Two stage treatment of dairy effluent using immobilized Chlorella pyrenoidosa
© Yadavalli and Heggers; licensee BioMed Central Ltd. 2013
Received: 24 March 2013
Accepted: 4 November 2013
Published: 19 December 2013
Dairy effluents contains high organic load and unscrupulous discharge of these effluents into aquatic bodies is a matter of serious concern besides deteriorating their water quality. Whilst physico-chemical treatment is the common mode of treatment, immobilized microalgae can be potentially employed to treat high organic content which offer numerous benefits along with waste water treatment.
A novel low cost two stage treatment was employed for the complete treatment of dairy effluent. The first stage consists of treating the diary effluent in a photobioreactor (1 L) using immobilized Chlorella pyrenoidosa while the second stage involves a two column sand bed filtration technique.
Whilst NH4 +-N was completely removed, a 98% removal of PO4 3--P was achieved within 96 h of two stage purification processes. The filtrate was tested for toxicity and no mortality was observed in the zebra fish which was used as a model at the end of 96 h bioassay. Moreover, a significant decrease in biological oxygen demand and chemical oxygen demand was achieved by this novel method. Also the biomass separated was tested as a biofertilizer to the rice seeds and a 30% increase in terms of length of root and shoot was observed after the addition of biomass to the rice plants.
We conclude that the two stage treatment of dairy effluent is highly effective in removal of BOD and COD besides nutrients like nitrates and phosphates. The treatment also helps in discharging treated waste water safely into the receiving water bodies since it is non toxic for aquatic life. Further, the algal biomass separated after first stage of treatment was highly capable of increasing the growth of rice plants because of nitrogen fixation ability of the green alga and offers a great potential as a biofertilizer.
Agro-food industries like the dairy and brewery sectors produce effluents with high organic load. These effluents contain high BOD, COD and nutrients such as phosphates, ammonia and/or nitrate, pose a serious threat to the water quality and should be appropriately treated before discharging into the environment . There are several physical, chemical and biological methods for the treatment of effluents derived from the agro-food industries. Biological methods are proved to be much eco friendly when compared with other types of treatments [2, 3].
Research suggests that N- and P-rich wastewater is also considered as valuable substrate for cultivation of microalgae which is a type of biological treatment . Microalgae are mostly suspension-type microorganisms and efficient solar driven cell factories that can be useful as treating agents for wastewater. The cultivation of algae in wastewaters offers the combined advantages of treating the wastewaters and simultaneously producing algal biomass, which can further be exploited for protein complements and food additives for aquaculture, animal and human feed, energies such as biogas and fuels and biofertilizer . Little information is available on the use of Chlorella pyrenoidosa as a potential agent for treatment of various types of waste waters among the several microalgae used to treat effluents .
Separation of algal biomass from the treated water discharge is the key for the success of waste water treatment. Numerous efforts have been devoted to developing a suitable technology for harvesting microalgae. The use of industrial filtration and centrifugation is not cost effective for wastewater treatment. In this context, immobilization of algal cells has been proposed for overcoming the harvest problem as well as retaining the high-value algal biomass for further processing .
Among all unicellular algal species, Chlorella is a common and effective species for the immobilization and nutrient removal purposes . Uptake rate of phosphorous was low when compared with Nitrogen for Chlorella sp from waste waters [9, 10]. There are various methods of immobilization out of which calcium alginate is a very simple and cost-effective method, to entrap microbial cells as alginate beads.
Many green algae are also capable of using atmospheric dinitrogen (N2) as the source of nitrogen. This important characteristic of nitrogen fixation has enhanced the agro production when green alga is used in agriculture . Many studies have been reported on the use of dried green alga to inoculate soils as a means of aiding fertility. Algalization is the term applied to the use of a defined mixture of algal species to inoculate soil. A 15-20% increase in rice grain yield was observed because of algalization [12, 13].
Reduction of BOD and COD is one of the major challenges in wastewater treatment. If effluent with high BOD is discharged into environment, it will increase the bacterial growth in the water while reducing the dissolved oxygen levels in the water that may prove lethal to aquatic life. Much like BOD, a high COD can also deprive aquatic organisms of oxygen needed for survival .
In this study, we made an attempt to treat the diary effluent by a novel, low cost photobioreactor using immobilized C. pyrenoidosa and evaluate the potential of dairy effluent as a nutrient medium for microalgae growth. The efficacy of sand bed filtration to reduce/remove some of the organic compounds present in the dairy effluent was assessed. The treated dairy effluent is used to check the survival of zebrafish (Danio rario) whose gene sequence is identical with humans to assess the biosafety to aquatic life. In addition, the biomass produced is analyzed for its composition, and amenable to be used as fertilizer.
Materials and methods
Chlorella pyrenoidosa sp. (NCIM NO: 2738) was obtained from National Centre for Industrial Microorganisms (NCIM), Pune, India. Stock culture of Chlorella pyrenoidosa was grown photoautotrophically in BG11 media at 28°C under continuous light illumination in four 100 ml borosil flasks. Each liter of the BG11 medium contained NaNO3-1.5 g, K2HPO4-0.04 g, MgSO4•7H2O-0.075 g, CaCl2•2H2O-0.036 g, Citricacid-0.006 g, NaCO3-0.02 g, H3BO3-0.00286 g, MnCl2•4H2O-0.00181 g, ZnSO4•7H2O-0.00022 g, Na2MoO4•2H2O-0.00039 g, CuSO4•5H2O-0.00008 g, Co(NO3)2•6H2O-0.00005 g, (NH4)6Mo7O24.4H2O-0.003 g, Na2EDTA-0.00001 g. The inoculum was prepared by transferring the cells from stock culture, and incubated aseptically in a 1000 ml flask containing 700 ml of fresh BG11 media under continuous illumination of 34 μmol m-2 s-1 at 28°C for four days on an orbital shaker set at 120 rpm. A 4 day old culture was used as inoculum at 10% volume for the preparation of immobilized microalgal beads. Gel entrapment is done using 2% Sodium alginate and 0.1 M Calcium chloride. The average diameter of the algal beads inoculated was 0.556 m. Untreated dairy effluent is collected from Vyshnavi dairy, Khammam, Andhrapradesh, India.
