Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate.

Brown and black rice substrates were applied for sugar syrup production by the hydrolysis of raw starch degrading enzyme (RSDE) from Laceyella sacchari LP175 (300 U/mL) and commercial glucoamylase (GA, 2.0 U/mL) at 50 °C for 12 h using a simplex centroid mixture design. Results indicated that 300 g/L of substrates, consisting of 255 g/L Leum Pua glutinous rice and 45 g/L Black Jasmine rice, gave the highest sugar syrup production at 124.6 ± 2.52 g/L with 2.00 ± 0.05 mg GAE/mL of total phenolic content (TPC), equivalent to 0.42 ± 0.01 g/g rice sample and 6.67 ± 0.15 mg GAE/g rice sample, respectively. The obtained sugar syrup was used as the substrate for production of bacterial cellulose (Nata) by Komagataeibacter xylinus AGR 60 in a plastic tray at room temperature for 9 days. The fermentation medium containing 200 mL of rice syrup (25 g/L), 2.0 g of ammonium sulfate [(NH4)2SO4] and 0.4 mL glacial acetic acid yielded 1.1 ± 0.08 cm thickness with 8.15 ± 0.12 g of dry weight. The obtained bacterial cellulose from colored rice was characterized compared with bacterial cellulose from the conventional coconut juice by scanning electron microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR) which demonstrated that the sugar syrup from colored rice could use as substrate for a novel bacterial cellulose as a healthy product in the future through microbial enzyme technological process. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

Production and development of vinegar fermentation from broken Riceberry rice using raw starch-degrading enzyme hydrolysis.

Broken Riceberry rice was used as a substrate for sugar syrup production by the hydrolysis of raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA, Thailand). Response surface methodology (RSM) with a central composite design (CCD) showed that an optimized substrate concentration of 250 g/L yielded 13°Brix of total soluble solid (TSS) content when incubated at 50 °C for 12 h. The major product from the broken Riceberry rice hydrolysis was glucose with lesser amounts of maltose and maltotriose. Maximum alcohol content (16% w/v) for broken Riceberry rice wine was obtained after fermentation with two mixed strains of Saccharomyces cerevisiae for 10 days. Scanning electron micrographs showed that yeast strains could grow on the solid residue of broken Riceberry rice that supported yeast cell survival under stress conditions. Broken Riceberry rice wine was used as the substrate for vinegar fermentation by Acetobacter aceti TISTR 354. Maximum acetic acid concentration was achieved at 5.4% when incubated at room temperature for 6 days, containing 10.92 mg/L and 965.53 ± 7.74 mL sample/g DPPH of anthocyanin content and antioxidant assay, respectively. Our finding revealed the feasibility of broken Riceberry rice substrate for sugar syrup, wine and vinegar production by raw starch-degrading enzyme hydrolysis which increased the value of low-cost agricultural crops through biotechnological processes.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Poly(DL-lactide)-degrading enzyme production by immobilized Actinomadura keratinilytica strain T16-1 in a 5-L fermenter under various fermentation processes

Background: Poly(DL-lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results: Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions: This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).

Polycladomyces subterraneus sp. nov., isolated from soil in Thailand.

A thermophilic poly(L-lactide)-degrading Gram-stain-positive filamentous bacterial strain that develops single spores on the aerial mycelium was isolated from forest soil at Srinagarind Dam, Kanchanaburi Province, Thailand. The results of a polyphasic taxonomic study showed that our isolate had characteristics typical of members of the genus Polycladomyces. The isolate grew aerobically at an optimum temperature of 50-55 °C and optimal pH 6-7. Meso-diaminopimelic acid was present as the diagnostic diamino acid in the peptidoglycan but no characteristic sugars are detected. The predominant menaquinone was MK-7. The diagnostic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine diphosphatidylglycerol, phosphatidylglycerol and phosphatidylserine. The predominant cellular fatty acid was iso-C15 : 0. The DNA G+C content of strain KSR 13T was 53.4 mol%. The 16S rRNA gene sequence analysis also indicated that strain KSR 13T belonged to the genus Polycladomyces, being most closely related to Polycladomyces abyssicola JIR-001T (99.2 %). The DNA-DNA relatedness values that distinguished KSR 13T from P. abyssicola JIR-001T were 17.8-32.1 %, which were significantly below the 70 % cutoff value recommended for species delineation. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, the new isolate is proposed as a novel species and named Polycladomyces subterraneus sp. nov. The type strain is KSR 13T (=BCC 50740T=NBRC 109332T).

Microbispora siamensis sp. nov., a thermotolerant actinomycete isolated from soil.

An actinomycete, strain DMKUA 245(T), isolated from soil, was investigated using a polyphasic approach. The isolate formed longitudinally paired spores on the tips of short sporophores that branched alternately from aerial hyphae. The morphological and chemotaxonomic properties clearly demonstrated that the new isolate belonged to the genus Microbispora. 16S rRNA gene sequence analysis supported the assignment of the novel strain to the genus Microbispora. The gene sequence similarity values between the novel strain and the closely related species Microbispora corallina, Microbispora rosea subsp. rosea, Microbispora rosea subsp. aerata and Microbispora amethystogenes were 98.4 %, 97.4 %, 97.0 % and 96.9 %, respectively. The DNA-DNA hybridization values and some physiological and biochemical properties indicated that strain DMKUA 245(T) could be distinguished from its phylogenetically closest relatives. Based on these genotypic and phenotypic data, strain DMKUA 245(T) represents a novel species in the genus Microbispora for which the name Microbispora siamensis sp. nov. is proposed. The type strain is strain DMKUA 245(T) (=BCC 14407(T)=NBRC 104113(T)). In addition, DNA-DNA relatedness values in reciprocal hybridization experiments showed that M. amethystogenes was a separate genomic species from M. rosea subsp. rosea. A combination of genotypic and phenotypic data supported the classification of M. amethystogenes as a separate species.