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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.


Rice straw is an obvious choice to be used as residual low-cost feedstock for the bio-based economy, especially in South- and South-East Asian countries that are the main producers of rice, the third most important grain crop in the world after wheat and corn (Binod et al. 2010). For every ton of rice grain, 700–1500 kg of rice straw is produced, and over 700 million tons of rice straw is produced worldwide (Bakker et al. 2013). In Vietnam, the total rice planted area is about 7.5 million ha, with a total of 40 million tons of rice and about 50 million tons of rice straw being produced annually (Diep et al. 2015). Some of the rice straw is collected for cooking, animal feed, roof covering or mushroom production. With improvement in the living conditions of farmers, such use of rice straw has become less common and 25–60% (depending on the region) is left for burning in the fields or considered as waste material (Bakker et al. 2013; Nguyen et al. 2016; Le et al. 2020;). Rice straw burning is estimated to release 11 tons of CO2-equivalent per hectare of land in addition to a large amount of NOx, a precursor to photochemical smog, and fine dust (Sarkar et al. 2012; Bakker et al. 2013). Valorization of rice straw would thus offset such emissions. Many studies claim that 25–35% of the straw may be available for biofuels and other products after leaving that is needed to conserve soil quality and for competitive uses (Bakker et al. 2013).

There have been many studies on the use of rice straw for the production of second-generation biofuel, and more recently even to other products like insulation materials, composites, biodegradable plastics and chemicals (Abraham et al. 2016; Bilo et al. 2018; Goodman 2020; Overturf et al. 2020). The biodegradable bio-based plastics that are increasingly attracting interest to replace the fossil-based plastics are the microbial polyesters—polyhydroxyalkanoates (PHAs) (Bedade et al. 2021; Naser et al. 2021; Yadav et al. 2021); their high rate of biodegradability even in marine environments lowers the risk of their accumulation as microplastics (Suzuki et al. 2021). As a major part of the production cost of PHA is attributed to the carbon source, use of rice straw could provide a potential low-cost alternative besides lowering the environmental impact caused by its burning (Obruca et al. 2015; Heng et al. 2016).

Rice straw contains over 70% carbohydrates in the form of cellulose and hemicellulose, access to which requires pretreatment, as for all lignocelluloses, for the removal of the lignin network (Binod et al. 2010; Sarkar et al. 2012). Unlike other agricultural residues, the straw also contains high ash content that poses a challenge in thermal processes leading to a high tendency for fouling the combustion systems. Various methods have been tested for pretreatment of rice straw (Hendriks and Zeeman 2009; Agbor et al. 2011; Tsegaye et al. 2019; 2020; Guo et al. 2020; Kurokochi and Sato 2020; Zang et al. 2020). Production of PHAs by Bacillus firmus and Cupriavidus necator using the hydrolysate of acid or alkali-pretreated rice straw with good yields has been reported (Sindhu et al. 2013; Ahn et al. 2015, 2016). Mild alkaline pretreatment has been widely applied on rice straw because of its simplicity, efficiency and relatively low cost. It increases the surface area by swelling the biomass particles and increasing carbohydrate accessibility to enzymes, while reducing the degree of polymerization and crystallinity of the cellulose (Tsegaye et al. 2019; Li et al. 2020). As very little of the carbohydrate is solubilized, most of it can be converted to sugars during the subsequent hydrolysis step (Hendriks and Zeeman 2009; Binod et al. 2010; Agbor et al. 2011; Ashoor and Sukumaran 2020).

The present report describes a study in which rice straw was used both as a source for isolation of PHA-producing bacteria, as well as the carbon source for the production of the polymer by the isolated bacteria. Decomposing rice straw was used for bacterial isolation, while dried straw pretreated with alkali followed by enzymatic hydrolysis was used as carbon source for PHA production. The effect of pretreatment with three different alkaline reagents (aqueous ammonia, sodium hydroxide and calcium hydroxide) at different temperatures on lignin removal and the sugar recovery from the enzymatic step was compared to find the best conditions for preparing the carbon source for PHA production.

Materials and methods

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 temperatures 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 1.5 L (129.3 mg protein/mL, 30.7 cellobiose units/mL and 63.8 filter paper units/mL), Novozyme 188 (102.2 mg protein/mL, 626.4 CBU/mL), and Pentopan Mono BG (2500 U/g) 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 hydrolyzed 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 temperature of 65 °C. The glucose concentration was used to calculate glucan-to-glucose conversion as follows:

$${\text{Glucan}}\;{\text{conversion}}\left( \% \right) = {\text{glucose}}\;{\text{liberated }}({\text{g}}) \times 0.{\text{9}} \times 100/{\text{initial}}\;{\text{cellulose}}(g)$$

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 s. 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, while the column temperature at 60 °C for the first 5 min 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.

