Biochemical Engineering Journal (v.74, #C)

BEJ Keywords (II).

Display Omitted► (R)-Epichlorohydrin from 1,3-dichloro-2-propanol was prepared by two-step biocatalysis. ► Haloalcohol dehalogenase and epoxide hydrolase were successfully immobilized on perlites. ► A special reactor was designed for eliminating the racemisation of epichlorohydrin. ► Two-phase solution was used to reduce the spontaneous chemical hydrolysis and reverse reaction of epichlorohydrin.Recombinant Escherichia coli cells harbouring haloalcohol dehalogenase and epoxide hydrolase were successfully immobilized by adsorption onto perlite and used to prepare (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis in a specially designed reactor. Two-phase solution was used as the reaction system in order to improve the yield of epichlorohydrin. In the two-phase system containing 40% (v/v) cyclohexane, the yield of racemic epichlorohydrin formed in the first step was 73%, and the yield of (R)-epichlorohydrin with enantiomeric excess (ee) ≥99% increased from 19.2% to 25.1% in the second step. Ultimately, the yield of (R)-epichlorohydrin reached 26.4% by optimization of the flow rate of air and amount of immobilized cells. To our knowledge, this was the first report on production of (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase.
Keywords: Haloalcohol dehalogenase; Epoxide hydrolase; Two-phase system; (R)-Epichlorohydrin; Immobilized cells;

Effects of photobioreactors design and operating conditions on Stichococcus bacillaris biomass and biodiesel production by Giuseppe Olivieri; Immacolata Gargano; Roberto Andreozzi; Raffaele Marotta; Antonio Marzocchella; Gabriele Pinto; Antonino Pollio (8-14).
► Autotrophic cultures of Stichococcus bacillaris were carried to produce biodiesel. ► Two photobioreactor configurations were investigated. ► Effects of pH, CO2 concentration and hydrodynamics were investigated. ► Biomass and FAME productivity were optimized: 0.26 g/(L d) and 0.032 g/(L d).Indoor cultures of Stichococcus bacillaris were carried out to investigate the effect of pH, CO2, and hydrodynamics on biomass and biodiesel productivity in a 0.6 L vertical bubble column (VBC) and a 1.7 L inclined bubble column (IBC) photobioreactor. The temperature and the irradiance level were set at 23 °C and 300 μE/(m2  s), respectively. The pH of the liquid culture was in the range 3.0–8.5. The CO2 concentration in the gas phase was increased up to 15%, typical of exhaust gas from power plants. The increased CO2 concentration in the gas phase stimulated the microalgal growth. Tests carried out at different pH and spreading a 5% CO2 gas stream showed that the biomass productivity was maximum at a pH of about 7.0. Acid and alkaline conditions can also be adopted with an approximate 25% decrease in biomass productivity. The comparison between a IBC and a VBC reactor operated under the same conditions pointed out that the performances of IBC were higher than those of VBC: 0.26 gbiomass/(L d) and 0.032 gFAME/(L d) for IBC, and 0.124 gbiomass/(L d) and 0.010 gFAME/(L d) for VBC. IBC was characterized by an intensive liquid circulation that promotes a continuous renewal of microalgal cells in the photic zone.
Keywords: Biodiesel; Microalgae; Photobioreactor; Bubble column; Stichococcus bacillaris;

► Diverse organic wastes present high potential as substrates for protease production. ► Our findings consolidate solid-state fermentation as a waste valorisation process. ► Respiration indices are useful to determine the maximum enzyme activity. ► Soy fibre exhibits the highest potential as a source of protease. ► Alkaline pH and mesophilic temperature were optimal for protease activity.The production of alkaline proteases by solid-state fermentation (SSF) was evaluated. The effect of three agro-industrial residues was examined: coffee husk, hair waste from the tanning industry and soy fibre residues. Soy fibre presented the highest yield for protease production at the laboratory scale (37 °C, 100 g samples). Consequently, experiments with soy fibre (F) and soy fibre with 10% compost as an inoculum (FC) were performed for 14 days in 4.5 L bench-scale aerobic near-adiabatic reactors. The highest activity occurred under thermophilic conditions and a high respiration activity of 47,331 ± 1391 U/g dry matter for F and 18,750 ± 1596 U/g dry matter for FC, which are much higher values than those reported in other studies. Alkaline proteases showed maximum stability at pH 11 and temperatures of 43.8 °C (F) and 30 °C (FC), which was determined by a full factorial experimental design. Storage assays demonstrated that 90% of enzyme activity was preserved for three months by lyophilising or freezing the samples at −80 °C.
Keywords: Solid-state fermentation; Alkaline protease; Respiration activity; Experimental design; Bench scale production;

