Enhanced production of lactic acid from pretreated rice straw using co-cultivation of Bacillus licheniformis and Bacillus sonorenesis.

Lactic acid (LA) production from sugar mixture derived from lignocellulosic rice straw employing co- culture system of thermotolerant and inhibitor tolerant Bacillus licheniformis DGB and Bacillus sonorenesis DGS15 was carried out. In minimal media, both the strains of Bacillus DGB and DGS15 worked together by efficiently utilising glucose and xylose respectively. Response Surface Methodology (RSM) was used for optimisation of pretreatment of rice straw to achieve maximum yield of 50.852 g/L total reducing sugar (TRS) from 100 gm of rice straw biomass. Pretreatment of rice straw resulted in its delignification, as confirmed by FTIR spectroscopy, since the peak at 1668 cm-1 disappeared due to removal of lignin and scanning electron microscopy (SEM) revealed disruption in structural and morphological features. Crystallinity index (CrI) of treated rice straw increased by 15.54% in comparison to native biomass. DGB and DGS15 individually yielded 0.64 g/g and 0.82 g/g lactic acid respectively, where as their co-cultivation led to effective utilisation of both glucose and xylose within 15 h (70%) and complete utilisation in 48 h, producing 49.75 g/L LA with a yield of 0.98 g/g and productivity of 1.036 g/L/h, and resulting in reduction in fermentation time. Separate hydrolysis of rice straw and co-fermentation (SHCF) of hydrolysates by Bacillus spp. enhanced the production of lactic acid, can circumvent challenges in biorefining of lignocellulosic biomass.

Polyhydroxyalkanoate production from rice straw hydrolysate: Insights into feast-famine dynamics and microbial community shifts.

Activated sludge contains a versatile microbiome capable of converting wastes into valuable chemicals like polyhydroxyalkanoates (PHA). This study investigated the influence of repeated feast and famine phases on PHA production as well as the corresponding microbial population dynamics using waste activated sludge (WAS) as inoculum. Hydrolysate derived from rice straw was employed as a substrate for PHA production. The 16sRNA analysis results revealed that Corynebacteriaceae (40%), Bacillaceae (23%), and Pseudomonas (5%) were the primary contributors to PHA synthesis. Notably, Bacillaceae and Pseudomonas thrived in all the feast and famine phases. The achieved PHA concentration was 3.5 ± 0.2 g/L, and its structure and composition were assessed using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The analysis revealed that the PHA consists of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV), specifically identified as Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV).

Cultivation of Navicula sp. on rice straw hydrolysate for the production of biogenic silica.

Rice straw hydrolysate (RSH) prepared at room temperature was found to be rich in silica (140 ± 4.1 mg L-1) and other nutrients (nitrate-N: 160 ± 4.3 mg L-1, total dissolve phosphate: 164 ± 6.7 mg L-1, ammoniacal-N: 439.8 ± 17 mg L-1). The aim of this work was to study four RSH dilutions (10, 30, 50, 70% v/v) to cultivate Navicula sp. with modified ASN-III as a control. The best result was achieved in 30% RSH in terms ofdoubling time (d = 1.49 days) and growth rate (µmax = 0.46 day-1). Compared to control, specific growth rate and biomass productivity were increased by 2.93 folds and 1.85 folds, respectively. Cultivation in 5 L reactor with optimized 30% RSH yielded frustule (54.2 ± 1.9%), carbohydrate (12.4 ± 1.2%), lipid (18.9 ± 1.4%), and protein (8.2 ± 0.6%). The residual solid fraction showed 18.99% increased theoretical methane yield than raw rice straw. Overall, the present process offers a sustainable solution to manage rice straw residue and recover nanoporous silica.

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 xylA Xc and xylB Cg 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.

Improving probiotic spore yield using rice straw hydrolysate.

Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO4 and yeast extract were screened by Plackett-Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO4 of 0·78 g l-1 and yeast extract of 1·2 g l-1 . The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.