Analyses of sugars, PHA and CDW were performed in triplicates.

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). PHA yield (g/g) was calculated as concentration of the polymer divided by the amount of sugar used. PHA productivity (g/L/h) was calculated as PHA concentration divided by the cultivation time.

Results and discussion

Isolation and identification of PHA-producing bacteria from decomposing rice straw

More than 100 bacterial colonies were isolated from decomposing rice straw, among which seven isolates were found to show significant PHA accumulation by Nile blue staining, and were used in this study. The phylogenetic characterization of the seven isolates based on their 16S rRNA gene sequences showed them to belong to genus Bacillus (Fig. 1). The sequence of strain VK24 showed a high level of similarity (98.2%) with that of B. thuringiensis LDC 507. Two strains VK33 and VK38 clustered together and showed the highest similarity of 99% and 100%, respectively, with B. anthracis IHB B 7021. Three other strains VK91, VK92 and VK98, also clustered together and showed similarity of 100% with B. cereus DBA1.1. Strain VK164 showed 100% sequence identity with B. paranthracis NWPZ-61 (100%). According to earlier reports, the bacterial populations degrading the straw under anoxic conditions comprised mainly Clostridium species with Bacillus species as one of the minor groups (Weber et al. 2001), while Bacillus species were predominant during composting of rice straw (Hefnawy et al. 2013). In fact, Bacillus species is used as an inoculant in rice straw composting for which they play an important role in degradation of cellulose and hemicellulose (Zhang et al. 2021).

Fig. 1
figure 1

Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationships between the seven selected strains and other strains of the genus Bacillus. Bar 0.05 subtitutions per position

PHA production by several Bacillus species such as B. subtilis, B. firmus, B. cereus, B. thuringiensis isolated from different ecosystems and using different carbon sources has been reported earlier (Singh et al. 2009; Sindhu et al. 2013; Gowda and Shivakumar 2014; Odeniyi and Adeola 2017; Mohandas et al. 2018; Ponnusamy et al. 2019; Mohammed et al. 2020). Synthesis of both homo- and copolymers has been reported depending on the substrate (Singh et al. 2009; Mohapatra et al. 2017).

Mild alkaline pretreatment of rice straw and dry matter loss

Alkaline pretreatment of the dried rice straw was tested with 2% NaOH, 2% Ca(OH)2 and 20% aqueous NH3, respectively, at solid:liquid ratio of 1:10. These reagents have been used earlier for pretreatment of various biomass substrates including rice straw, rice hulls, switchgrass, corn stover, wood and bagasse (Chang et al. 1997, 1998; Park and Kim 2012; Sindhu et al. 2015; Heng et al. 2016; Kobkam et al. 2018; Tsegaye et al. 2019). Alkaline pretreatment solubilizes lignin by disrupting its structure and breaking the linkages with carbohydrate residues. It also removes acetyl and uronic acid substitutions on hemicellulose and helps to improve the accessibility of the polysaccharides to the hydrolytic enzymes (Carvalheiro et al, 2008; Bali et al. 2015; Kobkam et al. 2018; Tsegaye et al. 2019), but also resulting in their partial hydrolysis under strong alkaline conditions (Zhang and Cai 2008; Kobkam et al. 2018; Tsegaye et al. 2019).

In accordance with previous reports (Ko et al. 2009; Rodrigues et al. 2016; Sophonputtanaphoca et al. 2018), the amount of residual rice straw decreased with increase in temperature and time for the pretreatment; increase in temperature being a more effective parameter (Table 1). The highest straw recovery with 2% NaOH was 83-84% after 1 h treatment at 30–50 °C. Higher degree of solubilization was achieved during 1–5 h at 80 °C (Table 1), while removing 41–52% of lignin. In an earlier study, release of about 61% of hemicellulose and 36.2% of lignin was reported on treatment of chopped rice straw with 2% NaOH at solid:liquid ratio of 1:4, 85 °C for 1 h (Zhang and Cai 2008).

Table 1 Rice straw recovery after pretreatment with alkali at different incubation temperatures and times

Pretreatment with 2% Ca(OH)2 resulted in rather high recovery of residual straw at all temperatures although slight decrease was seen with increasing time period; the lowest recovery (84.6%) being after 1 h treatment at 121 °C. Ca(OH)2 is a weaker alkali with low solubility in water and hence requires much longer treatment time and higher temperature, and moreover the calcium ions form calcium–lignin complex that hinder proper lignin solubilization (Rodrigues et al. 2016).