► A novel hairy particle was synthesized by grafting PNIPAM brushes onto PS cores. ► PNIPAM brushes effectively suppress protein aggregation by hydrophobic interactions. ► Refolding yield of rhIFN-γ was greatly enhanced with the help of hairy particles. ► The hairy particles could be easily separated and recycled due to the PS cores.Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γin vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55 nm and 110 nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33 °C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50 μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15 °C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.
Keywords: Poly(N-isopropylacrylamide) brush; Recombinant human interferon gamma; Aggregation; Refolding; Protein recovery; Downstream processing;

Cold enzyme hydrolysis was investigated on the ethanol production by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing. An integrated statistical design, which incorporated single factor design, response surface methodology (RSM) and weighting coefficient method, was used to determine the optimum hydrolysis conditions leading to maximum biomass, ethanol concentration and starch utilization ratio. After the studied ranges of α-amylase, glucoamylase and liquefaction time were identified by single factor design, RSM was used to further optimize the hydrolysis conditions for each objective. The results showed that, under hydrolysis condition optimized with RSM, biomass, ethanol concentration and starch utilization ratio reached 4.401 ± 0.042 × 108  cells/ml, 14.81 ± 0.23% (wt.%) and 94.52 ± 0.53%, respectively. Finally, multi-objective optimization (MOO) was applied to obtain a compromised result of three desirable responses by weighting coefficient methodology. Biomass of 4.331 ± 0.038 × 108  cells/ml, ethanol concentration of 14.12 ± 0.21% (wt.%) and starch utilization ratio of 92.88 ± 0.21% were simultaneous obtained when hydrolysis at pH 5.9 for 114 min with 233 IU/gstarch α-amylase and 778 IU/gstarch glucoamylase. The optimized conditions were shown to be feasible and reliable through verification tests.
Keywords: Ethanol; Starch; Yeast; Cold enzyme hydrolysis; Optimisation; Response surface methodology;

Optimization of medium using response surface methodology for l-DOPA production by Pseudomonas sp. SSA by Sushama A. Patil; Shripad N. Surwase; Shekhar B. Jadhav; Jyoti P. Jadhav (36-45).
l-3,4-Dihydroxyphenylalanine (l-DOPA) is the most potent drug used for treatment of Parkinsonism. Statistical optimization of nutritional parameters for the production of l-DOPA by Pseudomonas sp. SSA has been carried out using response surface methodology (RSM). Four most significant medium constituents identified by initial screening method of Plackett–Burman (PB) were glucose, peptone, l-tyrosine and CuSO4. In order to investigate quantitative effects of the four variables selected from PB design on l-DOPA production, Box–Behnken design was subsequently employed for further optimization. The medium having glucose 1.69 g l−1, peptone 1.61 g l−1, l-tyrosine 4.11 g l−1 and CuSO4 0.03 g l−1 was found to be optimum for maximum l-DOPA production. The optimization strategies used lead to a 6.27-fold increase in l-DOPA yield (3.251 ± 0.12 g l−1) compared to non-optimized medium (0.572 ± 0.1 g l−1). l-DOPA produced was further characterized by spectroscopic techniques, such as HPTLC, HPLC and GC–MS.
Keywords: Pseudomonas sp. SSA; l-DOPA; Response surface methodology; Tyrosinase; HPTLC;

► Isolated indigenous strain USTBa was identified as Bacillus methylotrophicus. ► Growth-associated biosurfactant production from strain USTBa. ► Strain USTBa able to produce biosurfactant with excessive foam-forming properties. ► USTBa biosurfactant has stability at extreme pH, salinity and temperature. ► Biosurfactant with excellent surface tension reduction and emulsification activity.An efficient biosurfactant-producing bacterium was isolated and cultured from petroleum reservoir in northeast China. Isolate was screened for biosurfactant production using haemolytic assay, Cetyl Trimethyl Ammonium Bromide agar plate assay (CTAB) and the qualitative oil-displacement test. Based on partial sequenced 16S rDNA analysis of isolate, USTBa, identified as Bacillus methylotrophicus with 100% identity. This bacterium was able to produce a type of biosurfactant with excessive foam-forming properties. The maximum biosurfactant production was obtained when the cells were grown on minimal salt medium containing 2% (v/v) crude-oil as the sole source of carbon at 35 °C and 180 rpm after 192 h. This strain had a high emulsification activity and biosurfactant production of 78% and 1.8 g/L respectively. The cell free broth containing biosurfactant could reduce the surface tension to 28 mN/m. Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the presence of carboxyl, hydroxyl and methoxyl functional groups. Elemental analysis of the biosurfactant by Energy dispersive X-ray spectroscopy (EDS) reveals that the biosurfactant was anionic in nature. The strain USTBa represented as a potent biosurfactant-producer and could be useful in variety of biotechnological and industrial processes, particularly oil industry.
Keywords: Microbial growth; Purification; Biodegradation; Growth kinetics; Surface tension; Emulsification;

