Cultivating Microalgae in Domestic Wastewater for Biodiesel Production

The objective of this study was to evaluate the growth of nine species of microalgae (green and blue green microalgae) on domestic wastewater obtained from Zenein Wastewater Treatment Plant (ZWWTP), Giza governorate, Egypt. The species were cultivated in different wastewater treatments namely: without treatment; after sterilization; with nutrients with sterilization and with nutrients without sterilization. The experiment was conducted in triplicate and cultures were incubated at 25±1°C under continuous shaking (150 rpm) and illumination (2000 Lux) for 15 days. Algal growth parameters i.e., pH, electric conductivity (EC), optical density (OD), dry weight (DW) and chlorophyll-a (Ch-a) were measured at zero-time and at the end of the experimental period; while, the percentages of total lipids, biodiesel and the residual sediments (glycerine, pigments, etc) were determined in the harvested algal biomass. The data revealed that domestic wastewater with nutrients and with sterilization (T3) was promising for cultivating five algal species as compared to the synthetic media. Moreover, the sterilized-domestic wastewater (T2) was the selective medium for cultivating Oscillatoria sp. and Phormedium sp; however, T1 medium (wastewater without treatment) was the promising medium for cultivating Nostoc humifusum. Biodiesel production from the algal species cultivated in synthetic media was ranging between 3.90% (Wollea saccata) and 12.52% (Nostoc muscorum). On the other hand, the highest biodiesel production from algal biomass cultivated in wastewater was obtained by Nostoc humifusum (11.80%) when cultivated in wastewater without treatment (T1) and the lowest (3.80%) was recorded by Oscillatoria sp. when cultivated on the sterilized-domestic wastewater (T2). The results of this study suggest that cultivating microalgae on domestic wastewater combines nutrients removal and algal lipid production for potential use as a biodiesel feedstock. Additionally, using the domestic wastewater, as nutrient media for microalgae cultivation, is suitable and non-expensive method as compared to the conventional cultivation methods for sustainable biodiesel and glycerol production.


Introduction
The need of energy is increasing continuously, because of increases in industrialization and population.The basic sources of this energy are petroleum, natural gas, coal, hydro and nuclear (Kulkarni and Dalai, 2006).Renewable, carbon neutral transportation fuels are necessary for environmental and economic sustainability and biofuel from biomass is one of the most promising alternatives to petroleum fuels.(U.S. DOE, 2010).
Bioenergy is one of the most important components to mitigate greenhouse gas emissions and substitute of fossil fuels (Goldemberg, 2000).Biomass is one of the better sources of energy (Kulkarni and Dalai, 2006).Large-scale introduction of biomass energy could contribute to sustainable development on several fronts, environmentally, socially and economic (Turkenburg, 2000).Biodiesel (monoalkyl esters) is one of such alternative fuel, which is obtained by the transesterification of triglyceride oil with monohydric alcohols.It has been well-reported that biodiesel obtained from canola and soybean, palm, sunflower oil, algal oil as a diesel fuel substitute (Lang et al., 2002;Spolaore et al., 2006).
Biodiesel is a nontoxic and biodegradable alternative fuel that is obtained from renewable sources.Biodiesel fuel can be prepared from waste cooking oil, such as palm, soybean, canola, rice bran, sunflower, coconut, corn oil, fish oil, chicken fat and algae (Sharif et al., 2007) which would partly decrease the dependency on petroleum-based fuel.Biodiesel from oil crops are being produced in increasing amounts as renewable biofuels, but their production in large quantities is not sustainable (Chisti, 2008).
An alternative is offered by microalgae (Chisti and Yan, 2011).Microalgae are photosynthetic microorganisms that are able to rapidly generate biomass from solar energy, CO 2 and nutrients in bodies of water.This biomass consists of important primary metabolites such as sugars, oils and lipids, for which process path-ways exist for the production of high-value products including human and animal feed supplements, transport fuels, industrial chemicals and pharmaceuticals.Algae are capable of producing 30 times the amount of oils and lipids per unit area of land as compared to terrestrial oilseed crops (Sheehan et al., 1998).
Many microalgae are exceedingly rich in oil which can be converted to biodiesel using existing technology.More cultivated in Zarrouk medium (Zarrouk, 1966).While, Bold medium (Nichols and Bold, 1965) was used for the green alga Chlorella vulgaris.Cultures were incubated in a growth chamber under continuous shaking (150 rpm) and illumination (2000 Lux) at 25±1°C for 30 days.Tab. 1 shows the characterization (OD, DW, chlorophyll a and pH) of the algal inoculums APHA (1998).

