Bioresource Technology xxx (2014) xxx–xxx

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Bioresource Technology

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Cultivation of microalgae in dairy effluent for oil production and removalof organic pollution load

http://dx.doi.org/10.1016/j.biortech.2014.03.0280960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +91 471 2515368; fax: +91 471 2491712.E-mail address: rajeevs@niist.res.in (R.K. Sukumaran).

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivation of microalgae in dairy effluent for oil production and removal ofpollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.028

Ummalyma Sabeela Beevi, Rajeev K. Sukumaran ⇑Centre for Biofuels, Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Industrial Estate PO, Trivandrum 695019, India

h i g h l i g h t s

� Chlorococcum sp. RAP-13 can grow and adapt to dairy effluent.� It can produce significant amount of biomass and lipids using dairy effluent as medium.� Accumulates up to 42% cell weight as lipids in effluent supplemented with waste glycerol.� Algal cultivation reduces the organic pollution load of the effluent.� Fatty acid profile of algal oil indicates suitability for biodiesel and nutraceuticals.

a r t i c l e i n f o

Article history:Received 30 December 2013Received in revised form 4 March 2014Accepted 6 March 2014Available online xxxx

Keywords:MicroalgaeChlorococcumDairy effluentHeterotrophicBiodiesel

a b s t r a c t

Dairy effluent (DE) was evaluated for cultivation of the oleaginous micro alga Chlorococcum sp. RAP13under mixotrophic and heterotrophic modes. The alga grew better and accumulated more lipids underheterotrophic cultivation. Supplementation of biodiesel industry waste glycerol (BDWG) to DE enhancedthe biomass production as well as lipid accumulation. While the biomass yield was 0.8 g/L for mixo-trophic cultivation, it was 1.48 g/L and 1.94 g/L respectively when cultivated with 4% or 6% BDWG. Thecells accumulated 31% lipid when grown in mixotrophic mode, and heterotrophic cultivation with 4%or 6% BDWG resulted in a lipid accumulation of 39% and 42% respectively. Saturated fatty acids produc-tion was elevated in the DE, and the major fatty acid components of the algal oil were palmitic (16:0),oleic (18:1), stearic (18:0), linoleic (18:2) and linolenic (18:3) acids. DE quality improved with reductionin COD and BOD after algal cultivation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Algal oil is considered as one of the most potent resources forbiofuels and is limited mainly by the yield under phototrophiccultivation and the challenges in harvesting. Microalgae areconsidered the best organisms to produce oil, due to their abilityto photosynthetically fix atmospheric CO2, producing biomass moreefficiently and rapidly than terrestrial plants (Chisti, 2007). Themajor challenges in mass production of algal biomass are the costsof fertilizers, harvesting and the availability of a suitable source ofwater, besides the limitations imposed by lighting, especially whenthe algae are grown phototrophically (Danquah et al., 2009).Recent advances in systems biology, genetic engineering andbio-refinery approach present opportunities to expand algal lipidproduction from a craft to a major industrial process in near future(Wijffels and Barbosa, 2010).

Micro algal cultivation is either phototrophic, mixotrophic orheterotrophic depending on the algal strains’ adaptation with theirenvironment. Micro algae have immense potential for adaptingwith the fluctuating environmental conditions. While phototrophiccultivation of microalgae is advantageous in that it requires onlysunlight and CO2 for growth, it can be limited in terms of achievinghigh biomass yield and hence oil production. Heterotrophic growthof microalgae eliminates the requirement for light and hence offersthe possibility of increasing algal cell density and productivities.Therefore, micro algae adapted to heterotrophic growth on cheapcarbon substrates are of great significance in production of algaloil or other value added products. The limitations here would bethe sourcing of the cheap carbon source in sufficient quantities.