Density plot showing the distribution of Lactobacillus rhamnosus ATCC 7469 cells growing at a 0.62 h−1 steady-state in the presence of propidium iodide (PI) and 3,3′-dihexylocarbocyanine iodide (DiOC6(3)). Three main populations were distinguished. Healthy cells stained with DiOC6 holding polarized membranes. Cells with a depolarized cytoplasmic membrane (no staining). These cells are capable of recovering towards a fully growth state. Permeabilized cells with a disrupted cytoplasmic membrane stained with both DiOC6 and PI (dead cells).Display Omitted► We describe the heterogenic nature of steady state microbial cultures. ► High dilution rates results in the change from glucose to oxygen limitation. ► Oxygen limitation leads to a change of organic acid profile produced by L. rhamnosus. ► Flow cytometry reveals that organic acids affect L. rhamnosus membrane potential. ► Dilution rate increased, the proportion of L. rhamnosus cells with depolarized membrane steadily increased.Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h−1 and 0.6 h−1. At 0.45 h−1, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD+ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D  = 0.20 h−1, 8% at D  = 0.30 h−1, 14% at D  = 0.45 h−1 and 26% for D  = 0.62 h−1, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.
Keywords: Lactobacillus rhamnosus; Flow cytometry; Continuous culture; Steady state; Lactic acid;

Enzymatic hydrolysis of peptide bonds becomes possible after removing steric obstacles shielding polypeptide sites against enzymatic attack, i.e. after demasking of these sites. In a simple two-step model, proteolysis was regarded as a two-step process with consecutive demasking and hydrolysis stages. A new analytical procedure was suggested to determine three experimental kinetic parameters: demasking rate constant k d , degree of initially masked peptide bonds m and maximum hydrolysis rate constant k h . The approach was shown on the example of the hydrolysis of summary casein by chymotrypsin (25 °C, pH 7.5). Kinetic analysis includes the determination of an apparent Michaelis constant that we regard as a function of the degree of peptide bond hydrolysis. Two sets of hydrolysis rate constants were used to calculate the parameters of a two-step model. It was found that k d is lower than the hydrolysis rate constants for specific sites consisting of aromatic amino acid residues, and at least a half of peptide bonds is initially masked in casein. Using parameters of a two-step model, we calculated second-order rate constant and degrees of hydrolysis for specific peptide bonds as functions of the hydrolysis degree.
Keywords: Proteolysis model; Chymotryptic digestion; Enzymatic kinetics; Peptide bond demasking; Casein hydrolyzates;

Phosphorus (P) accumulation in a closed recirculating aquaculture system (RAS) was studied using a goldfish tank as a model. It was found that the accumulated P in this system was soluble inorganic phosphates (Pi) and the highest concentration was up to 8 mg P/L after 40 days of fish cultivation. Phosphorus in the water was increased linearly with the rate of 0.19 mg P/L/day. A mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 (ΔSphU) that lacks the SphU regulator of the Pho regulon could decrease Pi in the wastewater of RAS to the concentration below the P detection limit of 0.01 mg P/L at the rate of 2.07 ± 0.33 mg P/h g DW. This was corroborated by the increase of cellular polyphosphate and P content in the ΔSphU strain as revealed by fluorescence microscopy. After the first cycle of P removal, the cyanobacterial cells were recovered from wastewater by cell flocculation using chitosan. The flocculated cells could be reused for efficient P removal for the next 3 cycles.
Keywords: Recirculating aquaculture system; Cyanobacteria; Wastewater; Photosynthetic phosphorus removal system; Polyphosphate; Pho regulon;

