Microplastics a Novel Substratum for Polyhydroxyalkanoate (PHA)-Producing Bacteria in Aquatic Environments

Abstract

Polyhydroxyalkanoates (PHA) being biological polymers have attracted great attention. PHA have similar properties to that of synthetic plastic and are biodegradable. To discourage plastic pollution in the environment alternative solutions to the plastic pollution has to be readily available. High cost in production of PHA limits the production of these polymers at industrial scale. Bacteria are screened for PHA from diverse niches to meet the current requirements of cheap PHA production at industrial level. The microbial biofilm formed on the surface of microplastic could be a potential source in providing bacteria of economic importance. This paper is an attempt to search microplastic niche for potential PHA producers. PHA production variation was observed with different parameters such as type of carbon source, nitrogen source concentration and also time of incubation. Bacillus sp. CM27 showed maximum PHA yield up to 32.1% among other isolates at 48 h with 2% glucose as carbon source. Optimization of media leads to increase in PHA yield (37.69%) of CDW in Bacillus sp. CM27. Amino acid sequence of Bacillus sp.CM27 showed the presence of PhaC box with sequence, G-Y-C-M-G-G having cysteine in the middle of the box. The extracted polymer was confirmed by FTIR spectroscopy.

Fig. 1

The phylogenetic tree of PHA-producing bacteria isolated from microplastic and its nearest neighbours derived from the maximum likelihood method based on the partial sequence of the 16S rRNA gene. GenBank accession numbers are indicated in parenthesis. Type strains are used as reference strains with superscript T. Bootstrap values (based on 1000 replication) > 50% are shown at branch points. Bar, 0.05 means the nucleotide substitution per position

Fig. 2

The selected screened isolates accumulating PHA grown in MBM media with 2% glucose for 48 and 72 h of incubation. PHA content in bacteria was tested by Slepecky and Law’s method wherein crotonic acid is measured at 235 nm. The results are averages of ± standard deviations from three separate experiments. *Statistical analysis was carried out by one-way ANOVA test, *p value (≤ 0.05)

Fig. 3

Effect on PHA production in Bacillus sp. CM27 during Optimizing incubation time and nutrient composition of Marine Basal Medium. Fig. a., Effect of Incubation condition on PHA production in Bacillus sp. CM27. Fig. b. Effect of different Carbon source on PHA production in Bacillus sp. CM27. Fig. c.,Effect of Yeast Extract Concentration on PHA production in Bacillus sp. CM27. Fig. d, Effect of Tryptone Concentration on PHA production Bacillus sp. CM27. Analysis was carried out by converting PHA to crotonic acid by Slepecky and Law’s method measured at 235 nm. The results are averages of ± standard deviations from three separate experiments. Fig. a, b and d *Statistical analysis was carried out by one-way ANOVA test, * p value (≤ 0.05). Fig. c. *Statistical analysis was carried out by one-way ANOVA test, *p value (= 0.08)

Fig. 4

Multiple alignment of PhaC from Bacillus sp. C19, Priestia megaterium, Bacillus iocasae, Anoxybacillus calidus Cerasibacillus terrae, and Bacillus cereus with reported PHA synthases of Bacillus sp.CM27. Marked rectangular area in sequence alignment showed the conserved PhaC box