Aqueous NH3 is considered to be highly selective for lignin removal; it cleaves C–O–C bonds in lignin as well as ether and ester bonds in the lignin–carbohydrate complex (Binod et al. 2010), and has a significant swelling effect on lignocellulose. Residual straw recovery was intermediate to that obtained with NaOH and Ca(OH)2. Pretreatment with 20% aqueous NH3 for 15 h at 80 °C resulted in 29.5% of the weight loss of straw (Table 1), and 63% lignin removal (data not shown), which agrees well with the study by Park and Kim (2012) reporting removal of up to 66% lignin along with 22.9% hemicellulose and 11% cellulose from the rice straw treated with 15% aqueous NH3 (solid:liquid ratio of 1:10) at 60 °C for 24 h.

The alkaline-pretreated rice straw was much softer than the untreated one, indicating removal of large amounts of lignin and hemicellulose. As seen in Fig. 2, the rice straw pretreated with NaOH was distorted and separated from the initially connected structure, thus increasing the surface area (Fig. 2A) in comparison to the untreated straw with rigid, smooth and highly ordered fibrils (Fig. 2B).

Fig. 2
figure 2

SEM micrographs of rice straw before (A) and after (B) pretreatment with 2% NaOH at 80 °C for 5 h

Enzymatic hydrolysis for releasing sugars from the polysaccharides

The effect of pretreatment with different alkaline reagents on the efficiency of enzymatic hydrolysis step was then evaluated (Additional files 1: Figures S1–S3). Both pretreated and untreated rice straw samples (1 g) were incubated in 4% w/v suspension at pH 5.0, 50 °C with an enzyme cocktail comprising 1% v/v cellulase preparation derived from Trichoderma reesei (Celluclast 1.5L), 0.4% v/v β-glucosidase from Aspergillus niger (Novozyme 188) and 0.2% w/v 1,4-β-xylanase from Thermomyces lanuginosus (Pentopan Mono BG). Glucose and xylose were the major products of hydrolysis besides a small amount of arabinose. Maximum yield of total reducing sugars from untreated rice straw was only 34.2%. In case of the straw pretreated with NaOH (Additional files 1: Figure S1), up to 72.2% of total reducing sugar was obtained for the sample subjected to pretreatment at 30 °C for 7.5 h while longer pretreatment time gave lower yield (Additional files 1: Figure S1A). Increasing the pretreatment temperature to 50 °C (2.5 or 5 h) and 80 °C (3 h) resulted in a slight increase in the sugar yield to about 74% (Additional files 1: Figure S1B) and 77% (Additional files 1: Figure S1C), respectively. In case of Ca(OH)2-pretreated rice straw, the highest sugar yield of 60–66% was obtained for the samples pretreated at 30 °C for 48 h (Additional file 1: Figure S2A), or 80 °C for 24 h (Additional files 1: Figure S2B), while significantly lower yield was noted when the pretreatment was performed at 121 °C (Additional files 1: Figure S2C). Pretreatment with aqueous ammonia gave the highest sugar yield ranging between 77 and 87% upon enzymatic hydrolysis (Additional file 1: Figure S3A, B), the highest yield being obtained for the straw pretreated at 80 °C for 15 h (Additional file 1: Figure S3B). However, a much longer time (30 h) for enzymatic treatment was needed to obtain the maximal sugar release as compared to that for the samples treated with Ca(OH)2 and NaOH which took about 15 h for the maximal release.

Table 2 shows that the glucan conversion efficiency obtained in this study was in the same range as that in previous studies. A few studies have reported slightly higher yields for the straw pretreated either at a higher temperature, longer time or lower solid loading (Ko et al. 2009; Kim and Han 2012; Kim et al. 2014; Tsegaye et al. 2019).

Table 2 Comparison of the glucan-to-glucose conversion of cellulose in rice straw treated with different pretreatment methods followed by enzymatic hydrolysis