Streptomyces mobaraensis transglutaminase is one of the most useful transglutaminases. ► The pld-signal and prepro-domain of S. cinnamoneus transglutaminase encouraged the secretion of MTG. ► We successfully produced a large amount of MTG using a genetically modified strain of S. lividans. Streptomyces mobaraensis transglutaminase (MTG) is one of the most useful transglutaminases due to its rather broad substrate specificity and independence of Ca2+. To achieve efficient production of active-form MTG using Streptomyces lividans as a host, we created three vector constructs consisting of the signal peptide sequence (pld signal) derived from the phospholipase D gene of Streptomyces cinnamoneus, prepro-domain of S. cinnamoneus transglutaminase, and the sequence encoding mature MTG, and then generated three over-expressing S. lividans strains. We successfully demonstrated that S. lividans can be used as a host for the efficient production of mature, active-form MTG.
Keywords: Enzyme production; Recombinant DNA; Protease; Filamentous bacteria; Streptomyces mobaraensis transglutaminase; Streptomyces lividans;

Addition of carboxymethyl cellulose to alginate gel significantly improved the yield of lactase immobilization and the total and specific activity of the fixed enzyme. It was due to a decrease in protein loss during the gel bead formation in the enzyme immobilization procedure and an increase in specific surface area of the gel beads. When the weight ratio of carboxymethyl cellulose to sodium alginate in the gel support was 1.0:1.5, the yield of lactase immobilization achieved 58.2% and this value was 14.2% higher than the yield of lactase immobilization in alginate gel. The immobilized lactase in the combined alginate–carboxymethyl cellulose gel exhibited higher thermal and pH stability than the fixed enzyme in the conventional alginate gel. However, the Michaelis–Menten constant (K m ) increased from 99.57 mM (fixed lactase in alginate gel) to 107.24 mM (fixed lactase in alginate–carboxymethyl cellulose gel) while the apparent turnover number (K cat ) and the specificity constant (K cat /K m ) of the both immobilized biocatalysts were statistically similar.
Keywords: Alginate; Carboxymethyl cellulose; Enzyme activity; Immobilization; Immobilized enzyme; Lactose;

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K m and v max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.
Keywords: S-(+)-2-Chlorophenylglycine; Immobilized penicillin G acylase; Biocatalysis; Bioconversion; Packed bed bioreactors; Optimization;

Hypoxia efficiently induces differentiation of mouse embryonic stem cells into endodermal and hepatic progenitor cells by Takeshi Katsuda; Takumi Teratani; Mohammad Mahfuz Chowdhury; Takahiro Ochiya; Yasuyuki Sakai (95-101).
► Effect of hypoxia on early hepatic differentiation of mouse ES cells was studied. ► Hypoxia amplified the endodermal and early hepatic differentiation signals. ► The differentiating cells did not consume oxygen even under hypoxia. ► Oxygen may negatively affect the normal early hepatic differentiation of ES cells.Although ES cells have potential impact on cell-based therapy for liver diseases, their efficient differentiation into functional hepatocytes remains difficult. One possible approach to this issue is to control the culture environment to recapitulate the in vivo liver development. Given that the embryo is exposed to hypoxic condition in its early stage, hypoxic condition seems suitable for inducing early differentiation of ES cells. However, no studies have evaluated the effect of hypoxia on endodermal or early hepatic differentiation of ES cells. Here we investigated the potential role of hypoxia as a regulatory factor in endodermal and subsequent early hepatic differentiation of mouse ES cells. Under the stimulus of retinoic acid, a typical endodermal morphology emerged more dominantly under hypoxia (5% O2) than normoxia. Accordingly, quantitative RT-PCR revealed significant increase in Foxa2 and GATA4, essential transcription factors for endoderm specification. In addition, hypoxia amplified the effects of hepatic induction factors FGF1, FGF4 and HGF, which was evidenced by dramatically increased ALB-GFP-positive cell fraction, as well as increased gene expression levels of ALB, AFP, and CK18. Furthermore, the hepatic progenitor cells induced under hypoxic condition were efficiently differentiated into mature hepatocytes with ALB secretion ability. We also found that the differentiating cells did not consume oxygen even under normoxia, suggesting that oxygen per se might have a negative effect on the normal endodermal and early hepatic differentiation of ES cells. These results will provide useful insight into efficient production of hepatic progenitor cells, and contribute to stem cell-based liver tissue engineering.
Keywords: Embryonic stem cell; Endodermal differentiation; Hepatic differentiation; Hypoxia;

