Ecosafe bioremediation of dairy industry effluent using Streptomyces indiaensis ACT 7 and Streptomyces hygroscopicus ACT 14 and application for seed germination of Vigna radiata

Marine actinomycetes strains of Streptomyces indiaensis ACT 7 and Streptomyces hygroscopicus ACT 14 were efficiently utilized for the bioremediation of dairy industry effluent. The effluent characterization before and after the treatment were observed. The maximum reduction of TS, TDS, TSS, biological oxygen demand (BOD), chemical oxygen demand (COD), chloride, oil and fat content were observed for effluent treatment by mixed consortium. It showed the reduction percent of 92.57 and 90.73 for BOD and COD, respectively. The untreated and treated effluent was used for the germination of Vigna radiata. The seed germination of 100 and 70% were observed for treated and untreated effluent studies. The seedling length (27.2 cm) and vigour index (2720) was maximum for V. radiata treatment with treated dairy effluent.


INTRODUCTION
There has been a substantial increase in generation of industrial wastewater due to the exponential increase in the number of industries.The waste water is discharged either into open land or nearby aquatic ecosystems that results in the contamination of ground water and causes severe effects to the aquatic life.The release of effluents into our environment, coupled with the growth of Industrialization has resulted in massive and destructive operations in our ecosystems (Manu et al., 2012).Indis-criminate discharge of untreated or partially treated waste water directly or indirectly into aquatic bodies may render water resources unwholesome and hazardous to man and other living systems (Olorunfemi et al., 2010).The untreated effluent also causes harmful effects to the agricultural field and effect the growth of plants.Dairy effluent has high organic loads as milk is its basic constituent with high levels of biological oxygen demand (BOD), chemical oxygen demand (COD) and oil content (Manu et al., 2012).The dairy effluent affects the aesthetic value of the receiving water and causes damage to aquatic life (Hur et al., 2010).Its high BOD depletes the dissolved oxygen content of the aquatic system and in due course, creates anaerobic conditions.Also, the foul smell of the effluent is a nuisance to the public and it becomes the breeding ground for flies and mosquitoes (Piyush and Rathore, 2001).The removal of organic matter from the waste water using chemicals is more expensive and cause severe effects to the environment.Bioremediation is adopted to remove toxicants using novel microbes.Effluents emanating from dairy processing industries can be treated by using effective microbes.A mixed bacterial culture comprising Pseudomonas aeruginosa LP602, Bacillus sp.B304 and Acinetobacter calcoaceticus LP009 were used for the treatment of lipid rich wastewater (Mongkolthanaruk and Dharmasasthiti, 2002).Silambarasan et al. (2012) used Pithophora sp. for the treatment of dairy industry effluent.In the present study, marine actinomycetes were isolated from the marine sediment samples and exploited for the dairy industry effluent treatment and the treated effluent can be efficiently used for the germination studies of Vigna radiata.

Collection of marine sediment sample and Isolation of Actinomycetes
The marine sediment samples were collected by using a core sampler at a depth of 3 m from two different points of Tiruchendur coastal area (Latitude: 8°29.80N Longitude: 78°07.73E), Tuticorin District, Tamil Nadu, East Coast of India.The actinomycetes were isolated from the sediment samples by serial dilution plate method (Ellaiah, 1996) using actinomycetes isolation agar and starch casein nitrate agar medium (Kuster and Williams, 1964).

Effluent sample collection
The untreated dairy effluent sample of 10 L was collected from a dairy industry located in Coimbatore, Tamil Nadu, India.The sample was transferred into a sterile container and transported to the laboratory immediately.The effluent was stored at 4°C for further analysis.

Inoculum preparation
A loopful of cells from the isolated pure cultures were inoculated separately in different flasks with 100 ml of inorganic starch broth (Shirling and Gottlieb, 1996).The flasks were incubated at 40°C in an incubator shaker at 180 rpm for 72 h.A volume of 50 ml of inoculum was used for each 500 ml of effluent treatment.

Effluent treatment by isolates
The dairy effluent was treated by using selective isolates.The effluent was filtered and autoclaved at 121°C for 15 min to make them sterile from other microorganisms.The different parameters like BOD, COD, chloride, TS, TDS, TSS and oil content (APHA, 1998) of untreated effluent were analyzed.A volume of 500 ml of the effluent sample was taken in 3 sets of the flask.To this, 100 ml of nutrient solution and 50 ml of inoculum was added.Each strain was added as single inoculums (Streptomyces indiaensis ACT 7; Streptomyces hygroscopicus ACT 14) and mixed Streptomyces spp.(S. indiaensis ACT 7 and S. hygroscopicus ACT 14) to the effluent sample amended with nutrients.The microbial treatment was given for 20 days.The flasks were kept in an incubator shaker with 180 rpm/min.All the experiments were done in triplicates.