Immobilization of Laccase on Magnetic Nanoparticles and Application in the Detoxification of Rice Straw Hydrolysate for the Lipid Production of Rhodotorula glutinis.

The production of microbial lipid using lignocellulosic agroforestry residues has attracted much attention. But, various inhibitors such as phenols and furans, which are produced during lignocellulosic hydrolysate preparation, are harmful to microbial lipid accumulation. Herein, we developed a novel detoxification strategy of rice straw hydrolysate using immobilized laccase on magnetic Fe3O4 nanoparticles for improving lipid production of Rhodotorula glutinis. Compared with free laccase, the immobilized laccase on magnetic nanoparticles showed better stability, which still retained 76% of original activity at 70 °C and 56% at pH 2 for 6 h. This immobilized laccase was reused to remove inhibitors in acid-pretreated rice straw hydrolysate through recycling with external magnetic field. The results showed that most of phenols, parts of furans, and formic acids could be removed by immobilized laccase after the first batch. Notably, the immobilized laccase exhibited good reusability in repeated batch detoxification. 78.2% phenols, 43.8% furfural, 30.4% HMF, and 16.5% formic acid in the hydrolysate were removed after the fourth batch. Furthermore, these detoxified rice straw hydrolysates, as substrates, were applied to the lipid production of Rhodotorula glutinis. The lipid yield in detoxified hydrolysate was significantly higher than that in undetoxified hydrolysate. These findings suggest that the immobilized laccase on magnetic nanoparticles has a potential to detoxify lignocellusic hydrolysate for improving microbial lipid production.

Valorization of xylose-rich hydrolysate from rice straw, an agroresidue, through biosurfactant production by the soil bacterium Serratia nematodiphila.

Biosurfactants, amphiphilic compounds that reduce interfacial tension in oil-aqueous mixtures, are used in the petroleum, pharmaceutical, food, and agriculture industries. Fermentative production of biosurfactants requires expensive sugar or lipid substrates. Lignocellulosic biomass is a relatively cheap and abundant agricultural residue that can be used as an alternative substrate. Currently, several million tonnes of rice and wheat straw are generated globally as agricultural residues, most of which is disposed by open-field burning thereby leading to severe environmental pollution. This study aimed to produce biosurfactants in xylose-rich hydrolysates generated from rice straw. The hydrolysate is also a byproduct of 2G biofuel processes that often goes underutilized. A soil bacterium capable of growing and producing biosurfactants in rice straw hydrolysates, which typically contain growth-inhibitory compounds such as furfural and hydroxymethyl furfural, was isolated. Interestingly, the organism, identified as Serratia nematodiphila, exhibited higher glycolipid formation (4.5 ± 0.6 gL-1) in xylose-rich hydrolysate than in glucose-rich enzymatic hydrolysate (3.1 ± 0.2 gL-1) despite the higher bacterial cell density observed with the latter. The biosurfactants were thermostable and possessed promising emulsifying property and anti-microbial activity against bacteria and yeast. Further optimization of C:N resulted in a 2.8-fold increase in glycolipid production from xylose-rich hydrolysates. This study demonstrates the production of glycolipid biosurfactants from lignocellulosic biomass, a low-cost substrate and offers a plausible strategy for the management of these residues. Further, it also provides insights into the generation of additional high-value compounds in a bioethanol biorefinery to improve its commercial feasibility.

Cost-effective pigment production by Monascus purpureus using rice straw hydrolysate as substrate in submerged fermentation.