An economic process for algal oil based fuel also depends onsource of water, fertilizers and organic carbon source used forcultivation (Yang et al., 2006). As fresh water is projected to be avaluable commodity in the future, spending of this important re-source for algal cultivation may be considered as a luxury. Use ofindustrial effluents or sea water could be a viable alternative as

organic

2 S.B. Ummalyma, R.K. Sukumaran / Bioresource Technology xxx (2014) xxx–xxx

many effluents such as those from food processing industries arerich in nitrogen and phosphorus, besides having high carbon con-tent. Increase in population and industrialization in developingcountries like India, generate large amount of effluent that mustbe treated before being discharged into natural water bodies. Dairyindustry is ubiquitous all over the world, but their manufacturingprocesses vary tremendously. This sector generates huge volumeof wastewater and its pollution is primarily organic (Briao andTavares, 2007). Dairy industry generates about 0.2–10 L of effluentper litre of milk processed (Vourch et al., 2008). In general, theliquid waste stream from dairy industry has high organic contentwith high levels of protein, nitrogen, phosphorous, dissolvedsugars and nutrients. Organic waste present in the dairy effluentis a serious environmental threat due to the high COD and BODand problems associated with rapid putrefaction. There are a largenumber of studies on the treatment of industrial, municipal andagricultural waste waters by micro algal culture systems (Samoriet al., 2013; Zhu et al., 2013). Micro algae can grow in effluentsand produce valuable biomass while they remove organic contentand minerals for building the biomass. Cultivation of micro algae indairy effluent offers several advantages such as (1) biomassproduction utilizing the organic carbon, nitrogen and mineralswithout need for any additional nutrients (2) reduction of CODand BOD of the effluent (3) oxygenation of the treated effluentand (4) possibility to extract high value products like, lipids,proteins and carbohydrates for fuel, pharmaceutical/nutraceuticaland chemical industries. Integrated strategies to enhance the costeffectiveness and environmental sustainability of algal cultivationinvolve combining the benefits of biofuel production, CO2 mitiga-tion and wastewater treatment.

The present study was undertaken to evaluate the use ofuntreated dairy effluent as a source of nutrition for the intensivemicro algal cultivation. The fresh water micro alga Chlorococcumsp. RAP-13 was used for this investigation to evaluate biomassand lipid accumulation in dairy effluent (DE). The culture wasstudied for growth and lipids production under mixotrophic andheterotrophic conditions, the latter with supplementation ofbiodiesel industry waste glycerol (BDWG). The suitability of the ex-tracted oil for biofuel production was assessed by measuring typeand relative proportions of fatty acids by GC analysis. Removal oforganic pollution load was monitored as reduction in of BOD andCOD of effluent.

2. Methods

All solvents and reagents were either HPLC grade or AR gradefrom either Merck India or Sigma–Aldrich, India. Biodiesel industrywaste glycerol (BDWG) was a kind gift from Prof. K.B.Ramachandran, Indian Institute of Technology, Chennai, India.

2.1. Sample collection

Dairy effluent for this study was obtained from milk processingfactory of Kerala co-operative milk marketing federation (MILMA)at Ambalathura, Trivandrum India. Fresh Dairy effluent was col-lected in sterilized containers before being sent to the effluenttreatment plant, and was brought to the laboratory. The dairyeffluent (DE) was stored at 4 �C until used.

2.2. Organism and growth condition

Chlorococcum sp. RAP-13, a fresh water alga isolated in ourlaboratory was used for the study. Stock culture was inoculatedinto dairy effluent and was incubated for 15 days with an approx-imately 13 h light and 11 h dark cycle at 30 ± 2 �C and with

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivatiopollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech

100 rpm agitation. Micro algal culture so adapted to DE was usedfor the mixotrophic and heterotrophic cultivation experiments.Mixotrophic cultivation experiments were performed in 500 mlErlenmeyer flasks, each containing 200 ml of untreated sterilizedDE. Each flask was inoculated with 10% (v/v) of an inoculum con-taining 3 � 106 cells/ml produced as above and was incubated un-der static conditions in an environmental chamber equipped withfluorescent lamps (Illumination �3000 lux) with temperature anddiurnal light cycle as above. For heterotrophic cultivation, the dairyeffluent was supplemented with 2%, 4% or 6% (v/v) of BDWG. Ster-ilized medium (200 ml) taken in 500 ml flasks were inoculated at10% v/v level with the same inoculum as above and the cultivationwas carried out as for heterotrophic growth studies but withoutlight. All experiments were carried out in triplicates.

