Endo-Exoglucanase Synergism for Cellulose Nanofibril Production Assessment and Characterization.

A study to produce cellulose nanofibrils (CNF) from kraft cellulose pulp was conducted using a centroid simplex mixture design. The enzyme blend contains 69% endoglucanase and 31% exoglucanase. The central composite rotational design (CCRD) optimized the CNF production process by achieving a higher crystallinity index. It thus corresponded to a solid loading of 15 g/L and an enzyme loading of 0.974. Using the Segal formula, the crystallinity index (CrI) of the CNF was determined by X-ray diffraction to be 80.87%. The average diameter of the CNF prepared by enzymatic hydrolysis was 550-600 nm, while the one produced by enzymatic hydrolysis and with ultrasonic dispersion was 250-300 nm. Finally, synergistic interactions between the enzymes involved in nanocellulose production were demonstrated, with Colby factor values greater than one.

Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest.

Research background: This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach: Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions: The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Novelty and scientific contribution: Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

Improving propionic acid production from a hemicellulosic hydrolysate of sorghum bagasse by means of cell immobilization and sequential batch operation.

Propionic acid (PA) is an important organic compound with extensive application in different industrial sectors and is currently produced by petrochemical processes. The production of PA by large-scale fermentation processes presents a bottleneck, particularly due to low volumetric productivity. In this context, the present work aimed to produce PA by a biochemical route from a hemicellulosic hydrolysate of sorghum bagasse using the strain Propionibacterium acidipropionici CIP 53164. Conditions were optimized to increase volumetric productivity and process efficiency. Initially, in simple batch fermentation, a final concentration of PA of 17.5 g⋅L-1 was obtained. Next, fed batch operation with free cells was adopted to minimize substrate inhibition. Although a higher concentration of PA was achieved (38.0 g⋅L-1 ), the response variables (YP/S = 0.409 g⋅g-1 and QP = 0.198 g⋅L-1 ⋅H-1 ) were close to those of the simple batch experiment. Finally, the fermentability of the hemicellulosic hydrolysate was investigated in a sequential batch with immobilized cells. The PA concentration achieved a maximum of 35.3 g⋅L-1 in the third cycle; moreover, the volumetric productivity was almost sixfold higher (1.17 g⋅L-1 ⋅H-1 ) in sequential batch than in simple batch fermentation. The results are highly promising, providing preliminary data for studies on scaling up the production of this organic acid.

Statistical analysis of the crystallinity index of nanocellulose produced from Kraft pulp via controlled enzymatic hydrolysis.

Enzymatic hydrolysis processes can change the physical characteristics of nanocellulose derived from Kraft pulp. Among these attributes are its crystallinity index and dimensions. In this study, we determined the optimal conditions under which nanocellulose could be produced enzymatically with the greatest increase of the crystallinity index relative to its initial state. Application of Central Composite Rotatable Design statistical analysis to the experiments was employed to direct an increase the crystallinity index in 10% at the 24-H hydrolysis time. Upon establishment of ideal levels of starting material and enzyme, reactions were carried out at hydrolysis times of 24, 48, and 72 H under these ideal parameters. The effectiveness of deagglomeration was demonstrated by measuring the hydrodynamic diameter of the particles by dynamic light scattering. Scanning electron microscopy was performed on four samples, the original material, kraft pulp, and hydrolyzed biomaterials at 72 H in the ideal parameters. The hydrolyzed material with the best statistical data, revealing a fiber diameter of 180 nm, disclosing to be biomaterial with nanocellulose dimensions.

Insights from genome of Clostridium butyricum INCQS635 reveal mechanisms to convert complex sugars for biofuel production.

Clostridium butyricum is widely used to produce organic solvents such as ethanol, butanol and acetone. We sequenced the entire genome of C. butyricum INCQS635 by using Ion Torrent technology. We found a high contribution of sequences assigned for carbohydrate subsystems (15-20 % of known sequences). Annotation based on protein-conserved domains revealed a higher diversity of glycoside hydrolases than previously found in C. acetobutylicum ATCC824 strain. More than 30 glycoside hydrolases (GH) families were found; families of GH involved in degradation of galactan, cellulose, starch and chitin were identified as most abundant (close to 50 % of all sequences assigned as GH) in C. butyricum INCQS635. KEGG metabolic pathways reconstruction allowed us to verify possible routes in the C. butyricum INCQS635 and C. acetobutylicum ATCC824 genomes. Metabolic pathways for ethanol synthesis are similar for both species, but alcohol dehydrogenase of C. butyricum INCQS635 and C. acetobutylicum ATCC824 was different. The genomic repertoire of C. butyricum is an important resource to underpin future studies towards improved solvents production.

Adsorption of praziquantel enantiomers on chiral cellulose tris 3-chloro, 4-methylphenylcarbamate by frontal analysis: Fisherian and Bayesian parameter estimation and inference.