Monascus pigments (MPs), the secondary metabolites produced by the fungal strains of Monascus spp., hold commercial importance in not only the food and meat industries, but also therapeutic, cosmetic, and textile industries. To reduce the cost of MPs production, the utilization of rice straw hydrolysate as a substrate in submerged fermentation was investigated. The atmospheric and room temperature plasma (ARTP) mutation system was employed to develop a mutant strain Monascus purpureus M630, with high total extracellular Monascus pigments (exMPs) production of 34.12 U/mL in submerged fermentation with glucose-based medium. The results revealed that M. purpureus M630 produces 8.61 U/mL and 20.86 U/mL of exMPs in rice straw hydrolysate alone or in combination with glucose fermentation medium, respectively. Furfural (Fur) and 5'-hydroxymethyl furfural (5'-HMF), produced during pretreatment and hydrolysis of rice straw; are generally inhibitory for microbial growth and fermentation. Our findings revealed that M. purpureus M630 develops the tolerance and adaptation mechanisms in response to 5'-HMF and Fur during growth and MPs biosynthesis in rice straw hydrolysate. In conclusion, we report that rice straw hydrolysate can serve as an efficient and low-cost substitute for the MP production through submerged fermentation by Monascus spp.

Modulation of culture medium confers high-specificity production of isopentenol in Bacillus subtilis.

Enthusiasm for mining isoprenoid-based flavors, pharmaceuticals, and nutraceuticals from GRAS (Generally Regarded as Safe) status microbial hosts has increased in the past few years due to the limitations associated with their plant-based extraction and chemical synthesis. Bacillus subtilis, a well-known GRAS microbe, is a promising alternative due to its fast growth rate and the ability to metabolize complex carbon sources. The study focused on the high-specificity production of isopentenol in B. subtilis by modulating the culture medium. Media modulation led to a 2.5 folds improvement in isopentenol titer in the wild-type strain. In the recombinant strain, optimization of physico-chemical factors, coupled with overexpression of the nudF enzyme resulted in a maximum isopentenol titer of ∼6 mg/L in a shake flask. The recombinant strain produced ∼5 mg/L isoprenol (∼80% of the total isopentenol production) and ∼1.8 mg/L prenol (∼65% of the total isopentenol production) by utilizing sorbitol and pyruvate as the carbon sources, respectively. Replacement of glucose with sorbitol and pyruvate reduced the production of the undesired metabolites and enhanced high-specificity production of isopentenol. Upon replacement of the carbon source with a low-cost substrate, a non-detoxified rice-straw hydrolysate, the engineered strain produced 2.19 mg/L isopentenol. This proof-of-concept study paves the path for the high-specificity production and cost-effective recovery of isopentenol from industrially competent microbial strains with engineered isoprenoid pathways.

Metabolic engineering of Acinetobacter baylyi ADP1 for removal of Clostridium butyricum growth inhibitors produced from lignocellulosic hydrolysates.

BACKGROUND: Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars. Selective inhibitor removal can be achieved with biodetoxification where microorganisms catabolize the inhibitors without consuming the sugars. We engineered the strictly aerobic Acinetobacter baylyi ADP1 for detoxification of lignocellulosic hydrolysates by removing the gene for glucose dehydrogenase, gcd, which catalyzes the first step in its glucose catabolism. RESULTS: The engineered A. baylyi ADP1 strain was shown to be incapable of consuming the main sugar components of lignocellulosic hydrolysates, i.e., glucose, xylose, and arabinose, but rapidly utilized acetate and formate. Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate. The engineered strain tolerated glucose well up to 70 g/l, and the consumption of glucose, xylose, or arabinose was not observed in prolonged cultivations. The engineered strain was applied in removal of oxygen, a gaseous inhibitor of anaerobic fermentations. Co-cultivation with the A. baylyi ADP1 gcd knockout strain under initially aerobic conditions allowed the strictly anaerobic Clostridium butyricum to grow and produce hydrogen (H2) from sugars of the enzymatic rice straw hydrolysate. CONCLUSIONS: We demonstrated that the model organism of bacterial genetics and metabolism, A. baylyi ADP1, could be engineered to be an efficient biodetoxification strain of lignocellulosic hydrolysates. Only one gene knockout was required to completely eliminate sugar consumption and the strain could be used in production of anaerobic conditions for the strictly anaerobic hydrogen producer, C. butyricum. Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.

Production of poly-ß-hydroxybutyrate by Bacillus cereus PS 10 using biphasic-acid-pretreated rice straw.

