Production enhancement and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10.

Application of halophiles can decrease the cost of polyhydroxyalkanoate (PHA) production or bioplastic which are an alternative to the petroleum-derived plastic. Extremely halophilic archaeon, Natrinema ajinwuensis RM-G10 accumulated 61.02±0.68% PHA of its cell dry mass at 72h in repeated batch cultures yielding 0.210±0.001gL-1h-1 volumetric productivity after selection of the best cultivation conditions. Transmission electron microscopy showed the presence of PHA granules inside the archaeal cells. Characterization by gas chromatographic analysis, gas chromatographic- mass spectrophotometric analysis, thermogravimetric analysis, differential scanning calorimetric analysis, X-ray diffraction analysis, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy revealed the polymer to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 13.93mol% 3-hydroxyvalerate content and having 35.45% crystallinity, -12.3°C glass transition temperature, 143°C and 157.5°C melting temperatures and 284°C degradation temperature. This is the first report on production enhancement (on a small scale) and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10 and the Natrinema genus in general.

Streptomyces euryhalinus sp. nov., a new actinomycete isolated from a mangrove forest.

A Gram-positive, aerobic, non-motile actinomycete (strain MS 3/20T) was isolated from the sediment of the Sundarbans mangrove forest in India. On International Streptomyces Project (ISP) medium 2, the isolate produced yellowish brown to red aerial hyphae that carried spiny-surfaced spores in a retinaculum-apertum arrangement. Whole-cell hydrolysate of the strain contained LL-diaminopimelic acid and galactose. Predominant menaquinones were MK-9(H8) and MK-9(H6). Diagnostic polar lipids were glycolipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, unidentified phospholipid and unidentified amino lipid. The major fatty acids were anteiso-C15:0 (17.53%), iso-C16:0 (23.89%) and anteiso-C17:0 (10.29%). The strain showed 100% 16S ribosomal RNA (rRNA) gene sequence similarity with Streptomyces variabilis NBRC 12825T, Streptomyces erythrogriseus LMG 19406T, Streptomyces griseoincarnatus LMG 19316T and Streptomyces labedae NBRC 15864T. However, strain MS 3/20T could be distinguished from these and seven other closely related species based on low levels of DNA-DNA relatedness (27.2-53.8%), supported by the unique banding pattern obtained from random amplified polymorphic DNA-PCR amplification and the distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain MS 3/20T in comparison with its phylogenetic relatives. Disparate morphological, physiological and chemotaxonomic features, principally growth in NaCl, further corroborated the distinction of strain MS 3/20T from other phylogenetic relatives. Strain MS 3/20T is therefore suggested to be a novel species of the genus Streptomyces, for which the name Streptomyces euryhalinus sp. nov. is proposed. The type strain is MS 3/20T (=CICC 11032T=DSM 103378T).

Polymerase chain reaction-based screening method applicable universally to environmental haloarchaea and halobacteria for identifying polyhydroxyalkanoate producers among them.

The existing techniques for detection of polyhydroxyalkanoates (PHA) in halophilic archaea/bacteria are either imprecise or require prior PHA production before screening. The proposed method involves amplification of the approximately 280-300 bp conserved region of Class III PHA synthase (phaC) gene of halophiles using the primers codehopCF and codehopCR (Han et al. Appl Environ Microb 76:7811-7819, 2010). In this study, the best reaction condition was ascertained after repeated trials. This developed method was tested on nine haloarchaeal and halobacterial type strains and 28 environmental halophilic archaea and bacteria isolated from the salt pans of the east and west coasts of India. 29 strains were found to be phaC-positive, while eight were found to be phaC-negative although they appeared PHA positive through conventional Nile Red staining. 16S rRNA-based phylogenetic analysis identified 9 haloarchaeal and 9 halobacterial species as novel PHA producers. Multiple sequence alignment of the phaC gene-derived amino acid sequences showed that only 7 amino acid residues were conserved within all four classes of phaC enzymes, whereas 61 amino acids were identical among the phaC enzyme specific to the haloarchaeal and halobacterial strains presently investigated. All phaC-positive strains produced PHA in standard nutrient deficient medium, whereas the phaC-negative strains did not accumulate any PHA as detected by gas chromatography and nuclear magnetic resonance analyses, thus proving the precision of the developed method and elimination of false positives seen with the traditional Nile Red staining procedure.