Praziquantel (PZQ) is an anthelmintic chiral pharmaceutical utilized in schistosomiasis treatment, commonly sold as a racemate, whose primary active molecule is the enantiomer L-(-)-PZQ. The development of new pharmaceutical formulations contenting L-(-)-PZQ has mobilized worldwide efforts from the academy and private companies. Several processes have been proposed to produce pure L-(-)-PZQ, including racemate resolution by preparative chromatography. The design of complex chromatographic processes such as SMB requires accurate information about the adsorption isotherm models and other system parameters and well-quantified uncertainties. We obtained the adsorption isotherms of both PZQ enantiomers using the Frontal Analysis (FA) technique. The associated uncertainties and model confidence bands were calculated from Fisherian and Bayesian approaches. Parameter uncertainties from both methods presented reasonable agreement. Bayesian inference allowed calculating conservative confidence intervals for the parameters, the isotherm curves and the experimental profiles related to FA. Predicted confidence intervals varied from 5.6% to 14% for parameters, 3.9% to 7.1% for the isotherms and 2.02% to 2.22% for the concentration on FA profiles. The estimated nuisance factor agreed with the experimental relative standard deviation and could be applied to predict experimental variances when the same is absent.

Usefulness of a real-time PCR platform for G+C content and DNA-DNA hybridization estimations in vibrios.

The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA-DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T(m) (°C). The T(m) and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R(2) = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT(m) values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT(m) values. For instance, the ΔT(m) of the sister species Vibrio harveyi LMG 4044(T) and Vibrio campbellii LMG 11216(T) was 5.2 °C, whereas the ΔT(m) of Vibrio coralliilyticus LMG 20984(T) and Vibrio neptunius LMG 20536(T) was 8.75 °C. The mean ΔT(m) values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT(m) values (R(2) = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT(m) in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT(m). The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.

Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by Actinobacillus succinogenes.

Succinic acid, a four-carbon diacid, has been the focus of many research projects aimed at developing more economically viable methods of fermenting sugar-containing natural materials. Succinic acid fermentation processes also consume CO(2), thereby potentially contributing to reductions in CO(2) emissions. Succinic acid could also become a commodity used as an intermediate in the chemical synthesis and manufacture of synthetic resins and biodegradable polymers. Much attention has been given recently to the use of microorganisms to produce succinic acid as an alternative to chemical synthesis. We have attempted to maximize succinic acid production by Actinobacillus succinogenes using an experimental design methodology for optimizing the concentrations of the medium components. The first experiment consisted of a 2(4-1) fractional factorial design, and the second entailed a Central Composite Rotational Design so as to achieve optimal conditions. The optimal concentrations of nutrients predicted by the model were: NaHCO(3), 10.0 g l(-1); MgSO(4), 3.0 g l(-1); yeast extract, 2.0 g l(-1); KH(2)PO(4). 5.0 g l(-1); these were experimentally validated. Under the best conversion conditions, as determined by statistical analysis, the production of succinic acid was carried out in an instrumented bioreactor using sugarcane bagasse hemicellulose hydrolysate, yielding a concentration of 22.5 g l(-1).

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity.

The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pre-treatments are essential. The hydrolysis of cellulose - major component of LCB - demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail was performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp. LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp. LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52 g/L solids and 30 mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78 g/L solids and 19 mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp. LMI03, and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum.

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis.

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L(-1)) and beta-glucosidase (1,835 U L(-1)) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58 degrees C and pH values of around 4.9 contributed to maximum enzyme activity. At 37 degrees C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L(-1)). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of beta-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L(-1) h(-1)) when incubated with corn cob than with Celluclast, GC 220 and Spezyme (312, 454 and 400 mg L(-1) h(-1), respectively).

Evaluation of the Enzymatic Arsenal Secreted by Myceliophthora thermophila During Growth on Sugarcane Bagasse With a Focus on LPMOs.