Poly-ß-hydroxybutyrate (PHB) has attracted a great deal of attention in recent years due to its potential use for production of fully degradable bioplastics, however, high cost of PHB production is the major bottleneck for its wide range industrial applications. In the current study rice straw hydrolysate (RSH) was employed as a cost-effective substrate for PHB production. RSH was prepared based on biphasic acid-pretreatment of rice straw i.e. first phase treatment with 1% sulphuric acid at 121 °C for 45 min, followed by second phase treatment using 5% sulphuric acid at 121 °C for 60 min (solid:liquid ratio, 1:10). RSH turned out be an efficient substrate for PHB production from a recently isolated Bacillus cereus PS 10, and yielded higher PHB amount than that obtained with glucose (8.6g/L in glucose based medium vs 10.61 g/L in RSH based medium) after response surface methodology (RSM) based optimization. Design of experiments based on RSM was used to optimize three process variables i.e. amount of RSH and NH4Cl, and medium pH, and enhanced PHB yield (23.3%) was obtained. PHB produced was investigated by differential scanning calorimetry and X-ray diffraction powder analysis.

Ethanol fermentation by xylose-assimilating Saccharomyces cerevisiae using sugars in a rice straw liquid hydrolysate concentrated by nanofiltration.

Concentrating sugars using membrane separation, followed by ethanol fermentation by recombinant xylose-assimilating Saccharomyces cerevisiae, is an attractive technology. Three nanofiltration membranes (NTR-729HF, NTR-7250, and ESNA3) were effective in concentrating glucose, fructose, and sucrose from dilute molasses solution and no permeation of sucrose. The separation factors of acetate, formate, furfural, and 5-hydroxymethyl furfural, which were produced by dilute acid pretreatment of rice straw, over glucose after passage through these three membranes were 3.37-11.22, 4.71-20.27, 4.32-16.45, and 4.05-16.84, respectively, at pH 5.0, an applied pressure of 1.5 or 2.0 MPa, and 25 °C. The separation factors of these fermentation inhibitors over xylose were infinite, as there was no permeation of xylose. Ethanol production from approximately two-times concentrated liquid hydrolysate using recombinant S. cerevisiae was double (5.34-6.44 g L(-1)) that compared with fermentation of liquid hydrolysate before membrane separation (2.75 g L(-1)).

Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata.

In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FW of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources.

Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01.

Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

Development of semi-defined rice straw-based medium for butanol production and its kinetic study.

Rice straw is one of the potential economic feedstock for biobutanol production through ABE fermentation. However, the rice straw hydrolysate-based fermentation medium needs to be supported with nutritional elements. In this study, an attempt is made to optimize the rice straw hydrolysate-based fermentation medium employing Clostridium acetobutylicum MTCC 481 using Taguchi design of experiments (DOE) statistical model. Initially, a set of 12 nutrient components viz. MgNO3·6H2O, FeNO3, NH4NO3, yeast extract, PABA, biotin, PABA + biotin mixture, CaCl2, KCl, NaCl, MgSO4 and CH3COONa were screened through classical (one-variable-at-a-time) method. Based on the results, four components (PABA, yeast extract, MgSO4 and CH3COONa) were found to have significant impact, and were further subjected to statistical optimization through Taguchi DOE method. These experiments revealed that RSH supported with 3 g L-1 of yeast extract and 4 mg L-1 PABA to RSH was the most optimum fermentation medium. Experiments using 2 L bioreactor with this optimum fermentation medium showed nearly complete utilization of soluble sugars with the production of 8.7 g L-1 of total solvents and 6 g L-1 of butanol. The experimental data were fitted to kinetic models reported in the literature to determine the kinetic parameters of the fermentation process. An interesting result was revealed from this analysis that the under optimized fermentation medium, the kinetic parameters for both shake flask and bioreactor level were similar. This essentially means that effect of scale of operation is rendered insignificant when fermentation medium is under optimum conditions.