การคัดเลือกแบคทีเรียสังเคราะห์แสงสีม่วงที่ไม่สะสมซัลเฟอร์ที่ย่อยโปรตีนและมีฤทธิ์ยับยั้งแบคทีเรียก่อโรคกุ้ง vibrio spp.

Abstract

The aims of this study were to select purple nonsulfur bacteria (PNSB) with ability to produce proteolytic enzyme and antivibrio activity, including affecting factors on proteolytic enzyme production of the selected PNSB. Overlay diffusion method was used to test antivibrio activity against shrimp pathogenic Vibrio spp. by 22 PNSB isolates. It was found that 12 PNSB isolates (54.55%) were able to inhibit shrimp pathogenic Vibrio spp. However, there was only one PNSB strain (4.55%), PS342b, had ability to inhibit all vibrios tested (6 isolates). Observation of inhibited shrimp pathogenic Vibrio spp. cells collected from clear zone and around clear zone using a scanning electron microscope found altered cells with many holes. Almost PNSB tested (81.82%, 18 isolates) degraded gelatin. No inhibition of shrimp pathogenic vibrios was found for co-culture between vibrios and 12 PNSB strains under conditions of microaerobic light and aerobic dark when compared with controls. Moreover, culture supernatant of 12 PNSB isolates did not inhibit shrimp pathogenic Vibrio spp. However, concentrated culture supernatants at 20 times by freeze-dry method had ability to inhibit some isolates of shrimp pathogenic vibrios. There was only 1 isolate namely PS342b was selected according to its abilities to digest protein and inhibit shrimp pathogenic Vibrio spp.; and this strain was identified as Rhodovulum sulfidophilum using 16S rRNA. Rhodovulum sulfidophilum PS342b was further studied optimal medium and conditions for proteolytic enzyme production. Glutamate-malate (GM medium) supplemented with 1.5% NaCl was the most suitable medium by giving specific growth rate and doubling time as 0.336 h and 2.066 h.; while optimal shaking speeds were between 150 and 200 rpm. GM medium containing 1.5% NaCl with replacement of (NH4)2HPO4 by 1% gelatin was used for proteolytic production under aerobic-dark conditions at 150 rpm. The use of response surface methodology with central composite design (CCD), optimum conditions for the production of proteolytic enzymes were pH 7.90, 1.30% NaCl and 29.50°C as the highest enzymatic activity up to 15.40 units per ml. The verification test confirmed the predicted values from the CCD.