Estimation of Chlorophyll
On 7th, 15th, 30th, 45th, and 60th day, 100 mg of leaves of the plants was weighed and homogenized with 2 ml of 80-% acetone. Then the volume was made up to 10 ml using distilled water. The contents were centrifuged at 5000 rpm for 15 minutes and the supernatants were taken for the absorbance analysis using UV ‘ Visible Spectrophotometer (Hitachi U 3210), at 663 nm and 645 nm. The chlorophyll contents were calculated by using the formula:
Total chlorophyll (mg/g tissue)
20.2 (A645) + 8.02 (A663) X V/1000 X W
A = Absorbance at specific wave lengths (645 nm and 663 nm)
B = Final volume of chlorophyll extract in 80% acetone and
W = Fresh weight of tisssue extracted.
Chloropyll ‘ a
12.7 (A663) ‘ 2.69 (A645) X V/1000 X W
Chlorophyll – b
22.9 (A645) ‘ 4.68 (A663) X V/1000 X W
Strain properties and identification
The agarolytic strain is a Gram-positive rod shaped bacterium, motile; it is also catalase, oxidase and lysine decarboxylase positive, and oxidase and Phosphatase negative. The preliminary identification results showed that the isolated strain was in accordance with Bacillus, according to Bergey’s Manual of Systematic Bacteriology. The results are shown in Table 1.
The 16S rDNA sequence of the agaro degrading strain RV3 was compared to available sequences in public databases. Fig. 1 shows an un rooted tree of the Bacillus species. The isolated strain and B. subtilis strain ZJ-06 formed a robust clade. Based on data results, we assigned of our strain as B. subtilis st. RV3. The 16S rDNA sequence of Bacillus subtilis strain RV3 was submitted to the GenBank database under the accession number GQ413935.
Purification of agarase
The enzyme has high binding affinity to DEAE-cellulose when loaded at low salt concentrations. The enzyme was released gradually from the DEAE-cellulose column by washing with 1.5 M NaCl. Further purification of the agarase was accomplished by gel filtration using Sephadex. The specific enzyme activity was increased from 0.22 units/mg protein to 5.3 units/mg protein after purification (Table S1).
Agarase from B. subtilis st. RV3 has a molecular weight of 65 kDa, as analyzed by a comparison with the of protein standard markers (Fig. 2).
Physiochemical properties of the enzyme
Agarase enzyme purified in the present investigation was found to be active in a broad range of temperature ranging from 10~60??C. However, optimum temperature was found to be 30??C (Fig. 3a). The pH report of agarase of B. subtilis strain RV3 had a maximum activity at pH 5 and 7.5 (Fig. 3b). Agarase activity was assayed in the presence and absence of metal ions. It was observed that none of the cations exhibited activation in the enzyme activity (Table.2).
The agarase stored at 20??C exhibited a decline in agarase activity by 20% within three days, which was totally lost within 6 days whereas the one stored at room temperature (30??C) exhibited a 48% decline in enzyme activity within 3 days which was completely lost within 6~7 days of incubation. In frozen condition (- 20??C), the agarase remains stable even after 10 days of incubation period (Fig. S1).
The kinetic parameters of agarase were Km = 4 ?? 0.17 mM, Kcat = 150 ?? 10 S -1 and Kcat / Km = 52 ?? 10 S -1.mM -1.
Effect of efficient agarolytic bacteria treated agar industry waste extracts on growth of Vigna radiata
Agar industry waste extract of different concentrations were prepared using distilled water. The extracts were then treated with immobilized bacteria of Bacillus subtilis for 10 days at 30??C. All the agar industry waste extracts (treated) were applied to the V. radiata seed and plant. Among the different treated extracts and concentration applied, the maximum growth of root length 17.3 ?? 0.56 was observed in 30% Bacillus subtilis treated extract on 60th day. All the parameters of root length were shown in table 3.
The effect of treated agar industry waste extract on the shoot length was shown in table 4. There was an increase in the shoot length from day 7 to 60 and the maximum shoot length 52.3 ?? 1.10 was recorded in 15% Bacillus subtilis treated extract. The concentration of treated extract over 20% had detrimental effect to plant growth.
The fresh weight of the plants was determined in all extract treated plants. In 15% Bacillus subtilis extract treated plant fresh weight had increased from 18.32 g/p to 18.81 g/p on day 60. In general, the growth of plants in all treatments was more or less similar (Table S2).
Number of leaves per plant of V. radiata was enumerated in all agar industry waste extract treated plants. There was an increase of leaves from 7th day to 60th day. The number of leaves was 24 on 60th day in 15% Bacillus subtilis treated extract. The number of leaves in control was only 15 (Table S3).
The experiment on leaf area of V. radiata on different treatment extracts were recorded in table 5. The maximum leaf area 352.25 cm2 was recorded in the 15% Bacillus subtilis treated extract on 45th day plant. The chlorophyll (a, b and total) of V. radiata grown in various treatment was estimated at different day intervals, and the results are displayed in table 6. Maximum chlorophyll content was observed in plants of 15% Bacillus subtilis treated extracts applied. The maximum total chlorophyll content was 22.97 mg/g on 45th day plant. A reduction in chlorophyll content was noticed on 60th day in all treatments. All the growth parameters of Vigna radiata applied with Bacillus subtilis treated agar industry waste extracts were shown in fig. 4