Characterization of wastewater sample
The effluent of the secondary treated sewage wastewater from Zenien Wastewater Treatment Plant (ZW-WTP), Giza Governorate, Egypt was used, after filtered using glass microfiber filter to remove large particles and indigenous bacteria, for algal cultivation experiment.The biological and physio-chemical analyses of the initial water sample were carried out following the methods described by APHA (1998) as shown in Tab. 2.

Experimental design
Algae were inoculated at 20% (V inoculation /V media ) in 500 ml Erlenmeyer flasks containing 200 ml liquid media (100% wastewater effluent supplemented with/without nutrients and with/without sterilization) and the treatments were as follows: T 1 : wastewater without nutrients or sterilization T 2 : wastewater with sterilization T 3 : wastewater + nutrients with sterilization T 4 : wastewater + nutrients without sterilization Wastewater effluent was supplemented with nutrients of NaNO 3 , K 2 HPO 4 and FeSO 4 .7H 2 O in amounts equal to those of the standard BG11, Bold and Zarrouk to be used as basal media while, the synthetic media of BG11, Bold and Zarrouk were used as control.The cultures were incubated at 25°C±1°C, under continuous shaking (150 rpm) and illumination (2000 Lux) for 15 days.
The experiment was carried out in triplicate and average values were recorded.

Algal growth analyses
The analyses of algal growth parameters i.e., pH, electric conductivity (EC), optical density (OD), dry weight (DW) and chlorophyll-a (Ch-a) were measured at zerotime and at the end of the experimental period (stationary phase of each one).than 50% of their biomass as lipids, sometimes even up to 80%, and oil levels of 20-50% are quite common (Chisti, 2007).Lipids production and biodiesel extraction from algae depend on algal species and extraction solvent system (Afify et al., 2010).
There is a unique opportunity to both treat wastewater and provide nutrients to algae using nutrient-rich effluent streams.By cultivating microalgae, which consume polluting nutrients in municipal wastewater, and abstracting and processing this resource, then the goals of sustainable fuel production and wastewater treatment can be combined (Andersen, 2005).Treated wastewater is rich in nitrogen and phosphorus, which if left to flow into waterways, can spawn unwanted algae blooms and result in eutrophication (Sebnem Aslan, 2006).These nutrients can instead be utilized by algae, which provide the co-benefit of producing biofuels and removing nitrogen and phosphorus as well as organic carbon (Mostafa and Ali, 2009).Wastewater treatment using algae has many advantages.It offers the feasibility to recycle these nutrients into algae biomass as a fertilizer and thus can offset treatment cost.Oxygen rich effluent is released into water bodies after wastewater treatment using algae (Becker, 2004).
This work aimed to evaluate the laboratory cultivation of nine algal strains belonging to Nostocales and Chlorellales in secondary treated municipal domestic wastewater for biomass and biodiesel production.

Chemicals and reagents
Pure chloroform, ether, acetone and methanol were purchased from E. Merch Co. (Germany), and dis¬tilled before use.

Preparation of algal inoculums
Cyanobacteria strains (Anabaena flous aquae, Anabaena oryzae, Nostoc humifusum, Nostoc muscorum, Oscillatoria sp., Spirulina platensis, Phormedium fragile and Wollea saccata) and the green alga strain Chlorella vulgaris were obtained from the Microbiology Department, Soils, Water and Environment Research Institute (SWERI), Agricultural Research Centre (ARC), Giza-Egypt.Cyanobacteria strains were maintained in BG11 medium (Rippka et al., 1979)  Cultures concentration was determined as optical density (OD) by spectrophotometer at 560 nm (Leduy and Therien, 1977).Chlorophyll-a was determined spectrophotometrically after extraction by absolute methanol as reported by Vonshak and Richmond (1988), pH values and algal dry weight were estimated according to Vonshak (1986).Electric conductivity (EC) was measured by conductivity meter (systronics 304) (Kar et al., 2008) Lipids extraction Dry extraction procedure according to Zhu et al. (2002) as a modification of the wet extraction method by Bligh and Dyer (1959) was used to extract the lipid in microalgae cells.Typically, cells were harvested by centrifugation at 8500 rpm for 5 min and washed once with distilled water.After drying the samples using freeze drier, the samples were pulverized in a mortar and extracted using mixture of chloroform:methanol (2:1, v/v).About 50 ml of solvents were used for every gram of dried sample in each extraction step.After stirring the sample using magnetic stirrer bar for 5 hr and ultrasonicated for 30 min., the samples were centrifuged at 3000 rpm for 10 min.The solid phase was separated carefully using filter paper (Advantec filter paper, no. 1, Japan) in which two pieces of filter papers were applied twice to provide complete separation.The extracted oil was evaporated under vacuum to release the solvent mixture solutions using rotary evaporator at 40-45°C.