2.3. Analytical procedures

Growth of algal cells in DE under both mixotrophic and hetero-trophic cultivation was monitored as change in optical density at680 nm compared to a un-inoculated DE sample. Morphologicalchanges of Chlorococcum sp. RAP-13 grown on dairy effluent wasobserved under phase contrast microscope. Lipid accumulationinside the cells grown in DE was monitored by Nile Red assayaccording to (Chen et al., 2009). Briefly, the cells were harvestedby centrifugation (8000 rpm, 10 min), washed with 0.2 Mphosphate buffer (pH 6. 8) and 40 ll Nile Red solution in acetone(containing 25% DMSO) was added. The cell suspension wasvortexed for 2 min and was incubated at room temperature(30 ± 2 �C) for 10 min. Fluorescence intensity of neutral lipid wasmeasured in a multimode reader (Tecan Infinite M200 pro,Switzerland) at a wave length of 575 nm.

2.4. Physicochemical analysis of dairy effluent

The physicochemical parameters of DE used for algal cultivationwere analyzed every 3 days starting from the third day of inocula-tion up to 12 days, for monitoring the reduction in organic pollu-tion load. The chemical oxygen demand (COD) and biochemicaloxygen demand (BOD) were analyzed according to the standardmethod described in (Clescerl et al., 1999). Changes in pH duringalgal growth were monitored by using a pH meter. The reductionin organic pollution load was expressed as percentage of the valuesfor control (un-inoculated fresh DE).

2.5. Biomass and lipid extraction

Micro algal biomass from both mixotrophic and heterotrophiccultures was harvested by centrifugation at 8000 rpm for 10 minon the 12th and 15th day of incubation. Biomass was washed withdistilled water to remove the salt and was lyophilized to removewater. Weights of the lyophilized cells were taken and were ex-pressed as mg/l. Total lipids were extracted from the dried biomassby solvent extraction method as described by Bligh and Dyer(1959). A 2:1 mixture of chloroform and methanol was added tothe biomass and sonicated for 10 min. Chloroform layer containinglipid fraction was separated using a separating funnel and 2 ganhydrous Na2SO4 was added for removing moisture. The lipidfraction was taken in a pre-weighed round bottom flask and the li-pid weight was determined after evaporation of chloroform in a ro-tary evaporator. Lipid yield was expressed as percentage of the drycell weight of algal biomass (% DCW).

2.6. Thin layer chromatography analysis

TLC analysis was carried out to identify the lipid components inthe extracted oil. TLC aluminum sheets coated with 0.2 mm

n of microalgae in dairy effluent for oil production and removal of organic.2014.03.028

S.B. Ummalyma, R.K. Sukumaran / Bioresource Technology xxx (2014) xxx–xxx 3

thickness silica gel 60F254 (E Merck, India) were spotted with 5 llof each lipid sample. Samples were separated using a solvent sys-tem with hexane, diethyl ether and acetic acid in the ratio of80:20:1. Lipids were developed in a glass chamber and the spotswere visualized using iodine vapors.

2.7. Fatty acids analysis

Fatty acid profile of the algal oil was determined as fatty acidmethyl esters (FAME). Transesterification reactions were per-formed with 2% HCl in dried methanol at 100 �C for 1 h. FAME wereextracted with hexane and dried over anhydrous Na2SO4. The sam-ples were analyzed in a gas chromatograph (6850N, Agilent Tech-nologies) equipped with FID detector and using an Agilent DB-225capillary column. The oven temperature was programmed as160 �C (2 min) – to – 230 �C at a ramping rate of 5 �C/min and finalhold at 230 �C for 20 min. The carrier gas (N2) flow rate was1 ml min�1 and the sample injection volume used was 1 ll. Theinjector and detector temperature were maintained at 230 �C and270 �C respectively. The peak area percentages were recorded witha standard HP-ChemStation� data system. The FAME was identi-fied by comparing their fragmentation pattern with internal stan-dards (Sigma Aldrich, India).

3. Results and discussion

3.1. Algal growth in dairy effluent

The diary effluent had an off white colour with offensive odourduring collection, and was highly alkaline with a pH of 10.6. Theeffluent was used for algal cultivation without any conditioningor pH adjustments. Chlorococcum sp.-RAP-13 could adapt to theeffluent and produce biomass under both mixotrophic and hetero-trophic cultivation in this medium. Growth was higher undermixotrophic mode compared to heterotrophically grown cellswhen only DE was used as the medium (Fig. 1). Supplementationof BDWG in dairy effluent as additional carbon source improvedthe algal growth under heterotrophic cultivation. Maximumgrowth for heterotrophic cultivation was obtained with 6% supple-mentation of BDWG in the DE (5.3-fold increase from 2nd to 15thday of incubation). Dairy effluent contained lesser amounts of sug-ars (0.02 g/L) compared to the normal levels used for heterotrophiccultivation of microalgae, even though the effluent was used with-out any dilution. Additional carbon source in the form of glycerolwould have provided a relatively easy to assimilate energy source,

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Fig. 1. Growth of Chlorococcum sp. RAP13 in dairy effluent. DE – dairy effluent, Ht –heterotrophic, BDWG – biodiesel industry waste glycerol.