PHA production from the rice straw hydrolysate

The seven bacterial isolates were cultured in the medium containing 20 g/L glucose or 20 g/L of reducing sugars obtained by saccharification of the ammonia-pretreated rice straw followed by enzymatic hydrolysis at pH 5.0 and re-adjusting the pH to 7.0. The CDW, PHA content and concentration obtained after 48 h of cultivation are summarized in Tables 3 and 4. All the seven strains grew well in the glucose medium with CDW ranging from 2.2 to 3.32 g/L, and the PHA content ranged between 42 and 73 wt% of the cell dry weight, the highest value being obtained for the strain B. cereus VK98 (Table 3 and Fig. 3A). The final cell mass obtained was generally lower in the straw hydrolysate medium, the only exceptions being the strains B. cereus VK92 and VK98 that gave CDWs of 5 g/L and 5.42 g/L, respectively (Additional file 1: Figures S4, S5). The PHA content was also highest in the B. cereus strains, 59.3 wt% in VK92, followed by 46.4 wt% in VK98 (Additional file 1: Figures S4, S5) and 43.8 wt% in VK 91 (Table 4). Figure 3A and B shows the TEM micrographs at 2 μm resolution of the PHA granules accumulated by the strain VK98 grown with glucose and straw hydrolysate, respectively. The polymer was primarily polyhydroxybutyrate (PHB) with traces of hydroxyvalerate. Glucose was fully consumed, but the bacteria did not utilize xylose. B. anthracis VK33, B. anthracis VK38, B. paranthracis VK164 exhibited PHA content of less than 10 wt%, while no PHA was detected in the cells of Bacillus sp. VK24. This suggests B. cereus VK92 and VK98 are resistant to the inhibitory compounds formed during the pretreatment and autoclaving. These include weak acids such as acetic acid, glycolic acid, formic acid and levulinic acids, and phenolic compounds, e.g., coumaric acid, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (Jönsson et al. 2013; van der Pol et al. 2016). VK92 and VK98 also exhibited higher final pH values (Table 4), which may suggest that these isolates are able to utilize some of the acids and transform them to other metabolic products or perhaps even to PHA with different monomer composition (Vu et al. 2021; Ahn et al. 2016). This needs, however, to be further investigated.

Table 3 Growth and PHA accumulation in seven Bacillus species in the medium containing 20 g/L glucose
Table 4 Growth and PHA accumulation by seven isolated Bacillus species in the medium containing 20 g/L reducing sugars produced by saccharification of aqueous ammonia-pretreated rice straw
Fig. 3
figure 3

TEM micrographs of PHA granules accumulated by strain VK98 on: A glucose-based culture medium and B rice straw hydrolysate-containing medium, respectively

Comparison of the results obtained in this study with earlier reports on PHA production from rice straw hydrolysate clearly shows the production parameters to be highly comparable (Table 5). Majority of the other studies have utilized acid-hydrolyzed rice straw. The highest accumulation of PHB (89% w/w) was reported in Bacillus firmus grown on pentose-rich hydrolysate with 0.75% xylose but at a much lower cell mass, hence yielding low PHB concentration (1.7 g/L) (Sindhu et al. 2013). On the other hand, Ralstonia eutropha (or C. necator), the most commonly used bacteria for PHA production, yielded the highest amount of PHB (9.88 g/L, 70.1% w/w) from the rice straw pretreated with NaOH followed by two-stage enzymatic hydrolysis (Saratale and Oh 2015) instead of the acid-hydrolyzed straw (Ahn et al. 2015, 2016). The high polymer concentration obtained in R. eutropha was primarily due to the very high concentration of the cell mass (15.5 g/L) but also high PHA accumulation in the cells.

Table 5 Comparison of PHA production by different bacterial strains from rice straw hydrolysates

Moderate PHA yield of 0.15 g/g was obtained with respect to the sugar consumed in the B. cereus isolates (Table 5), which was equivalent to 59.5 g PHA per kg rice straw in case of B. cereus VK92, considering also the losses during the pretreatment. It is possible to further improve the PHA content and productivity in the isolates by medium optimization, e.g., with respect to carbon:nitrogen ratio, phosphate level, aeration, etc., and by using fed-batch mode of cultivation under controlled conditions (Singh et al. 2021). Moreover, strategies to further improve the resource- and cost-efficiency need to be developed when utilizing rice straw as feedstock, e.g., by transformation of other components to other value added products.


This study shows the potential of utilizing the bacteria, involved in degradation of rice straw, as hosts for PHA production from reducing sugars liberated by pretreatment and hydrolysis of the rice straw lignocelluloses. Aqueous ammonia soaking pretreatment was found to be an efficient method for lignin removal from rice straw, providing suitable substrate for enzymatic digestibility of the polysaccharides. Further improvements in PHA content and productivity are possible by optimization of the culture medium and mode of cultivation. The pretreatment and the enzymatic hydrolysis, especially the latter, are the most cost-determining steps for PHA production from rice straw. In order to reduce these costs, it would be interesting to investigate PHA production in association with the Bacillus species that are involved in composting of rice straw through their polysaccharide-degrading activities (Hefnawy et al. 2013; Zhang et al. 2021), and even to consider developing a biorefinery by co-production of other chemicals and materials from the feedstock.

Availability of data and materials

The raw data supporting the conclusion of this article will be made available by the authors without undue reservation.


  1. Abraham A, Mathew AK, Sindhu R, Pandey A, Binod P (2016) Potential of rice straw for biorefining: an overview. Bioresour Technol 215:29–36.

