereby reduce the culturing costs.
The total carbohydrates and starch accumulation of the alga under different culture setups are presented in Fig. 3. Gradual increase in the total carbohydrates and starch content in the cell was observed pertaining to their growth rate. Among the different setups, alga grown in setup D accumulates maximum carbohydrates (380±2.3 mg g–1). Maximum starch content was reported in algal biomass obtained from setup E (184±1 mg g–1). Statistically there were no significant differences (p<0.001) between the carbohydrate and starch contents among the different setups. This shows the flexibility of adopting any of conditions in the perspective of carbohydrate accumulation but when combining the results of growth rate and carbohydrate accumulation it is clear that setup D (WW1 with 50% strength of Chu10) will be more suitable for cultivation of the alga for bioethanol.
| Fig. 3(a-e): | Time course of total carbohydrates and starch accumulation in different medium by Stigeoclonium sp. Data represents the Mean±Standard Deviation (w/v) of three separate trials, (a) A: Chu10 medium, (b) B: WW1, (c) C: WW2,(d) D: 50% Chu10 with WW1 and (e) E: 50% Chu10 with WW2 |
Jiang et al. (2011) examined the possibility of utilizing the mixture of municipal wastewater and seawater to cultivate Nannochloropsis sp. and reported that 50% of wastewater with seawater yielded maximum biomass, results of which were very similar to the present investigation.
Ethanol production: Ethanol fermentation was carried out with S. cerevisiae. They grow rapidly in glucose or sucrose rich medium to convert them in to ethanol. The produced ethanol is often released by the cell through the medium, thereby making the recovery process much easier. Since yeast can ferment only simple sugars, the microalgal biomass needs to be pretreated for the release and breakdown of intact complex carbohydrates to simple sugars. Miranda et al. (2012) concluded that utilization of acid for the disruption of cell wall was very efficient when compared to other physical methods and comparatively higher yield of sugar as well as lesser amount of harmful compounds were produced. Nguyen et al. (2009) demonstrated such process of using 3% sulphuric acid for extraction of sugars from Chlamydomonas reinhardtii to grow yeast and produce ethanol. Hence, in this study acid pretreatment was employed to extract the sugars from Stigeoclonium sp., The sugar extract was used as the fermentation medium for ethanol production. Often lower temperatures are recommended for ethanol production (Harun et al., 2009), hence the fermentation was carried out at 30°C. Fermentation was stopped at 24 h and the yield of ethanol was 0.195 g ethanol/g of algal biomass (19.5%). In a similar experiment, Nguyen et al. (2009) reported 29.2% ethanol production with pretreated algal biomass. Comparing with their data, the ethanol yield in this study is slightly lower. This is mainly due to the lower carbohydrate content per cell. Nevertheless, the alga Stigeoclonium sp., should be noted that they are efficient feedstocks for biodiesel (Praveenkumar et al., 2012a). With this in focus, work is currently being performed to integrate the production of both biodiesel and bioethanol sequentially from the same alga to establish a microalgal biorefinery.
CONCLUSION
In our study the ability of Stigeoclonium sp., to utilize wastewater nutrients was tested. The utility of cultivated algal biomass for ethanol production was investigated. The alga efficiently removed nutrients from wastewater and accumulates maximum biomass. Saccharomyces cerevisiae readily ferments the generated algal sugars to ethanol (0.195 g g–1). Thus, out of the present study an easy and economical way of generating biofuel is evident. The whole work emphasized an ecofriendly, economical and technical approach towards the production of bioethanol. The results obtained from this study were out of the preliminary examinations and furthermore, there are many possibilities to improve the ethanol production through optimization of rate determining steps and to integrate the process with biodiesel production to develop a microalgal biorefinery. The report demonstrates the technical feasibility of bioethanol production from microalgae cultivated from wastewater nutrients.
ACKNOWLEDGMENTS
The financial support from the Department of Biotechnology (DBT), Government of India is gratefully acknowledged (Project reference: BT/PR/6619/PBD/26/310/2013 and BT/IN/Indo-UK/SuBB/23/NT/2013).