Short Communication: Isolation and screening of polyhydroxylalkanoates producing microorganisms from Thailand

Pungsungvorn N, Wisetsing A. 2021. Short Communication: Isolation and screening of polyhydroxylalkanoates producing microorganisms from Thailand. Biodiversitas 22: 4963-4967. Polyhydroxylalkanotes (PHAs) are polyesters produced in nature by numerous microorganisms. They are biodegradable and are used in the production of bioplastics. In this study, one hundred twenty samples from different regions of Thailand were collected and screened for polyhydroxylalkanoates (PHAs) producing microorganisms. The samples were screened on YM agar containing 0.5 µg Nile-red/mL (YM-NR). Only one isolate of yeast (Y1) gave a positive result on PHA accumulation. The yeast isolate (Y1) was identified as Candida tropicalis by API 20C AUX kit and 18S rRNA nucleotide comparison. The yeast isolate Y1 produced 2.62% PHA when grown in synthetic N-limiting medium using rice straw hydrolysate as carbon source. The selected four bacteria (B1, B2, B3 and B4) were identified by BlastN of 16S rRNA as Enterobacter cloacae, Enterobacter carcerogenus, Escherichia coli and Klebsiella pneumonia, respectively. The selected yeast and bacterial strains gave PHA content of 2.62, 2.76, 5.38, 3.66 and 0.44%, respectively, in synthetic N-limiting medium using rice straw hydrolysate. Hence, these microorganisms could be used in PHA production from biomass in the future.

Rice Straw Hydrolysis and Cultivation of Xanthophyllomyces dendrorhous for Astaxanthin Production and Antioxidant Properties

Astaxanthin is a natural pigment with strong antioxidant activity and is widely supplied as dietary supplement. The red yeast Xanthophyllomyces dendrorhous is one of the potential sources for astaxanthin production. Rice straw was considered for utilization on cultivation and astaxanthin production. Rice straw was treated by autoclave-assisted alkaline pretreatment. and hydrolyzed by cellulase hydrolysis. X. dendrorhous TISTR5730 was cultured and accumulated as the astaxanthin on rice straw hydrolysate. The highest reducing sugar concentration of rice straw hydrolysate was 89.82±0.39g/L (0.71±0.01g/g) when using rice straw (3.3%) and cellulase loading (3 mL). High cell density X. dendrohous TISTR5730 cultivation on rice straw hydrolysate with 20 and 40 g/L of initial reducing sugar was investigated. 40 g/L was appropriate for biomass production while 20 g/L was suitable for astaxanthin accumulation. The highest astaxanthin content was 417.28±50.89 μg/g cell basis at 192 h. Astaxanthin productivity and yield coefficient were 0.01±0.00 mg/L/h and 0.11±0.01 mg/g sugar consumed. The antioxidant activities of astaxanthin were determined by DPPH and ABTS scavenging and FRAP reducing power. The produced astaxanthin represented the high antioxidant activities with IC50 of 9.30 and 1.67 μg/mL of DPPH and ABTS scavenging respectively and FRAP reducing power of 5.31±0.07 μg TEAC/mL. This research indicated that rice straw hydrolysate could be an alternative medium for astaxanthin production. Antioxidant activity of astaxanthin was proved and was feasible for further applications.

Polyhydroxyalkanoate production from rice straw hydrolysate obtained by alkaline pretreatment and enzymatic hydrolysis using Bacillus strains isolated from decomposing straw

Rice straw is an important low-cost feedstock for bio-based economy. This report presents a study in which rice straw was used both as a source for isolation of bacteria producing the biodegradable polyester polyhydroxyalkanoate (PHA), as well as the carbon source for the production of the polymer by the isolated bacteria. Of the 100 bacterial isolates, seven were found to be positive for PHA production by Nile blue staining and were identified as Bacillus species by 16S rRNA gene sequence analysis. Three isolates showed 100% sequence identity to B. cereus, one to B. paranthracis, two with 99 and 100% identity to B. anthracis, while one was closely similar to B. thuringiensis. For use in PHA production, rice straw was subjected to mild alkaline pretreatment followed by enzymatic hydrolysis. Comparison of pretreatment by 2% sodium hydroxide, 2% calcium hydroxide and 20% aqueous ammonia, respectively, at different temperatures showed maximum weight loss with NaOH at 80 °C for 5 h, but ammonia for 15 h at 80 °C led to highest lignin removal of 63%. The ammonia-pretreated rice straw also led to highest release of total reducing sugar up to 92% on hydrolysis by a cocktail of cellulases and hemicellulases at 50 °C. Cultivation of the Bacillus isolates on the pretreated rice straw revealed highest PHA content of 59.3 and 46.4%, and PHA concentration of 2.96 and 2.51 g/L by Bacillus cereus VK92 and VK98, respectively.

Production of Biopolyamide Precursors 5-Amino Valeric Acid and Putrescine From Rice Straw Hydrolysate by Engineered Corynebacterium glutamicum

The non-proteinogenic amino acid 5-amino valeric acid (5-AVA) and the diamine putrescine are potential building blocks in the bio-polyamide industry. The production of 5-AVA and putrescine using engineered Corynebacterium glutamicum by the co-consumption of biomass-derived sugars is an attractive strategy and an alternative to their petrochemical synthesis. In our previous work, 5-AVA production from pure xylose by C. glutamicum was shown by heterologously expressing xylA from Xanthomonas campestris and xylB from C. glutamicum. Apart from this AVA Xyl culture, the heterologous expression of xylAXc and xylBCg was also carried out in a putrescine producing C. glutamicum to engineer a PUT Xyl strain. Even though, the pure glucose (40 g L–1) gave the maximum product yield by both the strains, the utilization of varying combinations of pure xylose and glucose by AVA Xyl and PUT Xyl in CGXII synthetic medium was initially validated. A blend of 25 g L–1 of glucose and 15 g L–1 of xylose in CGXII medium yielded 109 ± 2 mg L–1 putrescine and 874 ± 1 mg L–1 5-AVA after 72 h of fermentation. Subsequently, to demonstrate the utilization of biomass-derived sugars, the alkali (NaOH) pretreated-enzyme hydrolyzed rice straw containing a mixture of glucose (23.7 g L–1) and xylose (13.6 g L–1) was fermented by PUT Xyl and AVA Xyl to yield 91 ± 3 mg L–1 putrescine and 260 ± 2 mg L–1 5-AVA, respectively, after 72 h of fermentation. To the best of our knowledge, this is the first proof of concept report on the production of 5-AVA and putrescine using rice straw hydrolysate (RSH) as the raw material.