    CAS  Article  PubMed  Google Scholar 

  2. Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB (2011) Biomass pretreatment: fundamentals toward application. Biotechnol Adv 29:675–685.

    CAS  Article  PubMed  Google Scholar 

  3. Ahn J, Jho EH, Nam K (2015) Effect of C/N ratio on polyhydroxyalkanoates (PHA) accumulation by Cupriavidus necator and its implication on the use of rice straw hydrolysates. Environ Eng Res 20:246–253.

    Article  Google Scholar 

  4. Ahn J, Jho EH, Kim M, Nam K (2016) Increased 3HV concentration in the bacterial production of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymer with acid-digested rice straw waste. J Polym Environ 24:98–103.

    CAS  Article  Google Scholar 

  5. Ashoor S, Sukumaran RK (2020) Mild alkaline pretreatment can achieve high hydrolytic and fermentation efficiencies for rice straw conversion to bioethanol. Prep Biochem Biotechnol 50:814–819.

    CAS  Article  PubMed  Google Scholar 

  6. Bakker RRC, Elbersen HW, Poppens RP, Lesschen JP (2013) Rice straw and wheat straw-potential feedstocks for the biobased economy. NL Agency

    Google Scholar 

  7. Bali G, Meng X, Deneff JI, Sun Q, Ragauskas AJ (2015) The effect of alkaline pretreatment methods on cellulose structure and accessibility. Chemsuschem 8(2):275–279.

    CAS  Article  PubMed  Google Scholar 

  8. Bedade DK, Edson CB, Gross RA (2021) Emergent approaches to efficient and sustainable polyhydroxyalkanoate production. Molecules 26:3463.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Bilo F, Pandini S, Sartore L, Depero LE, Gargiulo G, Bonassi A, Federici S, Bontempi E (2018) A sustainable bioplastic obtained from rice straw. J Clean Prod 200:357–368.

    CAS  Article  Google Scholar 

  10. Binod P, Sindhu R, Singhania RR, Vikram S, Devi L, Nagalakshmi S, Kurien N, Sukumaran RK, Pandey A (2010) Bioethanol production from rice straw: an overview. Bioresour Technol 101:4767–4774.

    CAS  Article  PubMed  Google Scholar 

  11. Carvalheiro F, Duarte LC, Girio GM (2008) Hemicellulose biorefineries: a review on biomass pretreatments. J Sci Ind Res 67:849–864

    CAS  Google Scholar 

  12. Chang VS, Burr B, Holtzapple MT (1997) Lime pretreatment of switchgrass. Appl Biochem Biotechnol 63–65:3–19.

    Article  PubMed  Google Scholar 

  13. Chang VS, Nagwani M, Holtzapple MT (1998) Lime pretreatment of crop residues bagasse and wheat straw. Appl Biochem Biotechnol 74:135–159.

    CAS  Article  Google Scholar 

  14. Cheng Y-S, Zheng Y, Yu CW, Dooley TM, Jenkins BM, VanderGheynst JS (2010) Evaluation of high solids alkaline pretreatment of rice straw. Appl Biochem Biotechnol 162:1768–1784.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Diep NQ, Sakanishi K, Nakagoshi N, Fujimoto S, Minowa T (2015) Potential for rice straw ethanol production in the Mekong Delta Vietnam. Renew Energy 74:456–463.

    Article  Google Scholar 

  16. Goodman BA (2020) Utilization of waste straw and husks from rice production: a review. J Bioresour Bioprod 5:143–162.

    CAS  Article  Google Scholar 

  17. Gowda V, Shivakumar S (2014) Agrowaste-based Polyhydroxyalkanoate (PHA) production using hydrolytic potential of Bacillus thuringiensis IAM 12077. Braz Arch Biol Technol 57:55–61.

    CAS  Article  Google Scholar 

  18. Gu Y, Zhang Y, Zhou X (2015) Effect of Ca(OH)2 pretreatment on extruded rice straw anaerobic digestion. Bioresour Biotech 196:116–122.

    CAS  Article  Google Scholar 

  19. Guo JM, Wang YT, Cheng JR, Zhu MJ (2020) Enhancing enzymatic hydrolysis and fermentation efficiency of rice straw by pretreatment of sodium perborate. Biomass Conv Biorefin.

    Article  Google Scholar 

  20. Hefnawy M, Gharieb M, Nagdi OM (2013) Microbial diversity during composting cycles of rice straw. Int J Adv Biol Biomed Res 1(3):232–245

    Google Scholar 

  21. Hendriks ATWM, Zeeman G (2009) Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 100:10–18.

    CAS  Article  PubMed  Google Scholar 

  22. Heng K-S, Hatti-Kaul R, Adam F, Fukui T, Sudesh K (2016) Conversion of rice husks to polyhydroxyalkanoates (PHA) via three-step process: optimized alkaline pretreatment, enzymatic hydrolysis, and biosynthesis by Burkholderia cepacia USM (JCM 15050). J Chem Technol Biotechnol 92:100–108.

