Experimental studies on lactose biodegradation of dairy effluent by K . lactis

Sumithra. It is inevitable to treat the dairy waste effluents since they are the most polluting ones due to its huge volumes discharged to the inland surface water streams.The COD range of the dairy effluents is about 2000-3000 mg/L. About ten organisms of different species have been tried for finding out their ability to degrade lactose. Among the ten organisms tested three strains were found to be most promising namely K. lactis, C. laurentii and L. plantarum. The strain K. lactis,degraded all the lactose during exponential cell growth phase and removed 67% COD in shake flask cultivation.Based on the above results, a batch and continuous process was performed with K. lactis to the optimize and scale up of the biodegradation process.The present studies are carried out to evaluate the performance of various microorganisms in degrading lactose present in dairy effluent and the highly degrading strain of K.lactis was identified. Copy Right, IJAR, 2016,. All rights reserved.


Introduction:-
The dairy industry revolves around the processing of raw milk to different products (e.g. yogurt, cheese, cream, milk, etc) [1] (Rivas et.al20100 .Its processing discharges vast amount of effluents which may result in severe pollution of environment. According to Carvalho et al. the water consumption, depending on production, is between 1-4times that of milk received [2] .Dairy wastes, due to their composition, volumes and fluctuating flow rates, represent a problem for municipal wastewater treatment plants therefore, treatment of this kind of effluent seems to be essential [3] .Several pretreatment technologies have been developed and applied to treat dairy effluent such as activated sludge system, anaerobic pond, oxidation pond, trickling filter, and combined trickling filter/activated sludge system [4] (Garrido et al., 2001;Perle et al., 1995). Pretreated effluent from these processes have considerable amount of biological and chemical oxygen demand (BOD &amp; COD), oil and grease and also some other pollutants. The pollution load is linked to the wastes organic content which is typically measured as COD and BOD [5] .Cheese whey, a dairy industry by-product [6] , stands out with a COD significantly greater than the other effluents. In order to eliminate these pollutants, conventional biological treatments of aerobic and anaerobic treatments or facultative digestion are the most commonly used.

Milk industry wastewater:-
The waste used is the permeate from Effluent treatment plant. It was obtained from Vijaya Dairy in Hyderabad. The wastewter was stored in sterile containers in a fridge or a cooling room at 4 o C and used within a week.

Media for stock cultures:-
Five different media (broth and agar) were prepared in 2 L (Table.1) Schott bottles. The pH of the media were adjusted by adding NaOH (1M) or HCl (1M). The media was sterilized by autoclaving (121 o C, 15 min).  and put to grow on agar plates and in broth. K. marxianus, K. lactis, C. laurentii and C. flavus were inoculated in 250 ml shake flasks containing 100 ml media. L. lactis and L. plantarumLp 39 were inoculated in 20 ml anaerobic tubes containing 17 ml media. The broth cultures were incubated for 24 h, while plates were incubated for 48 h. frozen cultures of L. plantarum BS and L. acidophilus were allowed to thaw before they were pre cultured in anaerobic tubes containing 17 ml media (M.11). The cultures were incubated for 24 h. Agar plates were incubated for 48 h. A fresh culture of S. cerevisiae and a frozen culture of P. pastoris were pre-cultured in closed 50 ml sterile bottles containing 15 ml media (M.393). The cultures were incubated for 24 h. Agar plates were incubated for 48 h.

Cultivation in nutrient solution with added lactose:-
Cultures of L. plantarum BS and L. acidophilus were inoculated in 25 ml anaerobic tubes containing media as described in Table 3 Table 2). Schott tubes with 10 ml medium containing salt premix, lactose (20 g/L) and yeast peptone (40 g/L).

Microbial growth measurements:-
Microbial growth was monitored by measuring optical density (OD) with Ultra spec 2100 pro spectrophotometer. During all measurements UPW was used as blank and when necessary also as dilutant. The absorbance was at all times measured at 600 λ. Cell dry weight was determined using a Mettler AE 240 analytical balance, a HeraeussepatechLabofuge 200 centrifuge, centrifuge tubes and a Termaks incubator. Suspended cells (5 ml) were added to two pre-weighed tubes. The tubes and their contents were weighed and centrifuged (2500 rpm, 12 min). The supernatant was discarded, and the cells were washed with ultrapure water prior to a second centrifugation (2500rpm, 12 min). The supernatant was again discarded, and the tubes were left to dry in the incubator at 105 o C overnight.

Chemical oxygen demand (COD) measurements:-
Samples for COD measurements were stored in Eppendorf tubes in a fridge at 4 o C.The COD content of the samples were determined by using a Spectroquant COD cell test kit from Merck.
pH measurements:-Measurements of pH were performed with a Radiometer Copenhagen PHM210 Standard pH meter according to manufacturer's instructions. The pH meter was periodically calibrated with buffers of pH 7 and 4.

Screening of organisms for waste biodegradation: -
For the preliminary screening of ability to degrade lactose, cultivation was performed in small-scale cultures. This enabled the efficient screening of a large number of different organisms at different growth conditions.

Organisms growth characteristics:-
Growth curve experiments were carried out on the three selected strains, K. lactis, C. laurentii and L. plantarumLp 39. This was done to get a better picture of their growth pattern and relationship between growth and lactose degradation. One set of experiments were performed on limited media (media I) and the other on nutrient media (media II and III) (media composition. The results for the cell growth, lactose degradation and pH changes are presented in Figure .1 for K. lactis, Figure 2.forC. laurentii and Figure .  Fermentation:-After a screening of 10 different organisms, K. lactis was chosen based on low nutrient demand, high lactose conversion rate and promising COD removal following batch culture tests. K. lactis' ability to grow and reduce the lactose and COD content of dairy waste at the larger scale was tested through fermentation in a 15 L fermenter. The fermenter was run for 98 h, with two different processes; (1) batch fermentation (0-41 h) and (2) continuously fed fermentation (49-98). Between 41-49 hours addition of yeast peptone was performed to investigate whether nutrients were limiting growth or waste biodegradation.

Conclusion:-
The screening performed in this project gave an indication of which kind of organism will contribute to the overall aim of dairy waste COD removal. S. cerevisiae and P. pastoris, as expected, did not grow on lactose as a carbon source. The Lactobacillus organisms and L.lactisproved to be nutrient demanding and their growth resulted in an inefficient COD reduction. C. flavus did not reach a high cell density, and did not completely mineralize lactose, resulting in a high concentration of lactose degradation by-products (galactose and/or glucose) in the growth media. Comparatively, K. marxianus was relatively inefficient at degrading the lactose. C. laurentii degraded lactose largely during cell stationary growth phase, and removed in excess of 30% COD in shake flask cultivation. K. lactis degraded all the lactose during exponential growth and removed in excess of 30% COD in shake flask cultivation. During batch fermentation K. lactis removed 67% of COD within the first 24 h. Results from trials with different flow rates (200, 300 and 500 ml/h) in continuously fed fermentation, pointed towards a flow rate close to 300 ml /h as optimum for this system.