The high demand for energy and the increase of the greenhouse effect propel the necessity to develop new technologies to efficiently deconstruct the lignocellulosic materials into sugars monomers. Sugarcane bagasse is a rich polysaccharide residue from sugar and alcohol industries. The thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophilum) is an interesting model to study the enzymatic degradation of biomass. The genome of M. thermophila encodes an extensive repertoire of cellulolytic enzymes including 23 lytic polysaccharide monooxygenases (LPMOs) from the Auxiliary Activity family 9 (AA9), which are known to oxidatively cleave the ß-1,4 bonds and boost the cellulose conversion in a biorefinery context. To achieve a deeper understanding of the enzymatic capabilities of M. thermophila on sugarcane bagasse, we pretreated this lignocellulosic residue with different methods leading to solids with various cellulose/hemicellulose/lignin proportions and grew M. thermophila on these substrates. The secreted proteins were analyzed using proteomics taking advantage of two mass spectrometry methodologies. This approach unraveled the secretion of many CAZymes belonging to the Glycosyl Hydrolase (GH) and AA classes including several LPMOs that may contribute to the biomass degradation observed during fungal growth. Two AA9 LPMOs, called MtLPMO9B and MtLPMO9H, were selected from secretomic data and enzymatically characterized. Although MtLPMO9B and MtLPMO9H were both active on cellulose, they differed in terms of optimum temperatures and regioselectivity releasing either C1 or C1-C4 oxidized oligosaccharides, respectively. LPMO activities were also measured on sugarcane bagasse substrates with different levels of complexity. The boosting effect of these LPMOs on bagasse sugarcane saccharification by a Trichoderma reesei commercial cocktail was also observed. The partially delignified bagasse was the best substrate considering the oxidized oligosaccharides released and the acid treated bagasse was the best one in terms of saccharification boost.

Lactic acid production from sugarcane bagasse hydrolysates by Lactobacillus pentosus: Integrating xylose and glucose fermentation.

Lactic acid, traditionally obtained through fermentation process, presents numerous applications in different industrial segments, including production of biodegradable polylactic acid (PLA). Development of low cost substrate fermentations could improve economic viability of lactic acid production, through the use of agricultural residues as lignocellulosic biomass. Studies regarding the use of sugarcane bagasse hydrolysates for lactic acid production by Lactobacillus spp. are reported. First, five strains of Lactobacillus spp. were investigated for one that had the ability to consume xylose efficiently. Subsequently, biomass fractionation was performed by dilute acid and alkaline pretreatments, and the hemicellulose hydrolysate (HH) fermentability by the selected strain was carried out in bioreactor. Maximum lactic acid concentration and productivity achieved in HH batch were 42.5 g/L and 1.02 g/L h, respectively. Hydrolyses of partially delignified cellulignin (PDCL) by two different enzymatic cocktails were compared. Finally, fermentation of HH and PDCL hydrolysate together was carried out in bioreactor in a hybrid process: saccharification and co-fermentation with an initial enzymatic hydrolysis. The high fermentability of these process herein developed was demonstrated by the total consumption of xylose and glucose by Lactobacillus pentosus, reaching at 65.0 g/L of lactic acid, 0.93 g/g of yield, and 1.01 g/L h of productivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2718, 2019.

Production of nanocellulose by enzymatic hydrolysis: Trends and challenges.

There is a great interest in increasing the levels of production of nanocellulose, either by adjusting production systems or by improving the raw material. Despite all the advantages and applications, nanocellulose still has a high cost compared to common fibers and to reverse this scenario the development of new, cheaper, and more efficient means of production is required. The market trend is to have an increase in the mass production of nanocellulose; there is a great expectation of world trade. In this sense, research in this sector is on the rise, because once the cost is not an obstacle to production, this material will have more and more market. Production of the cellulose fibers is determinant for the production of nanocellulose by a hydrolyzing agent with a reasonable yield. This work presents several aspects of this new material, mainly addressing the enzymatic pathway, presenting the hydrolysis conditions such as pH, biomass concentration, enzymatic loading, temperature, and time. Also, the commonly used characterization methods are presented, as well as aspects of the nanocellulose production market.

Cell immobilization on 3D-printed matrices: A model study on propionic acid fermentation.

This study uses three-dimensional (3D) printing technology as a tool for designing carriers for immobilization of microbial cells for bioprocesses. Production of propionic acid from glucose by immobilized Propionibacterium sp. cells was studied as a model system. For cell adsorption, the 3D-printed nylon beads were added to the culture medium during 3 rounds of cell cultivation. Cell adsorption and fermentation kinetics were similar irrespective of the bead size and lattice structure. The cells bound to 15 mm beads exhibited reduced fermentation time as compared to free cell fermentations; maximum productivity and propionic acid titer of 0.46 g/L h and 25.8 g/L, respectively, were obtained. Treatment of the beads with polyethyleneimine improved cell-matrix binding, but lowered the productivity perhaps due to inhibitory effect of the polycation. Scanning electron micrographs revealed the cells to be located in crevices of the beads, but were more uniformly distributed on PEI-coated carrier indicating charge-charge interaction.

Addition of Surfactants and Non-Hydrolytic Proteins and Their Influence on Enzymatic Hydrolysis of Pretreated Sugarcane Bagasse.