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivatiopollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.

thus supporting a higher biomass production. Microscopic obser-vation of the heterotrophically grown algal cells showed differ-ences in colour and morphology. The cells were brownish incomparison with the mixotrophically grown cells, which weregreen. Changes in pigment production in heterotrophic culturesof green algae are reported by many. It has been reported that un-der heterotrophic conditions, reduction in chlorophyll and carote-noids and a decrease in chlorophyll a/b ratio is part of the darkadaptation (Young, 1993). Phase contrast images showed that algalcells are aggregated when growing in DE. Cell aggregation was ob-served approximately from the 12th day onwards indicating thatthe onset of cell aggregation coincided with the stationary phase.Aggregate formation is an important quality of microalgae thathelps in self sedimentation, which in turn lowers the cost of cellharvesting. These results showed that the cultivation of Chlorococ-cum sp. RAP 13 in heterotrophic mode improved the cell density incomparison to the mixotrophic condition and there were visibledifferences in the cell properties. Similar results were obtainedfor dairy waste water treatment by the micro algae Botryococcus(Shen et al., 2008) and Chlamydomonas polypyrenoideum (Kothariet al., 2013).

3.2. Biomass and lipid production

Biomass and lipid production by Chlorococcum sp. RAP-13 wasevaluated for both mixotrophic and heterotrophic cultivation.The alga grew better and accumulated more lipid under heterotro-phic cultivation in DE with glycerol supplementation compared tomixotrophic cultivation. Under mixotrophic growth, the algal cellswere lemon green in color which gradually turned to dark green bythe 2nd week of cultivation. Heterotrophic cells, on the other handwere brownish. While the biomass yield was 0.8 g/L for mixo-trophic cultivation, it increased to 1.478 g/L and 1.94 g/L in hetero-trophic cultivation with 4% or 6% BDWG supplementationrespectively (Fig. 2). Further increase in glycerol concentration re-sulted in a decreased growth. Heterotrophic growth on raw DEsupported the lowest biomass yield 0.58 g/L which was lower thaneven the mixotrophic growth. It may be speculated that the avail-able carbon in raw DE may not be sufficient to support enhancedgrowth under heterotrophic cultivation of the alga and supplemen-tation of additional carbon source improves the biomass yieldsignificantly. For the economic production of algal biomass, hetero-trophic growth on a cheap carbon substrate is often suggested asan alternative to conventional low density phototrophic race waycultivation (Doucha and Livansky, 2012). The cost of carbon sourcerepresents �50% of the cost of medium in algal cultivation (Chenget al., 2009) and effluents like those from dairy industry canprovide a potent feedstock for cultivation of algae. Similarly,BDWG is a waste product from biodiesel industry, which may beused in algal cultivation without incurring high cost for carbonsupplementation.

Lipid accumulation in the cells enhanced with the increase insupplementation of BDWG in DE medium. While the cells accumu-lated 31% of their dry cell weight as lipid under mixotrophic culti-vation, the lipid content improved to 36, 39 and 42% respectivelyfor cells cultivated heterotrophically with 2%, 4% or 6% BDWGrespectively (Fig. 2). The lipid yields from heterotrophic cultiva-tions were 0.36, 0.58 and 0.8 g/L for the 2%, 4% and 6% glycerol sup-plementation respectively. Previous work on Chlorococcum sp.grown in municipal effluent had reported 30% lipid accumulationin the cell (Mahapatra and Ramachandra, 2013). Similarly, studieson green algae grown in dairy and municipal wastewaters hadyielded a maximum lipid content of 29% (Woertz et al., 2009). Re-cently, the micro alga C. polypyrenoideum was shown to accumu-late 42% of its dry cell weight as lipid when grown on dairyeffluent (Kothari et al., 2013). Oleaginous green algae typically

n of microalgae in dairy effluent for oil production and removal of organic2014.03.028

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Fig. 2. Growth and lipid production by Chlorococcum sp. RAP13 grown in dairy effluent. DE – dairy effluent, Ht – heterotrophic, BDWG – biodiesel industry waste glycerol, %DCW – lipid content as % dry cell weight. Biomass and lipid estimations were performed at the end of cultivation after harvesting the entire cells from each culture.