Transesterification and biodiesel production
Lipids extract was mixed with a mixture of catalyst (0.25 g NaOH) and 24 ml methanol, a process called transesterification, with stirring properly for 20 min.The Mixture was kept for 3 hrs in electric shaker at 3000 rpm.(National Biodiesel Board, 2002).After shaking the solution was kept for 16 hr to settle the biodiesel and the sediment layers clearly.The biodiesel layer was separated from sedimentation by flask separator carefully.Quantity of sediments (glycerine, pigments, etc) was measured.Biodiesel was washed by 5% water many times until it becomes clear then biodiesel was dried by using dryer and finally kept under the running fan for 12 hr the produced biodiesel was measured (using measuring cylinder), pH was measured and stored for analysis.

Statistical analysis
Data were subjected to an analysis of variance, and the means were compared using the Least Significant Difference (LSD) test at the 0.05 levels, as recommended by Snedecor and Cochran (1982).

Results and discussion
The main nutrients, nitrogen and phosphorus, required for microalgae growth can be utilized from various wastewaters generated by different sources (Becker, 2004).The chemical composition of the secondary treated sewage wastewater from Zenien (Egypt) revealed that the initial sample has a great deficiency in all the required nutrients for algal optimal growth comparing with their synthetic media (Tab.2).

Algal growth parameters pH
The pH values of microalgal cultures were changed by the time from initial phase up to stationary phase depending on the algal species and the growth media.Changes in pH values of the algal cultures were ranged between 6.02 and 9.30 (Tab.3).However, the highest values (pH>9) at the stationary phase were observed with Spirulina platensis.Variation in pH can affect metabolism and growth of algae in a number of ways, including altering the equilibrium of inorganic carbon (C) species, changing availability of nutrients, and, at extremes, directly affecting cell physiology.Most algae have pH optima for growth and photosynthesis in the neutral to alkaline pH range, but species may be found growing in acid conditions as low as pH 1±0 (Albertano et al., 1971;Langworthy, 1978;Raven, 1990).Azov (1982) found that biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably Tab. 2. Chemical analyses of the initial domestic wastewater comparing with the algal synthetic media (Zarrouk,BG11 and Bold) Parameters Wastewater Standard synthetic media Zarrouk BG11 Bold found that optimal productivity of the cyanobacterium Anabaena variabilis were obtained at pH 8.2-8.4,being slightly lower at 7.4-7.8,decreasing significantly above pH 9, and at pH 9.7-9.9 the cells were unable to thrive (Mostert and Grobbelaar, 1987).However, many algal species accept higher pH values than that.In algal cultures, pH usually increases due to the photosynthetic CO 2 assimilation (Borowitzka, 1998;Chevalier et al., 2000).During algal growth, fluxes of inorganic and organic species occur between different compartments inside the cells and between the cells and the surrounding medium.In a closed system, a steady decrease in nutrients is accompanied by a change in pH and alkalinity.Changes in pH and alkalinity are the results of net fluxes of H + across the plasma membrane as well as influx of different dissociation states of weak electrolytes such as CO 2 /HCO 3 − and NH 4 + /NH 3 (Raven and De Michelis, 1979).

