CFD simulation of mixing for high-solids anaerobic digestion

A computational fluid dynamics (CFD) model that simulates mechanical mixing for high-solids anaerobic digestion was developed. Numerical simulations of mixing manure slurry which exhibits non-Newtonian pseudo-plastic fluid behavior were performed for six designs: (i) one helical ribbon impeller; (ii) one anchor impeller; (iii) one curtain-type impeller; (iv) three counterflow (CF-2) impellers; (v) two modified high solidity (MHS 3/39°) impellers; and (vi) two pitched blade turbine impellers. The CFD model was validated against measurements for mixing a Herschel-Bulkley fluid by ribbon and anchor impellers. Based on mixing time with respect to mixing energy level, three impeller types (ribbon, CF-2, and MHS 3/39°) stand out when agitating highly viscous fluids, of these mixing with two MHS 3/39° impellers requires the lowest power input to homogenize the manure slurry. A comparison of digestion material demonstrates that the mixing energy varies with manure type and total solids concentration to obtain a given mixing time. Moreover, an in-depth discussion about the CFD strategy, the influences of flow regime and impeller type on mixing characteristics, and the intrinsic relation between mixing and flow field is included.     

Agitation,aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus

Factorial design and response surface analyses were used to optimize the production of inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) by Kluyveromyces marxianus ATCC 16045, using sucrose as carbon source. Effects of aeration, agitation and type of impeller (disk turbine, marine, pitched blade) were studied in a batch stirred reactor. Two factorial designs 2² were carried out. Agitation speed varied from 50 to 550 rpm (revolution per minute), aeration rate from 0.5 to 2.0 vvm (air volume/broth volume·minute). It has been shown that the enzyme production was strongly influenced by mixing conditions, while aeration rate was shown to be less significant. Additionally, the increase in the agitation speed is limited by the death rate, which increases drastically at high speeds, lowering the enzyme production. Also, the impeller type has significant influence in the production, the disk impeller at 450 rpm and aeration at 1.0 vvm led to an activity of 121 UI/mL, while the pitched blade was shown to be the best impeller for this process, leading to the best production, 176 UI/mL, at 450 rpm and 1.0 vvm. The maximum shear stress for inulinase production was about 0.22 Pa, since higher values cause higher cell death rates, affecting the enzyme production. The same results were confirmed with another microorganism, which was also sensible to shear stress. Therefore, it has been concluded that in some cases, mainly when the microorganism is sensible to shear stress, the interaction between mass transfer and mechanical stress should be considered in scale up processes.     

Bioproduction of phenylethanoid glycosides by plant cell culture of Scrophularia striata Boiss.: from shake-flasks to bioreactor

Phenylethanoid glycosides (PeG) are a class of polyphenols found in some plants that have pharmaceutical effects as anti-inflammatories and anti-oxidants. The presence of PeG (acteoside) in the aerial parts of Scrophularia striata Boiss. has been demonstrated. Considerable progress has been made using plant cell cultures to stimulate formation and accumulation of secondary metabolites. The present study optimized phenylethanoid production from shake flasks to bioreactor using a cell culture of S. striata. The optimal conditions for production of cell biomass by scale-up to a bioreactor were determined to be a pH of 4.8, air flow rate of 0.5–1.5 l min⁻¹, and mixing speed of 110–170 rpm at 25 ± 1 °C in darkness. Growth parameters and PeG production were measured and compared with the results from the shake flasks. The results showed that cell biomass was high in the bioreactor (15.64 g l⁻¹ DW) and in the shake flasks (14.16 g l⁻¹ DW). The acteoside content in the bioreactor was 1404.20 μg g⁻¹ DW, which is threefold higher than in the shake flasks (459.71 μg g⁻¹ DW). The echinacoside concentration in the bioreactor was 1449.39 μg g⁻¹, 1.36-fold lower than in the shake flasks (1973.03 μg g⁻¹ DW). This study established an efficient way for production of acteoside, the major PeG, in a bioreactor.