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Fig. 3. Cellular lipid accumulation under various growth conditions and TLCprofiling of the extracted algal oil. (A) Accumulation of neutral lipids by the algaunder various modes of growth monitored as Nile Red fluorescence. (B) TLC profileof the oil produced by alga under different modes of growth. Lane information-1:control oil (tripalmitate), 2: DE –negative control, 3: oil from mixotrophic, 4:heterotrophic in DE, 5: heterotrophic in DE with 2% BDWG, 6: heterotrophic in DEwith 4% BDWG, 7: heterotrophic in DE with 6% BDWG.

4 S.B. Ummalyma, R.K. Sukumaran / Bioresource Technology xxx (2014) xxx–xxx

have an average total lipid content of 25% DCW in normal photo-trophic mode, which can be raised to 55% DCW when they aregrown under stress conditions or heterotrophically (Xu et al.,2006; Hu et al., 2008).

The intracellular neutral lipid accumulation of algal cells wasmeasured by the ‘Nile Red’ fluorescence emission. Staining wasperformed on cells starting from the 7th day of incubation. Thefluorescence intensity of cells increased in proportion to the dayof harvesting, indicating that the age of cells may be a critical factorin lipid accumulation with older cells accumulating more lipids(Fig. 3A). In the case of heterotrophically grown cells, lipid accumu-lation increased both with age and the amount of carbon supplied.Maximum accumulation of lipid was observed for 6% BDWG sup-plemented medium on the 15th day of incubation. TLC analysisof the extracted lipid indicated that neutral lipids form the majorfraction (Fig. 3B). The Tri Acyl Glycerides (TAG) spot was moreprominent in the oil from heterotrophic cells. This is advantageousif the oil is to be considered for biodiesel applications since TAGsare the raw materials for transesterification reactions to producebiodiesel (Chisti, 2007). The cultivation of Chlorococcum sp. RAP13on effluent medium with crude glycerol therefore may be consid-ered to provide added advantage of the value addition of DE andwaste glycerol.

3.3. Fatty acid profiling

GC analysis of FAMEs from the algal oil showed that saturatedfatty acid content was high in the oil regardless of the growthmode. Major fatty acid from Chlorococcum sp.-RAP-13 oil producedin DE was palmitic acid (44–54%) followed by oleic acid (21–27%)and stearic acid (2–16%). Linoleic acid (C18:2), linolenic acid(C18:3) and other fatty acids were low in the oil (Table 1).Saturated fatty acids were 61–69% depending on the conditionsfor growth, whereas unsaturated fatty acid content was 28–33%.Stearic acid content enhanced from 3.6% in the oil from mixo-trophic cells to 16.9% for the oil from heterotrophically grown cells.There are numerous studies targeting to maximize growth of mic-roalgae under cultivation and to enhance the level of oil or othervalue added products. However, the reliability of the algal oil forbiodiesel applications depends not only on the quantity of oil pro-duced, but also on the type and structure of the fatty acids present

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivation of microalgae in dairy effluent for oil production and removal of organicpollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.028

Table 1Fatty acid profile of the lipids produced by Chlorococcum sp. RAP13 cultivatedmixotrophically or heterotrophically in dairy effluent.

Fatty acid type Relative proportions (%)

Mixotrophic DE Heterotrophic DE Heterotrophic DEwith 6% BDWG

C12 6.91 9.00 5.90C14 2.40 3.96 1.82C16 53.96 46.25 44.30C16:1 – 1.59 1.23C16:2 – – 1.56C18 2.35 2.30 16.90C18:1 27.00 22.50 20.90C18:2 3.10 10.80 –C18:3 – – 4.70SFA 65.61 61.51 68.92USFA 30.00 33.30 28.39

SFA – saturated fatty acids, USFA – unsaturated fatty acids.

