Abstract
The screening of pollutant-degrading bacteria are limited due to most of bacteria in the natural environment cannot be cultivated. For the purpose of resuscitating and stimulating “viable but non-culturable” (VBNC) or uncultured bacteria, Micrococcus luteus proteins are more convenient and cost-effective than purified resuscitation-promoting factor (Rpf) protein. In this study, medium composition and culture conditions were optimized by using statistical experimental design and analysis to enhance protein production by M. luteus. The most important variables influencing protein production were determined using the Plackett-Burman design (PBD) and then central composite design (CCD) was adopted to optimize medium composition and culture conditions to achieve maximum protein yield. Results showed that the maximum protein yield of 25.13 mg/L (vs. 25.66 mg/L predicted) was obtained when the mineral solution, Lithium L-lactate, initial pH and incubation time were set at 1.5 ml/L, 8.75 g/L, 7.5 and 48 h, respectively. The predicated values calculated with the model were very close to the experimental values. Protein production was obviously increased with optimization fitting well with the observed fluorescence intensity. These results verified the feasibility and accuracy of this optimization strategy. This study provides promising information for exploring highly desirable pollutant-degrading microorganisms.
Highlights
To date, just over 7000 well-founded species of bacteria have been described (Epstein 2013), because most of bacteria in the natural environment cannot be cultivated
Evaluation of significant variables affecting protein production Plackett-Burman design (PBD) was applied to determine the relative significance of seven medium components and four culture conditions
The effect of each variable on protein production was estimated by the difference between the average of measurements made at the high level (+1) and the low level (-1) of the particular factor
Summary
Just over 7000 well-founded species of bacteria have been described (Epstein 2013), because most of bacteria in the natural environment cannot be cultivated. It is very common for bacteria to survive under extreme conditions by entering into a “viable but non-culturable” (VBNC) state, where the cells are intact and alive and can resuscitate when surrounding conditions are more favorable (Oliver 2005; Oliver 2010; Pawlowski et al 2011). These VBNC or uncultured bacteria can be studied by molecular ecology methods (Vartoukian et al 2010), to elucidate their related function and genotype, it is necessary to isolate these bacteria and study their microbiology in pure cultures (Kaeberlein et al.2002). Despite many studies on Rpf family proteins and their function in resuscitating VBNC bacteria and stimulating the growth of bacteria (Mukamolova et al 2002; Panutdaporn et al 2006; Su et al 2013), the mechanism of action remains unclear
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