EC (Electrical conductivity)
Electrical conductivity (EC) refers to the ability of an aqueous solution to carry electric current.This ability depends on the presence of ions, their total concentration, mobility, and valence, and the temperature of the solution.Most solutions composed of inorganic compounds are relatively good electrical conductors.Conversely, although some organic compounds dissociate in aqueous solutions which are very good electrical conductors, most molecules in organic compounds do not dissociate in aqueous solution (Yu et al., 2005).All treatments reduced Electric conductivity (EC) values (dsm -1 ) by the time from initial phase to stationary phase (Tab.4) and this may be due to the ability of algal species to consume the nutrients during the algal growth.The important of EC as control parameters, agreed with that expressed by Oswald (1988), on the fact that total dissolved salt one among the decision factors that define the relationship culture medium species in the cultivation of microalgae.Earlier, Oswald and Gotaas (1957) studied the use of micro algae for tertiary treatment of municipal wastewater (phycoremediation) to affected by the pH at which the cultures were maintained.Goldman et al. (1981) suggested that pH tolerance limits of the algae are governed either by chemical influence on the growth medium or by metabolic effects on the cells.In addition it was concluded that the maximum tolerable pH is not influenced by the availability of inorganic carbon.However, pH is the major determinant of the relative concentrations of the carbonaceous system species in water and could affect the availability of carbon for algal photosynthesis in intensive cultures.
The pH range for most cultured algal species is between 7 and 9, with the optimum range being 8.2-8.7.Complete culture collapse due to the disruption of many cellular processes (Reactive oxygen species (ROS), fluoride and phenols accumulation) in closed systems.The decreased algal production which followed pH increase in continuous algal cultures was due mainly to decreasing the free carbon dioxide concentration in the medium (Azov, 1982).
The decrease of pH value in some treatment may be due to the ability of species for utilization the alkaline elements and this led to decrease the concentration of these elements in culture media or formation of acids compounds eg: carbonic acid during cultivation and led to decrease of pH value as reported by Richmond (1986).
High pH may affect cellular enzyme function and change the speciation of metals (McKnight et al., 2001).Since enzyme activities are pH-dependent, changes in extracellular pH are likely to affect intracellular pH and hence enzymatic activities and enzyme structure in algae.The enhanced ROS production observed in Chattonella marina under high pH (9.0 and 9.5) may possibly be attributable to an increase in the activities of enzymes regulating ROS production.The possibility also exists that elevated pH may affect metal speciation and reduce the bioavailability of iron, which may in turn enhance ROS production (Liu et al., 2007).
Microalgal growth rate and species composition may also be affected by pH.As an example, Fontes et al. (1987)  Algal growth can be expressed as biomass yield.Yield, as an expression of organic production, is usually given in terms of dry weight of the organic mass produced over a period of time per unit volume.Optical density (OD) measurements can be translated into biomass yield in terms of dry weight or chlorophyll (Tadros, 1988).
Optical density measurement is an indication of cell growth; to obtain the exact cell mass at certain optical density dry mass test was performed.Biomass yield of algae in synthetic media (control) are generally much higher than those found in most of the treatments.Nostoc muscorum, Oscillatoria sp., Spirulina platensis and Anabaena oryzae reached the maximum values of OD (0.28, 0.80, 0.47 and 0.50 respectively), DW (181.12,513.28, 303.36 and 320.0 mg l -1 , respectively) and chlorophyll content (1.05, 2.99, 1.77 and 1.86 mg l -1 respectively) when cultivated in synthetic media (control).However, Anabaena flous aquae, Chlorella vulgaris and Wollea sp.reached the highest values of OD, DW and chlorophyll content when cultivated in media contain the wastewater + nutrients with sterilization (T 3 ).Otherwise, Nostoc humifusum reached the comprise several applications: (a) nutrient removal from municipal wastewater and effluents rich in organic matter; (b) nutrient and xenobiotic compounds removal with the aid of algae-based biosorbents; (c) treatment of acidic and metal wastewaters.
Experiments on municipal wastewater effluent resulted in 23% reduction in N and 82% reduction in P (Rectenwald and Drenner, 2000).Nitrogen removal by microalgae and cyanobacteria could be limited by several environmental factors, such as light, pH and availability of carbon source (Garbisu et al., 1992;Urrutia et al., 1995).Cation concentrations such as Ca 2+ , Mg 2+ and pH affected the removal efficiency of TP (Li et al., 2006).
However, cultivation of these microalgae in different wastewater treatments (T 1 ,T 2 , T 3 and T 4 ), the lipid concentration fluctuated (decrease or increase) e.g: the lipid content in Nostoc muscorum decreased from 16.8% to 12.5% when cultivated in synthetic media (control) and wastewater + nutrients with sterilization (T 3 ), respectively.However, total lipid concentration of Anabeana flous aquae increased from 5.5 to 7.4% when cultivated in the same treatments.This may be due to the variation between the algal species in a biotic stress response and the great deficiency in all required elements in secondary treated sew-maximum values of these growth parameters in wastewater without any treatment (T 1 ) while, Phormedium fragile reached the maximum when cultivated in wastewater after sterilization (T 2 ) (Tab.8).
This variation in microalgal growth depend on the ability of different species for grown in wastewater without any treatment (T 1 ) which have low concentration from nutrients as shown in Tab. 2, or cultivation in wastewater media after treatment by sterilization (T 2 ) to prevent microorganism grown or after enrichment with nutrients to increase the nutrient concentration (T 3 and T 4 ).
The results were in an agreement with the results obtained by Wang et al. (2009), who reported that, Chlorella sp. could adapt well in different wastewaters (municipal wastewater treatment plant, MWTP) with no lag phase observed.
It is well known that the developing of algal growth and nutrient removal efficiency could be increased depending upon many factors, including algal species, cell concentration, aeration, and retention time.Many authors reported that Chlorella is a common and effective species for the immobilization and nutrient removal purposes the nitrogenous compounds and partly to form lipids and biodiesel.The data obtained in this investigation were in good agreement with results published by Widjaja (2009), who reported that the green microalgae Chlorella vulgaris accumulated high lipid content when cultivated in nitrogen depletion condition (0.02 mg/l nitrate).The present results also went parallel with those obtained by Lardon et al. (2009) who found that the control of nitrogen stress during the culture and optimization of wet extraction led age water especially nitrogen; led to increase accumulation of carbon skeleton compounds e.g.lipids (Becker, 2004).Also, the present results were in agreement with those of Shalaby et al. (2010), who reported that under salt stress conditions (with normal nitrate concentration in culture media) the algal metabolism was altered with over production of carbon skeleton which were partly directed towards the production of substances with beneficial role in algal tolerance or defence mechanism as polyols, carbohydrate, methylated amino acids and protein in addition to able bio-fuel that has the potential to completely displace petroleum derived transport fuels.The author added that oil productivity of many microalgae greatly exceeds the oil productivity of the best producing oil crops.The maximum accumulation of biodiesel after cultivating the microalgae in domestic wastewater (Tab.10) was obtained by Nostoc humifusum (11.80%) when grown in wastewater without any treatment (T 1 ).However, this value was higher than that obtained when the same species was cultivated in the synthetic medium (control).These results may due to the deficiency of nitrogen concentration in waste water media than the control media and this led to accumulation of lipid and biodiesel.These results are in agreement with the results obtained by Afify et al. (2010).