    CAS  Article  Google Scholar 

  23. Huijberts GNM, van der Wal H, Wilkinson C, Eggink G (1994) Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria. Biotechnol Tech 8:187–192.

    CAS  Article  Google Scholar 

  24. Jampatesh S, Sawisit A, Wong N, Jantama SS, Jantama K (2019) Evaluation of inhibitory effect and feasible utilization of dilute acid pretreatment rice straw on succinate production by metabolically engineered Escherichia coli AS1600a. Bioresour Technol 273:93–102.

    CAS  Article  PubMed  Google Scholar 

  25. Jönsson L, Alriksson B, Nilvebrant N-O (2013) Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels 6:16.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Kim I, Han J-I (2012) Optimization of alkaline pretreatment conditions for enhancing glucose yield of rice straw by response surface methodology. Biomass Bioenergy 46:210–217.

    CAS  Article  Google Scholar 

  27. Kim I, Lee B, Song D, Han J-I (2014) Effects of ammonium carbonate pretreatment on the enzymatic digestibility and structural features of rice straw. Bioresour Technol 166:353–357.

    CAS  Article  PubMed  Google Scholar 

  28. Ko JK, Bak JS, Jung MW, Lee HJ, Choi IG, Kim TH, Kim KH (2009) Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes. Bioresour Biotech 100:4374–4380.

    CAS  Article  Google Scholar 

  29. Kobkam C, Tinoi J, Kittiwachana S (2018) Alkali pretreatment and enzyme hydrolysis to enhance the digestibility of rice straw cellulose for microbial oil production. KMUTNB Int J Appl Sci Technol 11(4):247–256.

    Article  Google Scholar 

  30. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Kurokochi Y, Sato M (2020) Steam treatment to enhance rice straw binderless board focusing hemicellulose and cellulose decomposition products. J Wood Sci 66:7.

    CAS  Article  Google Scholar 

  32. Le HA, Phuong DM, Linh LT (2020) Emission inventories of rice straw open burning in the Red River Delta of Vietnam: evaluation of the potential of satellite data. Environ Pollut 260:113972.

    CAS  Article  PubMed  Google Scholar 

  33. Lee SY, Wong HH, Choi J, Lee SH, Lee SC, Han CS (2000) Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. Biotechnol Bioeng 64:466–470.;2-T

    Article  Google Scholar 

  34. Lee C, Zheng Y, VanderGheynst JS (2015) Effect of pretreatment conditions and post-pretreatment washing on ethanol production from dilute acid pretreated rice straw. Biosyst Eng 137:36–42.

    Article  Google Scholar 

  35. Li X, Sha J, Xia Y, Sheng K, Liu Y, He Y (2020) Quantitative visualization of subcellular lignocellulose revealing of mechanism of alkali pretreatment to promote methane production of rice straw. Biotechnol Biofuel 13:8.

    CAS  Article  Google Scholar 

  36. Li J, Yang Z, Zhang K, Liu M, Liu D, Yan X, Si M, Shi Y (2021) Valorizing waste liquor from dilute acid pretreatment of lignocellulosic biomass by Bacillus megaterium B-10. Ind Crops Prod 161:113160.

    CAS  Article  Google Scholar 

  37. Mohammed S, Behera HT, Dekebo A, Ray L (2020) Optimization of the culture conditions for production of polyhydroxyalkanoate and its characterization from a new Bacillus cereus sp. BNPI-92 strain, isolated from plastic waste dumping yard. Int J Biol Macromol 156:1064–1080.

    CAS  Article  PubMed  Google Scholar 

  38. Mohandas SP, Balan L, Jayanath G, Anoop BS, Philip R, Cubelio SS, Bright Singh IS (2018) Biosynthesis and characterization of polyhydroxyalkanoate from marine Bacillus cereus MCCB 281 utilizing glycerol as carbon source. Int J Biol Macromol 119:380–392.

    CAS  Article  PubMed  Google Scholar 

  39. Mohapatra S, Maity S, Dash HR, Das S, Pattnaik S, Rath CC, Samantaray D (2017) Bacillus and biopolymer: Prospects and challenges. Biochem Biophys Rep 12:206–213.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Naser AZ, Deiab I, Darras BM (2021) Poly(lactic acid) (PLA) and polyhydroxyalkanoates (PHAs), green alternatives to petroleum-based plastics: a review. RSC Adv 11:17151.