     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Triarylmethane Dye Decolorization by Pellets of Pycnoporus sanguineus: Statistical Optimization and Effects of Novel Impeller Geometry

A study was carried out to optimize selected parameters for decolorization of a triarylmethane dye, such as crystal violet by white rot fungus, Pycnoporus sanguineus, pellets. The parameters studied were initial dye concentration (ppm), agitation speed (rpm), and process time (days) and were optimized using response surface methodology (RSM). It is shown that process time, agitation speed, and their interactions have significant effects on the decolorization process. Following the optimization, the decolorization study was extended to a stirred tank reactor (STR) process. Effects of different geometry of impellers on the decolorization process and power consumption were studied. Novel impeller geometries, such as 180° curved blade and 60° angled blade impellers, were used in the STR. The application of 180° curved blade impeller resulted in higher percentage of decolorization at a relatively less power consumption as compared with 60° angled blade impeller.     

Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l⁻¹, a product yield of 0.13 g g⁻¹ and a volumetric productivity of 0.54 g l⁻¹ h⁻¹, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP.     

Cellulolytic enzyme production from agricultural residues for biofuel purpose on circular economy approach

This study evaluated the production of cellulolytic enzymes from different agricultural residues. The crude enzyme extract produced was characterized and applied for saccharification of some agricultural residues. Maximum cellulolytic activities were obtained using soybean hulls. All enzymatic activities were highly stable at 40 °C at a pH range of 4.5–5.5. For stability at low temperatures, the enzyme extract was stored at freezing temperature and cooling for about 290 days without major loss of activity. The K_m values found for total cellulase (FPase), endoglucanase (CMCase), and xylanase were 19.73 mg ml⁻¹, 0.65 mg ml⁻¹, and 22.64 mg ml⁻¹, respectively, and V_max values were 0.82 mol min⁻¹ mg⁻¹, 0.62 mol min⁻¹ mg⁻¹, and 104.17 mol min⁻¹ mg⁻¹ to cellulose, carboxymethyl cellulose, and xylan, respectively. In the saccharification tests, the total amount of total reducing sugars (TRS) released from 1 g of soybean hulls catalyzed by the enzymes present in the crude enzyme extract was 0.16 g g⁻¹ dry substrate.

     

The influence of impeller type in pilot scale xanthan fermentations

The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.     

Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection

The aim of this work was to select an efficient impeller to be used in a stirred reactor for the enzymatic hydrolysis of sugar cane bagasse. All experiments utilized 100 g (dry weight)/l of steam-pretreated bagasse, which is utilized in Brazil for cattle feed. The process was studied with respect to the rheological behavior of the biomass hydrolysate and the enzymatic conversion of the bagasse polysaccharides. These parameters were applied to model the power required for an impeller to operate at pilot scale (100 l) using empirical correlations according to Nagata [16]. Hydrolysis experiments were carried out using a blend of cellulases, β-glucosidase, and xylanases produced in our laboratory by Trichoderma reesei RUT C30 and Aspergillus awamori. Hydrolyses were performed with an enzyme load of 10 FPU/g (dry weight) of bagasse over 36 h with periodic sampling for the measurement of viscosity and the concentration of glucose and reducing sugars. The mixture presented pseudoplastic behavior. This rheological model allowed for a performance comparison to be made between flat-blade disk (Rushton turbine) and pitched-blade (45°) impellers. The simulation showed that the pitched blade consumed tenfold less energy than the flat-blade disk turbine. The resulting sugar syrups contained 22 g/l of glucose, which corresponded to 45% cellulose conversion.     

Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus

Effects of pellet morphology on broth rheology are reported for pelleted submerged cultures of the lovastatin producing filamentous fungus Aspergillus terreus, growing in fluidized bed and stirred tank bioreactors. The pellet diameter and compactness were affected by the agitation intensity of the broth; however, the total biomass productivity was not affected. In fluidized beds and stirred tanks with agitation intensity of up to 300 rpm (impeller tip speed of 1.02 m s⁻¹), the fungal pellets were stable at diameters of up to about 2300 μm. In more intensely agitated stirred tanks (≥600 rpm; impeller tip speed of ≥2.03 m s⁻¹), the stable pellet size was only about ≤900 μm. The biomass concentration and the pellet diameter were the main factors that influenced the flow index and the consistency index of the power-law broths. Because the biomass productivity was the same in all experiments in a given type of reactor and the oxygen concentration was kept at ∼400% of air saturation, the pellet size and morphology were not influenced by oxygen mass transfer effects. Pellets were always dense in the core region and no necrosis of the biomass occurred.     