Conclusions
This study provided a proof-of-concept for a wastewater treatment process that combines nutrient removal and algal lipid production for potential use as a biodiesel feedstock.This study also contributed data on cultivation nine algal strains on secondary treated domestic wastewater for lipids production, in addition, the suitability of to maximum biodiesel production from the microalgal culture Chlorella vulgaris.
The greatest yields of biodiesel in synthetic media (Tab.9) were archived by Nostoc muscorum, Nostoc humifusum and Phormedium fragile (12.52, 10.2 and 10.1% respectively).The produced biodiesel have slightly alkaline pH values ranged between 6.9 and 8.1 in all preparations.The present results were in agreement with those of Afify et al. (2010) and Hossain and Salleh (2008).
The highest biodiesel production from Nostoc species (Nostoc muscorum and Nostoc humifusum) observed in this investigation was in good agreement with data reported by Chisti (2007) and Shalaby (2011) who demonstrated that the biodiesel from microalgae seems to be the only renew- the microalgae lipids for biodiesel production by transesterification.Overall, it could be concluded that using the domestic wastewater, as nutrient media for microalgae cultivation, is suitable and non-expensive methods when compared with the conventional cultivation methods for sustainable biodiesel and glycerol production.
except Spirulina platensis which was Tab. 3. Changes of pH values of different microalgal cultures in wastewater during the treatment period Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05 according to Duncan's multiple range test T 1 : wastewater without treatment; T 2 : wastewater after sterilization; T 3 : wastewater+ nutrients with sterilization T 4 : wastewater+ nutrients without sterilization Tab. 4. Changes of electric conductivity (dsm -1 ) values of different microalgal cultures in wastewater during the treatment period Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05 according to Duncan's multiple range test T 1 : wastewater without treatment; T 2 : wastewater after sterilization; T 3 : wastewater+ nutrients with sterilization T 4 : wastewater+ nutrients without sterilization Tab. 6. Dry weight (mg l -1 ) of different microalgal species cultivated in different wastewater treatments Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05 according to Duncan's multiple range test T 1 : wastewater without treatment; T 2 : wastewater after sterilization; T 3 : wastewater+ nutrients with sterilization T 4 : wastewater+ nutrients without sterilization Tab. 8. Correlation coefficient (r) among the growth parameters of different microalgae cultivated under various conditions (T 1 -T 4 ) *EC: Electric conductivity; OD: Optical density; Chl: Chlorophyll; DW: Dry weight Tab. 10.Total lipids, biodiesel and glycerine+pigments percentages of different microalgal species cultivated in different wastewater treatments Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05 according to Duncan's multiple range tests.T 1 : wastewater without treatment; T 2 : wastewater after sterilization; T 3 : wastewater+ nutrients with sterilization T 4 : wastewater+ nutrients without sterilization Tab. 9. Total lipids, biodiesel, glycerine+pigments percentages, color and pH of biodiesel from different microalgal species cultivated in standard synthetic media Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05 according to Duncan's multiple range tests T 1 : wastewater without treatment; T 2 : wastewater after sterilization; T 3 : wastewater+ nutrients with sterilization T 4 : wastewater+ nutrients without sterilization