    CAS  Article  Google Scholar 

  41. Nguyen HV, Nguyen CD, Tran TV, Hau HD, Nguyen NT, Gunmert M (2016) Energy efficiency, greenhouse gas emissions, and cost of rice straw collection in the Mekong River Delta of Vietnam. Field Crops Res 198:16–22.

    Article  Google Scholar 

  42. Obruca S, Benesova P, Marsalek L, Marova I (2015) Use of lignocellulosic materials for PHA production. Chem Biochem Eng Q 29:135–144.

    CAS  Article  Google Scholar 

  43. Odeniyi OA, Adeola OJ (2017) Production and characterization of polyhydroxyalkanoic acid from Bacillus thuringiensis using different carbon substrates. Int J Biol Macromol 104:407–413.

    CAS  Article  PubMed  Google Scholar 

  44. Overturf E, Ravasio N, Zaccheria F, Tonin C, Patrucco A, Bertini F, Canetti M, Avramidou K, Speranza G, Bavaro T, Ubiali D (2020) Towards a more sustainable circular bioeconomy. Innovative approaches to rice residue valorization: the RiceRes case study. Bioresour Technol Rep 11:100427.

    Article  Google Scholar 

  45. Park YC, Kim JS (2012) Comparison of various alkaline pretreatment methods of lignocellulosic biomass. Energy 47:31–35.

    CAS  Article  Google Scholar 

  46. Ponnusamy S, Viswanathan S, Periyasamy A, Rajaiah S (2019) Production and characterization of PHB-HV copolymer by Bacillus thuringiensis isolated from Eisenia foetida. Biotechnol Appl Biochem 66:340–352.

    CAS  Article  PubMed  Google Scholar 

  47. Rodrigues CIS, Jackson JJ, Montross MD (2016) A molar basis comparison of calcium hydroxide, sodium hydroxide, and potassium hydroxide on the pretreatment of switchgrass and miscanthus under high solids conditions. Ind Crop Prod 92:165–173.

    CAS  Article  Google Scholar 

  48. Saratale GD, Oh MK (2015) Characterization of poly-3-hydroxybutyrate (PHB) produced from Ralstonia eutropha using an alkaline-pretreatment biomass feedstock. Int J Biol Macromol 8:627–635.

    CAS  Article  Google Scholar 

  49. Sarkar N, Ghosh SK, Bannerjee S, Aikat K (2012) Bioethanol production from agricultural wastes: an overview. Renew Energy 37:19–27.

    CAS  Article  Google Scholar 

  50. Semwal S, Raj T, Kumar R, Christopher J, Gupta RP, Puri SK, Kumar R, Ramakumar SSV (2019) Process optimization and mass balance studies of pilot scale steam explosion pretreatment of rice straw for higher sugar release. Biomass Bionergy 130:105390.

    CAS  Article  Google Scholar 

  51. Sindhu R, Silviya N, Binod P, Pandey A (2013) Pentose-rich hydrolysate from acid pretreated rice straw as a carbon source for the production of poly-3-hydroxybutyrate. Biochem Eng J 78:67–72.

    CAS  Article  Google Scholar 

  52. Sindhu R, Pandey A, Binod P (2015) Alkaline treatment. In: Pandey A, Negi S, Binod P, Larroche C (eds) Pretreatment of biomass. Process and technologies. Elsevier, Amsterdam, pp 51–60.

    Chapter  Google Scholar 

  53. Singh M, Patel SK, Kalia VC (2009) Bacillus subtilis as potential producer for polyhydroxyalkanoates. Microb Cell Fact 8:38.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Singh S, Sithole B, Lekha P, Permaul K, Govinden R (2021) Optimization of cultivation medium and cyclic fed-batch fermentation strategy for enhanced polyhydroxyalkanoate production by Bacillus thuringiensis using a glucose-rich hydrolyzate. Bioresour Bioprocess 8:11.

    Article  Google Scholar 

  55. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory, Colorado

    Google Scholar 

  56. Sophonputtanaphoca S, Sirigatmaneerat K, Kruakrut K (2018) Effect of low temperatures and residence times of pretreatment on glucan reactivity of sodium hydroxide-pretreated rice straw. Walailak J Sci Tech 15:313–323.

    Article  Google Scholar 

  57. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbüchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80.

    CAS  Article  PubMed  Google Scholar 

  58. Suzuki M, Tachibana Y, Kasuya K (2021) Biodegradability of poly(3-hydroxyalkanoate) and poly(ɛ-caprolactone) via biological carbon cycles in marine environments. Polym J 53:47–66.

