Rice straw
Rice straw (Oryza sativa L.) was collected from a field in the rural region of Dong Anh province in Vietnam, dried in air, ground and sieved. The particles that passed through a sieve with mesh size of 0.5 mm but not through a sieve with 0.2 mm mesh size were collected. Rice straw composition was analyzed using a standard analytical procedure (National Renewable Energy Laboratory (NREL), Golden, CO, USA) (Sluiter et al. 2008), and was determined to contain 43.1 ± 1.2% glucan, 17.7 ± 0.5% xylan, 3.0 ± 0.1 arabinan, 2.6 ± 0.1% galactan, and 12.9 ± 0.2% acid-insoluble lignin on a dry weight basis.
Isolation of polyhydroxyalkanoate producing bacteria
Decomposing rice straw was collected from the same field as above, ground, suspended in 0.9% NaCl solution, and the supernatant serially diluted, prior to spreading 100 µL of the diluted sample on a solid medium [Meat-Peptone-Agar (MPA)] containing per liter: 5 g each of NaCl, meat extract, and peptone, and 20 g granulated agar. The plates were incubated at 35 °C for 48 h. Hundreds of colonies were picked and plated again on fresh MPA-agar medium. PHA producing bacteria were then detected by Nile blue A staining method (Spiekermann et al. 1999), for which the bacterial isolates were grown on the modified MPA medium containing per liter: 5 g NaCl, 1 g meat extract, 1 g peptone, 20 g glucose, 20 g granulated agar, and Nile red (Sigma) (dissolved in dimethylsulfoxide) with a final concentration of 0.5 µg dye per mL of the medium. The agar plates were incubated at 35 °C for 2 days and then exposed to ultraviolet light (312 nm). The colonies with fluorescent bright orange staining were chosen for further studies.
Phylogenetic characterization of the selected PHA producing bacteria
The genomic DNA of the seven selected strains was extracted by Thermo Scientific GeneJET Genomic DNA Purification Kit according to the manufacturer’s recommendations. The 16S rRNA gene was amplified using the universal primers, 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGCTT-3’). Sequencing of the amplified DNA fragment was performed at 1st Base (Singapore), and GenBank database was used to search for 16S rRNA gene similarities. Phylogenic analysis based on 16S rRNA gene was performed with the aid of MEGA X software (Kumar et al. 2018) using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei 1993). The almost complete sequences of the 16S rRNA gene of the bacterial strains were deposited in GenBank/EMBL/DDBJ databases and used in the analysis.
Alkaline pretreatment of dried rice straw
Three different alkaline solutions (2% sodium hydroxide, 2% calcium hydroxide and 20% aqueous ammonia) were tested for the pretreatment of rice straw at solid:liquid ratio of 1:10. The mixtures of 10 g dry weight rice straw and 100 mL alkaline solution were placed in 250 mL glass bottles and incubated at different temperature and time periods. Subsequently, the soaked rice straw was recovered by filtration, washed with clean water until neutral pH, and then dried at 105 °C for 24 h prior to enzymatic hydrolysis. Rice straw recovery was calculated based on the percent of amount of insoluble fraction recovered after pretreatment with respect to that before pretreatment.
Enzymatic hydrolysis of the pretreated straw
Both pretreated and untreated rice straw samples were used as substrates for enzymatic hydrolysis. Three enzymes including Celluclast 1.5L, Novozyme 188, and Pentopan provided by Novozymes (Bagsvaerd, Denmark) were used, and the optimum conditions for rice straw hydrolysis were determined after several trials. Pretreated or untreated rice straw (1 g) was mixed with 25 mL of sodium acetate buffer (pH 5.0 containing 1% (v/v) of Celluclast 1.5L, 0.4% (v/v) of Novozyme 188 and 0.2% (w/v) of Pentopan in 100 mL glass bottles at 50 °C in a shaker incubator at 180 rpm for 40 h. Samples were withdrawn at different time intervals for monomeric sugar (glucose, xylose and arabinose) analysis.
Polyhydroxyalkanoate production from rice straw hydrolysate using the bacterial isolates
The selected bacterial isolates were grown in 20 mL of liquid MPA medium in 100 mL Erlenmeyer flasks with rotary shaking at 180 rpm for 13 h. Subsequently, 1 mL of each culture was inoculated in 50 mL of modified MPA medium in 250 mL Erlenmeyer flasks. The medium contained per liter 5 g NaCl, 1 g meat extract, 1 g peptone, 20 g glucose or reducing sugars from hydrolysates, and the pH was initially adjusted to 7.0 using 0.05 M phosphate buffer. The cultures were incubated at 35 °C with rotary shaking at 180 rpm. Samples were withdrawn at 48 h of cultivation for determination of cell dry weight (CDW) and PHA content.
Analytical methods
The surface characteristics of the untreated and pretreated rice straw were analyzed by scanning electron microscopy (SEM) (S-4800, Hitachi, Tokyo, Japan).
The PHA granules in the bacterial cells were observed by transmission electron microscopy (TEM) using JEM-1010 TEM (Jeol Korea Ltd., Seoul, South Korea).
The contents of sugars (glucose, xylose and arabinose) in the enzymatically hydrolysed samples were determined using a HPLC system (Jasco, Tokyo, Japan) equipped with a reflective index detector (ERC, Taguchi, Japan). The sugars were separated on an Aminex HPX-87P column, using MilliQ water as mobile phase at a rate of 0.4 mL/min, and column temparature of 65 °C.
Cell dry weight (CDW) was determined by centrifuging 3 mL of the culture samples at 4 000 g for 10 min in pre-weighed centrifuge tubes, the pellet was washed once with 3 mL distilled water, centrifuged and dried at 105 °C until constant weight was obtained. The tubes were weighed again to calculate the CDW.
PHA quantification was performed using a gas-chromatographic method (Huijberts et al. 1994). For this, about 10 mg of freeze-dried cells was mixed with 1 mL of chloroform and 1 mL of methanol solution containing 15% (v/v) sulphuric acid and 0.4% (w/v) benzoic acid. The mixture was incubated at 100 °C for 3 h to convert the constituents to their methyl esters. After cooling to room temperature, 0.5 mL of distilled water was added and the mixture was shaken for 30 seconds. The chloroform layer was transferred into a fresh tube and used for GC analysis to determine the PHA content. Sample volume of 2 µL was injected into the gas chromatography column (VARIAN, Factor Four Capillary Column, CP8907). The injection temperature was set at 250 °C, detector temperature at 240 °C, and the column temperature at 60 °C for the first 5 minutes and then increased at 3 °C/min until 120 °C was reached. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 12 % valerate (Sigma) was used as a standard for calibration.
PHA content (weight percent, wt%) was calculated as the percentage of the ratio of PHA concentration to CDW, while residual cell mass (RCM) was defined as the CDW minus PHA concentration (Lee et al. 2000).