S.B. Ummalyma, R.K. Sukumaran / Bioresource Technology xxx (2014) xxx–xxx 5

in the oil. Hence the lipid content and fatty acid profile are key fac-tors to consider when selecting algae for fuel production. FAMEs ofsaturated fats tend to have poor low-temperature operation, whilebiodiesel containing high levels of polyunsaturated fatty acids(PUFAs) have a shorter shelf life due to their tendency to undergooxidation (Chen et al., 2012). Fatty acids indicated for good biodie-sel properties include C14:0, C16:0, C16:1, C18:0, C18:1 and C18:2(Schenk et al., 2008). When the requirements for biodiesel qualityconsidered, any single fatty acid methyl ester (FAME) cannot sat-isfy every requirement of biodiesel. A mixture of different fattyacids containing higher amounts of mono-unsaturated fatty acidssuch as oleate (18:1D9), fewer saturated and polyunsaturated fattyacids is considered to be better for reliable biodiesel (Durrett et al.,2008). Based on European Biodiesel Standards EN14214 for vehicleuse, content of methyl linolenate (18:3) and the poly unsaturatedfatty acids containing greater than four double bonds are shouldbe limited to a maximum of 12% and 1% respectively (Knothe,2005).

Previous studies have indicated that, by alternating the environ-mental conditions that the microalgae is exposed during growth, itis possible to change the biosynthesis of fatty acids significantly(Los and Murata, 2004). The change in fatty acid composition ofthe algal oil on cultivation in effluent may have a protective role,helping the alga to adapt to the change in environmental condi-tions. Chlorococcum sp. has the potential to change its fatty acidprofile depending up on the cultivation mode and change in themedium. Therefore, tuning of the culture conditions, especiallythe medium can be considered as a strategy for obtaining desiredfatty acid profile for the specific end application – either for biofuelor other industries like nutraceuticals. Algal oils are a rich source ofomega 3 and omega 6 fatty acids which are essential fatty acidsand having therapeutic and nutritional benefits in humans. Humanbody can synthesize long chain PUFAs (EPA, DHA) from 18 fattyacid precursors like stearic acids, oleic acids, linolenic acids (Pere-ira et al., 2004). Chlorococcum sp. RAP-13 grew on dairy effluentand produced high levels of 18:1 fatty acids (20–27%), 18:2 fattyacids (10%), and 18:3 fatty acids (4.7%) which make this micro algaloil a good source of good fats for both human and animalconsumption.

For any feasible technology for micro algal oil production,achieving high levels of oil and biomass is critical. A suitable algalstrain should have high lipid productivity either by having highbasal productivities or the ability to accumulate significantamounts of lipid in response to induction conditions. Otherdesirable features include the easiness of harvesting and oil extrac-tion, adaptability to the cultivation conditions and resistance toother organisms which might invade their culture systems. Open

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivatiopollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.

phototrophic cultivation systems also requires that the culturehas the machinery to efficiently harvest and convert solar radiationto oil and is highly adaptable to prevailing climatic conditions, atthe same time achieving high biomass levels in culture. Consider-ing the above, it is natural that a locally isolated micro algal strainwould adapt better to the culture system and provide a better bio-mass and oil yield (Rodolfi et al., 2009). Chlorococcum sp. RAP 13cultivation in DE may be considered as effective in producing bio-mass and oil useful for both biodiesel or nutraceutical applicationsand the culture was highly adaptable to growth in the effluentwithout any conditioning of the medium.

3.4. Reduction in pollution load of dairy effluent

Changes in organic pollution load were monitored as COD, BODand pH during the experiment to evaluate the effect on algal culti-vation on effluent treatment process. pH is one of the physicalparameters used to assess the quality of water. Initial pH of theDE was 10.6, which gradually reduced to the range 7.5–8.8 after al-gal growth on effluent. According to environment protection andpollution control board standards for industrial effluent dischargedinto water bodies, pH should be in the range of 5.5–9.0. Several fac-tors influence the pH of the medium such as algal growth (pH in-crease as a result of CO2 uptake), and the excretion of acidic orbasic metabolites from organic matter biodegradation (Gonzalezet al., 2008).

Primary parameters for monitoring effluent quality are COD andBOD. COD gives the total pollution load in the form of both organicand inorganic matter whereas BOD gives an estimate for biologi-cally degradable matter in the effluent. Initial COD and BOD ofthe DE were very high and beyond the permissible limits. Permis-sible limit of BOD and COD for municipal and industrial effluentdischarge is 30 mg/l and 250 mg/l respectively according to thestate pollution control board standards. COD of the dairy effluentdecreased from 984 mg/l to 60 mg/l after 12 days of algal culturein it. COD reduction was rapid during the initial period with 73%of COD removed on 3rd day while 93% of reduction was observedafter 12 days (Fig. 4). The slow reduction in COD for latter periodcould be attributed to presence of remaining carbon as some colloi-dal form or as slowly biodegradable material.