Submerged culture production of chitinase by Trichoderma harzianum in stirred tank bioreactors – the influence of agitator speed

The chitinase fermentation process utilizing chitin as the sole carbon source was investigated in a stirred tank bioreactor. Agitator speed of 224 rpm was found to be most suitable for cell growth as well as for chitinase production. Chitinase yield decreased rapidly at higher agitator speed, while decrease in cell yield at higher agitator speed was not rapid. Probably, mass transfer limitation was predominant in the fermentation process at lower agitator speed. Higher agitator speed appears to reduce chitinase production.     

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)₃ as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L⁻¹). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)₃ as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L⁻¹. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L⁻¹ (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L⁻¹ for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

     

Smoke-saturated Water and Karrikinolide Modulate Germination,Growth, Photosynthesis and Nutritional Values of Carrot (Daucus carota L.)

Plant-derived smoke is a positive regulator of seed germination and growth with regard to many plant species. Of the several compounds present in plant-derived smoke, karrikinolide or KAR₁ (3-methyl-2H-furo[2,3-c]pyran-2-one) is considered to be the major active growth-promoting compound. To test the efficacy of smoke-saturated water (SSW) and KAR₁ on carrot (Daucus carota L.), two separate pot experiments were simultaneously conducted in the same environmental conditions. SSW and KAR₁ treatments were applied to the plants in the form of aqueous solutions of variable concentrations. Prior to sowing, seeds were soaked in the solutions of SSW (25.8 µg L⁻¹_, 51.6 µg L⁻¹,103.2 µg L⁻¹ and 258.0 µg L⁻¹) and KAR₁ (0.015 µg L⁻¹, 0.150 µg L⁻¹, 1.501 µg L⁻¹ and 15.013 µg L⁻¹). Percent seed germination, vegetative growth, photosynthesis and nutritional values were the major parameters through which the plant response to the applied treatments was investigated. The results obtained indicated a significant improvement in all the plant attributes studied. In general, SSW (51.6 µg L⁻¹) and KAR₁ (1.501 µg L⁻¹) proved optimum treatments for most the parameters. As compared to the control, 51.6 µg L⁻¹ of SSW and 1.501 µg L⁻¹ of KAR₁ increased the percent seed germination by 58.0% and 54.4%, respectively. Over the control, the values of plant height and net photosynthetic rate were enhanced by 33.9% and 40.9%, respectively, due to 51.6 µg L⁻¹ of SSW, while the values of these parameters were increased by 25.2% and 34.0%, respectively, due to 1.501 µg L⁻¹ of KAR₁. In comparison with the control, treatment 51.6 µg L⁻¹ of SSW increased the contents of β-carotene and ascorbic acid by 32.7% and 37.9%, respectively, while the treatment 1.501 µg L⁻¹ M of KAR₁ enhanced these contents by 42.0% and 48.4%, respectively. This study provides an insight into an affordable and feasible method of crop improvement.

     

Bacterium Hafnia alvei secretes l-methioninase enzyme: Optimization of the enzyme secretion conditions

I isolated bacteria from blue cheese in order to find bacterial strains secreting l-methioninase enzyme, and optimized the conditions for the most efficient enzyme secretion. The efficient isolate, identified according to the 16S rRNA gene sequence analysis, was Hafnia alvei belonging to Enterobacteriaceae. I confirmed that the H. alvei strain harbored the methionase gene, mdeA (1194 bp). The environmental (pH, temperature) and nutritional (carbon and nitrogen sources and Mg concentration) factors influencing the l-methioninase production of H. alvei were optimized. The highest yield of l-methioninase enzyme was reached after 48 h of incubation when the acidity of the growing medium was adjusted to pH 7.5 and the temperature was 35 °C. The following concentrations of the supplements increased the l-methioninase yield in the medium: galactose (2.0 g L⁻¹), MgSO₄ (0.25 g L⁻¹), l-methionine as an inducer (2.0 g L⁻¹), and l-asparagine as an additional N source (1.5 g L⁻¹). I introduce a bacterial strain of H. alvei that is previously unreported to secrete l-methioninase enzyme and show that a carbon source is a mandatory supplement whereas l-methionine is not a mandatory supplement for l-methioninase enzyme production of H. alvei.     