Germination of seeds and growth of seedlings using effluent
Seeds were collected from pulses Research Center, Tamil Nadu Agriculture University (TNAU), Coimbatore.The germination of seeds and the growth of seedlings were examined using treated and untreated effluent of dairy industry.The Green gram (Vigna radiata) healthy seeds (20) were taken and surface sterilized with 0.1% mercuric chloride and kept in the sterilized Petri dish with filter paper.To this, 5 ml of effluent was added and observed for the germination and growth of seedlings (Dhanam, 2009).The germinated seeds were transferred on to the soil tray with a spacing of about 3 cm each.Seedlings irrigated with distilled water served as control.Seedlings irrigated with treated and untreated effluents were used for experiments.The various parameters like germination percentage, root and shoot length, seedling length, vigour index (Baki and Anderson, 1973), percent of phytotoxicity (Chou et al., 1978), effluent tolerant index (Turner and Marshal, 1972) were examined.

RESULTS AND DISCUSSION
From the marine sediment samples, 30 strains of actinomycetes were isolated.Among 30 isolates, 2 efficient strains were finally selected for bioremediation of dairy effluent.In this investigation, 16 S rRNA gene sequencing was carried out to identify the marine actinomycetes by comparing the similar sequences retrieved from databases using BLAST search.The sequences were submitted in Genbank under the accession numbers of JQ801298.1,JQ801299.1 for Streptomyces indiaensis ACT 7 and Streptomyces hygroscopicus ACT 14, respectively.
In the present study, the untreated dairy industry effluent was treated by S. indiaensis ACT 7, S. hygroscopicus ACT 14 and mixed consortium of Streptomyces spp.for 20 days.The different parameters like BOD, COD, chloride, TS, TDS, TSS and oil content were analyzed on 10 th and 20 th day.The impact of the untreated and treated dairy industry effluent was checked with the germination of V. radiata.The results are presented in Tables 1 to 4. S. indiaensis ACT 7, S. hygroscopicus ACT 14 and mixed consortium of Streptomyces spp.treatment showed the reduction of the following parameters on 20 th day: BOD was reduced to 81.27, 83.72 and 92.57% respectively.Similarly COD was reduced to 87.69, 86.35 and 90.73%.Chloride was reduced to 85.01, 82.84 and 94.14%.Oil content was reduced to 94.50, 95.31 and 98.87%.The higher amount of TS may be due to the maximum concentration of biodegradable organic matter in the    The phytotoxicity analysis was done by the germination and growth of V. radiata using untreated and treated dairy effluent (Table 4).V. radiata showed 70 and 100% of germination using untreated and treated effluent.Seedling length by untreated and treated effluent was 12.84 and 27.2 cm.The treated effluent increased the seedling length of V. radiata than control (20.60 cm).The seedling vigour index using untreated and treated effluent was 992.91 and 2720.The phytotoxicity was 42.67% for Vigna sp.irrigated with untreated effluent and the effluent tolerant index was 1.74.According to Dhanam (2009), the untreated dairy effluent showed 81% of germination and seedling length of 11.1 cm.According to Manu et al. (2012), the untreated effluent showed 82% of germination and seedling length of 15.94 cm.The germination percentage, seedling growth and other growth parameters were decreased at 100% concentration of untreated effluent in the present study.It might be due to high osmotic pressure caused by untreated effluent.Presence of high amounts of toxicants in the effluent inhibits the germination and seedling growth of V. radiata.The treated effluent should not contain the toxicants and enhanced the growth of seedling as compared to control.
The studies on bioremediation of dairy effluent by Streptomyces sp.showed an efficient method for the prevention of water pollution.The mixed consortium showed the maximum reduction of toxicants from dairy effluent than single isolates for treatment purpose.The treated effluent can be recommended for the agricultural irrigation purpose because it enhanced the germination percentage and seedling growth than control.Bioremediation of effluent by marine Streptomyces sp. and utilization of the effluent for the agricultural purpose is an efficient ecosafe method for the treatment of effluent.
Values are mean of three replicates.

Table 3 .
Biotreatment of dairy industry effluent using mixed consortium.

Table 4 .
(Piyush and Rathore, 2001)reated dairy effluent on seed germination of Vigna radiata.effluent.The high levels of TDS may be due to the presence of salt content in the effluent.TS were reduced to 73.00, 76.28 and 90.53%.TDS was reduced to 70.32, 72.93 and 90.42%.TSS was reduced to 83.41, 89.30 and 90.97%.Biotreatment of the industrial effluents using microbes showed high reduction of BOD and COD levels.It enhanced the removal of most of the toxicants in the effluent.Oil and fat contents from the effluent can create unsightly floating matter and films on water surfaces.It may also interfere with biological life forms in the surface waters.If the surface water is contaminated with oil and grease, oxygen transfer from the atmosphere is reduced and creates a very high oxygen demand on account of their biodegradability(Piyush and Rathore, 2001).
Values are mean of three replicates.dairy