BOD is an indicator of biologically degradable content. From theinitial value of 37 mg/L, the BOD was reduced gradually during thealgal growth in the effluent and reached 7 mg/l after 15 days ofincubation, which was 82% reduction (Fig. 4). Similar COD andBOD removal efficiencies of 88% and 89.60% respectively fromindustrial waste water using Chlorella vulgaris was reported byAzeez (2010). Apart from reducing the COD and BOD of DE signif-icantly, Chlorococcum sp. RAP13 could also remove the offensivesmell of the effluent rapidly. Biomass production in the alga relieson rapid utilization of the organic content in the effluent and canbe considered as an attractive, efficient and eco friendly meansfor treating this waste water, since in addition to removing the pol-lution load, algal cultivation adds value to the process by generat-ing commercially valuable products – the algal biomass and oil.

Existing technologies used for algal cultivation to generate oilfor biofuel application are largely limited in viability due to themany challenges associated with attaining high biomass and henceoil yields. Major bottlenecks include the source of water, fertilizersused for the algal cultivation and costs associated with harvesting.However, dual use of micro alga cultivation for wastewater treat-ment and production of value added compounds/biofuel is anattractive option in terms of reducing the energy cost and thenutrient and freshwater resource costs. The high biomass produc-tivity of Chlorococcum sp. RAP13 on dairy effluent suggests that thiscultivation method offer real potential as a viable means for algalbiomass generation along with phyco remediation and value

n of microalgae in dairy effluent for oil production and removal of organic2014.03.028

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6 S.B. Ummalyma, R.K. Sukumaran / Bioresource Technology xxx (2014) xxx–xxx

addition of this waste stream. The culture produced high amountof biomass (�2 g/L) and oil (42%) while heterotrophically grownwith waste glycerol supplementation, providing an added advan-tage of the value addition of this waste/byproduct from biodieselindustry by converting it back to usable oil for fuel applicationsin a cost effective green process.

Waste water as carbon source and source of nutrients for algalcultivation in the context of biofuel production is interesting, sinceit serves the dual goal of algal biomass & oil production as well aswaste water treatment. Algae are highly efficient in removing thepollution load from waste waters at the same time generatingsignificant amounts of biomass which can be cycled into fuelproduction (Markou and Georgakakis, 2011; Dalrymple et al.,2013). Several different types of waste streams including brewerywaste water (Mata et al., 2013), municipal waste water (Robertset al., 2013), mixed industrial waste waters (Wu et al., 2012) anddairy water (Kothari et al., 2013) have been successfully evaluatedfor algal cultivation for biodiesel production. The current studydemonstrates the potential of using untreated dairy effluent foralgal cultivation with efficient removal or organic pollution loadbesides production of algal biomass and oil production suitablefor biodiesel.

4. Conclusions

Chlorococcum sp. RAP13 grown in DE produced high amount ofbiomass and lipids, especially under heterotrophic growth withsupplementation of BDWG. The cells displayed differences ingrowth characteristics, lipid content and fatty acid profiles basedon differences in the growth conditions and carbon substrate. Bio-mass and lipid yields increased significantly under heterotrophiccultivation. The lipid profile of the algal oil makes it interestingas a candidate for biodiesel or nutraceuticals. Algal cultivation inthe DE could reduce the effluent’s pollution load significantly, indi-cating potential for its use as an effective effluent treatment pro-gram with value addition of the waste stream.

Acknowledgements

USB would like to acknowledge financial support from theUniversity Grants Commission Govt. of India in the form of a SeniorResearch Fellowship for this work, which is part of her Ph.D.program. We thank APNP division of CSIR-NIIST for fatty acidanalysis and MILMA Dairy, Thiruvananthapuram for providing us

Please cite this article in press as: Ummalyma, S.B., Sukumaran, R.K. Cultivatiopollution load. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech

Pre ETP dairy effluents for this study. We thank Prof. K.B.Ramachandran, IIT, Chennai for providing BDWG.

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