Poly(ethylene glycol) (PEG 4000) and bovine serum albumin (BSA) were investigated with the purpose of evaluating their influence on enzymatic hydrolysis of sugarcane bagasse. Effects of these supplements were assayed for different enzymatic cocktails (Trichoderma harzianum and Penicillium funiculosum) that acted on lignocellulosic material submitted to different pretreatment methods with varying solid (25 and 100 g/L) and protein (7.5 and 20 mg/g cellulose) loadings. The highest levels of glucose release were achieved using partially delignified cellulignin as substrate, along with the T. harzianum cocktail: increases of 14 and 18 % for 25 g/L solid loadings and of 33 and 43 % for 100 g/L solid loadings were reached for BSA and PEG supplementation, respectively. Addition of these supplements could maintain hydrolysis yield even for higher solid loadings, but for higher enzymatic cocktail protein loadings, increases in glucose release were not observed. Results indicate that synergism might occur among these additives and cellulase and xylanases. The use of these supplements, besides depending on factors such as pretreatment method of sugarcane bagasse, enzymatic cocktails composition, and solid and protein loadings, may not always lead to positive effects on the hydrolysis of lignocellulosic material, making it necessary further statistical studies, according to process conditions.

Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1.

Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L(-1)-10.9 g L(-1)). These results offer promising pathways for the optimization of processes for the production of rhamnolipids.

Design of an enzyme cocktail consisting of different fungal platforms for efficient hydrolysis of sugarcane bagasse: Optimization and synergism studies.

Lignocellulosic materials represent a very important and promising source of renewable biomass. In order to turn them into fermentable sugars, synergism among the different enzymes that carry out bioconversion of these materials is one of the main factors that should be considered. Experimental mixture design was performed to optimize the proportion of enzymes produced by native strains of Trichoderma harzianum IOC 3844, Penicillium funiculosum ATCC 11797, and Aspergillus niger ATCC 1004, resulting in a proportion of 15, 50, and 35%, respectively. This mixture was able to hydrolyze 25 g/L of pretreated sugarcane bagasse with 91% of yield after 48 h of enzymatic reaction. Synergism along the hydrolysis process, besides the influence of lignin, hemicellulose, and solids loading, were also studied. Response surface methodology (RSM) based on Central Composite Rotatable Design was used to optimize solids and protein loadings to increase glucose release and enzymatic hydrolysis yield. The optimum solid and protein loadings established with RSM were 196 g/L and 24 mg/g cellulose, respectively, and under these conditions (94.1 ± 8) g/L of glucose were obtained, corresponding to a hydrolysis yield of 64%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1222-1229, 2016.

Evaluation of the Fermentation Potential of Pulp Mill Residue to Produce D(-)-Lactic Acid by Separate Hydrolysis and Fermentation Using Lactobacillus coryniformis subsp. torquens.

Lactic acid is widely used in chemical, pharmaceutical, cosmetic, and food industries, besides it is the building block to produce polylactic acid, which is a sustainable alternative biopolymer to synthetic plastic due to its biodegradability. Aiming at producing an optically pure isomer, the present work evaluated the potential of pulp mill residue as feedstock to produce D(-)-lactic acid by a strain of the bacterium Lactobacillus coryniformis subsp. torquens using separate hydrolysis and fermentation process. Enzymatic hydrolysis, optimized through response surface methodology for 1 g:4 mL solid/liquid ratio and 24.8 FPU/gcellulose enzyme loading, resulted in 140 g L-1 total reducing sugar and 110 g L-1 glucose after 48 h, leading to 61 % of efficiency. In instrumented bioreactor, 57 g L-1 of D(-)-lactic acid was achieved in 20 h of fermentation, while only 0.5 g L-1 of L(+)-lactic acid was generated. Furthermore, product yield of 0.97 g/g and volumetric productivity of 2.8 g L-1 h-1 were obtained.

Production of 1,3-propanediol by Clostridium beijerinckii DSM 791 from crude glycerol and corn steep liquor: Process optimization and metabolic engineering.

1,3-Propanediol (1,3-PDO) production from crude glycerol, a byproduct from biodiesel manufacturing, by Clostridium beijerinckii DSM 791 was studied with corn steep liquor as an inexpensive nitrogen source replacing yeast extract in the fermentation medium. A stable, long-term 1,3-PDO production from glycerol was demonstrated with cells immobilized in a fibrous bed bioreactor operated in a repeated batch mode, which partially circumvented the 1,3-PDO inhibition problem. The strain was then engineered to overexpress Escherichia coli gldA encoding glycerol dehydrogenase (GDH) and dhaKLM encoding dihydroxyacetone kinase (DHAK), which increased 1,3-PDO productivity by 26.8-37.5% compared to the wild type, because of greatly increased specific growth rate (0.25-0.40h(-1) vs. 0.13-0.20h(-1) for the wild type). The engineered strain gave a high 1,3-PDO titer (26.1g/L), yield (0.55g/g) and productivity (0.99g/L·h) in fed-batch fermentation. Overexpressing GDH and DHAK was thus effective in increasing 1,3-PDO production from glycerol.