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production

Microbial production of trans-4-hydroxy-l -proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l -proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l⁻¹ Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l -proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb_(L)-DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l⁻¹, 87.4% and 1.38 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.     

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min⁻¹ at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).     

Effect of carbon sources on the growth and ethanol production of native yeast Pichia kudriavzevii ITV-S42 isolated from sweet sorghum juice

The importance of non-Saccharomyces yeast species in fermentation processes is widely acknowledged. Within this group, Pichia kudriavzevii ITV-S42 yeast strain shows particularly desirable characteristics for ethanol production. Despite this fact, a thorough study of the metabolic and kinetic characteristics of this strain is currently unavailable. The aim of this work is to study the nutritional requirements of Pichia kudriavzevii ITV-S42 strain and the effect of different carbon sources on the growth and ethanol production. Results showed that glucose and fructose were both assimilated and fermented, achieving biomass and ethanol yields of 0.37 and 0.32 gg⁻¹, respectively. Glycerol was assimilated but not fermented; achieving a biomass yield of 0.88 gg⁻¹. Xylose and sucrose were not metabolized by the yeast strain. Finally, the use of a culture medium enriched with salts and yeast extract favored glucose consumption both for growth and ethanol production, improving ethanol tolerance reported for this genre (35 g L⁻¹) to 90 g L⁻¹ maximum ethanol concentration (over 100%). Furthermore Pichia kudriavzevii ITV-S42 maintained its fermentative capacity up to 200 g L⁻¹ initial glucose, demonstrating that this yeast is osmotolerant.

     

Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisk impeller

An immobilized enzyme bioreactor consisting of an agar gel-coated multidisk impeller was developed for the hydrolysis of highly viscous chitosan solutions, and the operating conditions for the production of physiologically active chitosan oligosaccharides (pentamers and hexamers) were investigated. Chitosanase was directly immobilized on the agar gel-coated multidisk impeller by a multipoint attachment method. The high stability of the immobilized enzyme was confirmed by means of five repetitions of a batch hydrolysis reaction. When the enzyme activity at the support surface was relatively high, the yield of the target products was higher at an impeller speed of 2 s⁻¹ than at a speed of 1 s⁻¹. However, no significant increase in yield was observed at impeller speeds higher than 2 s⁻¹ in reactions at either of the two substrate concentrations tested (5 and 20 kg/m³). When the surface enzyme activity was low, the impeller speed did not affect the yield of the target products. The maximum yield of pentamers and hexamers increased as the surface enzyme activity decreased, and high yields (>30%) were obtained at activities below 160 U/m². From the viewpoint of productivity, the optimal surface-enzyme activity was about 340 U/m², and at that activity, the yield of target products was 22%. This yield was higher than that reported for conventional acid hydrolysis. To maximize both the productivity and the yield of the target products, the surface area for the immobilized enzyme should be increased. Our results suggest that it may be possible to obtain high yields of pentamers and hexamers of chitosan oligosaccharides from highly viscous chitosan solutions with this reactor.     

Enzymatic production of trans-4-hydroxy-l-proline by proline 4-hydroxylase

Trans-4-hydroxy-l -proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging for industrial production of Hyp due to heavy environmental pollution and low production efficiency. To establish a green and efficient process for Hyp production, the proline 4-hydroxylase (DsP4H) from Dactylosporangium sp. RH1 was overexpressed and functionally characterized in Escherichia coli BL21(DE3). The recombinant DsP4H with l -proline as a substrate exhibited K_m, k_cat and k_cat/K_m values up to 0.80 mM, 0.52 s⁻¹ and 0.65 s⁻¹·mM⁻¹ respectively. Furthermore, DsP4H showed the highest activity at 35°C and pH 6.5 towards l -proline. The highest enzyme activity of 175.6 U mg⁻¹ was achieved by optimizing culture parameters. Under the optimal transformation conditions in a 5-l fermenter, Hyp titre, conversion rate and productivity were up to 99.9 g l⁻¹, 99.9% and 2.77 g l⁻¹ h⁻¹ respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.