    CAS  Article  Google Scholar 

  59. Takano M, Hoshino K (2018) Bioethanol production from rice straw by simultaneous saccharification and fermentation with statistical optimized cellulase cocktail and fermenting fungus. Bioresour Bioprocess 5:16.

    Article  Google Scholar 

  60. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526.

    CAS  Article  PubMed  Google Scholar 

  61. Tsegaye B, Balomajumder C, Roy P (2019) Alkali delignification and Bacillus sp. BMP01 hydrolysis of rice straw for enhancing biofuel yields. Bull Nat Res Cent 43:136.

    Article  Google Scholar 

  62. Tsegaye B, Balomajumder C, Roy P (2020) Organosolv pretreatments of rice straw followed by microbial hydrolysis for efficient biofuel production. Renew Energy 148:923–934.

    CAS  Article  Google Scholar 

  63. van der Pol EC, Vaessen E, Weusthuis RA, Eggink G (2016) Identifying inhibitory effects of lignocellulosic by-products on growth of lactic acid producing micro-organisms using a rapid small-scale screening method. Bioresour Technol 209:297–304.

    CAS  Article  PubMed  Google Scholar 

  64. Vu DH, Wainaina S, Taherzadeh MJ, Dan Åkesson D, Ferreira JA (2021) Production of polyhydroxyalkanoates (PHAs) by Bacillus megaterium using food waste acidogenic fermentation-derived volatile fatty acids. Bioengineered 12:2480–2498.

    CAS  Article  PubMed  Google Scholar 

  65. Weber S, Stubner S, Conrad R (2001) Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl Environ Microbiol 67:1318–1327.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  66. Yadav B, Talan A, Tyagi RD, Drogui P (2021) Concomitant production of value-added products with polyhydroxyalkanoate (PHA) synthesis: a review. Bioresour Technol 337:125419.

    CAS  Article  PubMed  Google Scholar 

  67. Zang Q, Cai W (2008) Enzymatic hydrolysis of alkali-pretreated rice straw by Trichoderma reesei ZM4-F3. Biomass Bioenergy 32:1130–1135.

    CAS  Article  Google Scholar 

  68. Zhang W, Liu J, Wang Y, Sun J, Huang P, Chang K (2020) Effect of ultrasound on ionic liquid-hydrochloric acid pretreatment with rice straw. Biomass Conv Bioref.

    Article  Google Scholar 

  69. Zhang S, Xia T, Wang J, Zhao Y, Xie X, Wei Z, Zhang X, Song C, Song X (2021) Role of Bacillus inoculation in rice straw composting and bacterial community stability after inoculation: unite resistance or individual collapse. Bioresour Technol 337:125464.

    CAS  Article  PubMed  Google Scholar 

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The authors are grateful to the Swedish Research Council for funding the study.


The authors thank the Swedish Research Council (Swedish Research Links grant, 348-2012-6169) for supporting this work.

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DVT: conceptualization, conducted all of the experimental work, analyzed and interpreted all data, writing—original draft, reviewing and editing. NTC: helped to conduct some parts of the experimental work. RHK: funding acquisition, writing—reviewing and editing, supervision. All authors read and approved the final manuscript.

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Correspondence to Rajni Hatti-Kaul.

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Additional file 1: Fig S1

. Yield of reducing sugars obtained after enzymatic hydrolysis of NaOH pretreated rice straw at (A) 30°C, (B) 50°C, and (C) 80°C, respectively, for different time periods. The enzymatic treatment was performed at 50°C. Fig S2. Yield of reducing sugars obtained during enzymatic hydrolysis of rice straw pretreated with Ca(OH)2 for different time periods at (A) 30°C, (B) 80°C, and (C) 121°C, respectively. The enzymatic treatment was performed at 50°C. Fig S3. Yield of reducing sugars obtained after enzymatic hydrolysis of rice straw pretreated with aqueous ammonia for different incubation times at (A) 50°C, and (B) 80°C, respectively. The enzymatic treatment was performed at 50°C. Fig S4. Cell growth and PHA accumulation by the strain B. cereus VK92 in culture media using (A) glucose and (B) rice straw hydrolysate, as carbon source at 35°C. Fig S5. Cell growth and PHA accumulation by strain B. cereus VK98 in culture media using (A) glucose and (B) rice straw hydrolysate, as carbon source at 35°C.

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Van Thuoc, D., Chung, N.T. & Hatti-Kaul, R. Polyhydroxyalkanoate production from rice straw hydrolysate obtained by alkaline pretreatment and enzymatic hydrolysis using Bacillus strains isolated from decomposing straw. Bioresour. Bioprocess. 8, 98 (2021).

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  • Bacillus species
  • Polyhydroxyalkanoate
  • Rice straw
  • Mild alkaline pretreatment
  • Enzymatic hydrolysis