Specimens of zebra fish (4–5 cm long; weight 1.25 ± 0.25 g) were used for toxicity tests. These were procured from a local aquarium shop, Hyderabad were collected from. The fish were transported from the farm in oxygenated polythene bags to the laboratory and immediately transferred into 20 L capacity containing well –aerated unchlorinated ground water. The fish were allowed to acclimatize for 15 days before the experiments. They were fed with rice bran during the acclimation period. Only healthy and active fishes were used for experiments. The temperature, pH, dissolved oxygen, used for acclimatization were 29°C, 7.2 ± 0.2, 5.0-6.5 mg/L respectively.
Sand bed filtration
Estimation of lipid, carbohydrate and protein
The carbohydrate content was analyzed based on the procedure published by Miao and Wu . In short, 0.1 g dried algal pellet was acidified by adding 20 ml 2.5 M HCl. The acidified solution was then hydrolyzed at 100°C for 30 min and neutralized to pH 7. The volume was adjusted to 100 ml. The filtered sample was subjected to 3,5-dinitrosalicylic acid (DNS) assay .
Lipid extraction was performed in a way that was similar to the procedures reported by earlier studies [17, 18]. Briefly, 1.0 g dried cells were ground in a mortar and pestle. The dried powder is then transferred to a 50 ml glass centrifuge tube. Chloroform was then added to the tube to make the chloroform/ methanol (2:1, v/v). The tube was vortexed for 5 min and was allowed to stand for 24 h. After that, the tube was centrifuged at 4,000 g for 15 min to remove the algal solids. The supernatant was collected and the solvent was vaporized using Rotovap at 65°C. Oil left in the flask without solvent was weighed to calculate lipid content.
Protein estimation is done using Lowry’s method . Briefly, 0.1 g algal powder is taken and 2 ml of 5% TCA is added. The mixture is centrifuged at 2000 rpm for 10 min. Solution containing pellet is dissolved in 1.5 ml of 0.1 N NaOH. From the above prepared solution 0.2 ml is taken into 20 ml test tube and 5 ml of alkaline copper solution is added. After incubation at room temperature for 15 min, 0.5 ml of Folin-Ciocalten reagent is added. Again the mixture is incubated for 30 min and OD values were taken at 540 nm.
Results and discussion
Biochemical analysis of algal biomass
Algal biomass analysis
Dairy water analysis
Nitrogen and phosphate removal
Water analysis of three different samples
Control (untreated dairy effluent)
Dairy effluent after algal bead treatment
Dairy effluent after sand bed filtration
Acceptable limits as per APPCB
BOD(3 @ 27°C)
Effect on pH
The untreated dairy effluent has an acidic pH value initially but after algal beads treatment and sand bed filtration the treated effluent is shifted to alkaline pH range. NH4 +-N could be lost via ammonia volatilization while PO4 3--P was removed by chemical precipitation, because of which alkaline pH was recorded in the two stage treatment system.
BOD and COD removal
Relative toxic potential of dairy effluent to Danio rario
Untreated Dairy effluent
Dairy effluent after algal bead treatment
Dairy effluent after Sand bed filtration
Algal biomass as biofertilizer
Lengths of root and shoot in rice plant
Control (without biofertilizer)
Test (with biofertilizer)
Green alga can photosynthesize and fix nitrogen, and these abilities, together with great adaptability to various soil types, make them ubiquitous. Ammonia can be taken up by green alga through passive diffusion or as ammonium (NH4 +) by a specific uptake system .
The present study demonstrated that two stage treatment was very effective in removing NH4 +-N and phosphate from wastewater. A complete removal of NH4 +-N (100%) and 99% reduction of PO4 3--P was achieved within 96 h of treatment. The completely treated dairy effluent was proved to be non toxic for the zebra fish because of the drastic decrease in BOD and COD values (98% and 96% respectively). The algal biomass was higher in protein content (44%) and when the same was used as biofertiliser for rice seeds a 35% increase in growth of the rice plant was observed. Hence the process proved to be useful in treating wastewater for aquatic life and as a biofertiliser.
YR has done her Masters in Chemical engineering currently holds the position of Associate Professor in Biotechnology and pursuing her Ph.D in the area of algal biofuels. HVNG has completed Graduation in Biotechnology and is working along with YR for his Postgraduate Project work.
The authors thank the Management of Sreenidhi Institute of Science and Technology (SNIST) for their financial support in carrying out this in-house funded project. Special thanks to Vyshanavi DairyPvt Ltd., Khammam, Andhrapradesh, India for providing the dairy effluent for conducting the experiments. The authors also wish to show their gratitude towards Dr. V. Sesha Srinivas, Head department of Biotechnology SNIST, Dr. P. Ravindrababu, Associate Professor, Department of Biotechnology, SNIST and Mr. G. Praveen Kumar for their constant support during the project.
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