Bioxidative dissolution of cinnabar by iron-oxidizing bacteria by Y.J. Wang; Y.J. Yang; D.P. Li; H.F. Hu; H.Y. Li; X.H. He (102-106).
Cinnabar leaching in chemobiological system and process parameters was investigated. The effect of temperature, initial pH of the solution, dilution rate and iron was studied. The results indicate a good leaching characteristic of cinnabar in the chemobiological reactor, where a maximum mercury concentration of 1.38 g/L was achieved.Display Omitted► Process parameters and characteristic of cinnabar bioleaching were investigated. ► The optimized conditions for bioleaching were established. ► A maximum Hg concentration of 1.38 g/L was achieved. ► Iron concentration was the most effective parameter. ► Bioprocess of A. ferrooxidans plays a major role in cinnabar dissolution.In the present study, bioleaching characteristic of cinnabar and process parameters were investigated. The effects of temperature, initial pH of the solution, dilution rate and iron on the cinnabar bioleaching were examined. The relationship between the redox potential and extraction of mercury was evaluated. Based on the S/N ratio, the optimized conditions for bioleaching were established. Iron of the solution was the most effective parameter in comparison with others. A maximum mercury concentration of 1.38 g/L was achieved. The indirect mechanism plays a major role in bioleaching. Some jarosite crystal deposits could be found when the iron concentration was 4.0 g/L and initial pH was 1.9. The bioxidation of sulfur could inhibit the hydrolyzation of ferric iron. Regulating pH value and ion concentration in medium could control the formation of the precipitation.
Keywords: Bioconversion; Cinnabar; Leaching; Immobilization; Optimization; Bioreactors;

Display OmittedThis study investigated biodegradation, transformation and sludge/water partition of two natural estrogens – estrone (E1) and 17β-estradiol (E2) - in a pilot-scale step-feed anoxic/oxic (A/O) wastewater treatment system. The results show that the total amounts of E1 and E2 (E1 + E2) in the water phase were approximately 9–23% of those in the sludge phase in the reactor tanks. The estrogens accumulated in the return sludge led to the increase in the estrogen concentration in the first-stage anoxic zone. The biodegradation of E1 and E2 in the first-stage aerobic zone was highly efficient. The sludge/water partition coefficients (Log  K d ) of E1 and E2 were gradually increased from 2.3 and 2.6 in the first stage to 3.2 and 3.3 in the third stage, respectively, and Log K d of E2 was higher than that of E1 in each A/O stage. In addition, the Log K d values of E1 and E2 were larger in the aerobic zones than in the anoxic zones. Under six operation conditions tested, there was a linear relationship between the concentrations of E1 and E2 in the water phase, indicating the transformation between E1 and E2. The calculated mutual conversion equilibrium constant (K) for the transformation between E1 and E2 was significantly higher in the anoxic zones (0.38–0.81) than in the aerobic zones (0.08–0.24).
Keywords: 17β-Estradiol; Estrone; Step-feed; Anoxic/Oxic; Sludge/water partition; Biodegradation;

Metabolic pools of phenolic acids in Salvia miltiorrhiza are enhanced by co-expression of Antirrhinum majus Delila and Rosea1 transcription factors by Donghao Wang; Yin Song; Yuqin Chen; Wei Yao; Zhen Li; Wenchao Liu; Sisi Yue; Zhezhi Wang (115-120).
► The increased phenylpropane metabolites resulted in the enhancements of antioxidant activities. ► Rosmarinic acid and salvianolic acid B were significantly accumulated during different growth stages. ► Transgenic lines with the similar transcription profiles of AnRosea1 and AnDelila had higher contents of metabolites.Phenolic acids are universally distributed in plant species, where they participate in numerous bioactivities. However, their low abundance in Salvia miltiorrhiza is a perplexing problem. We examined the heterologous expression of two transcription factors, Delila (DEL) and Rosea1 (ROS1), from Antirrhinum majus in genetically modified S. miltiorrhiza. Productions of rosmarinic acid (RA) and salvianolic acid B (Sal B) were significantly elevated during normal growth stages. Contents of other phenylpropanoid metabolites were enhanced and antioxidant activity was markedly stronger. Both SmCHS and SmRAS were stimulated by co-expression of DEL and ROS1. Because levels of phenylpropanoid metabolites were increased while both were co-expressed in S. miltiorrhiza, we believe that this will be a useful strategy for improving the contents of phenolic acids (especially RA and Sal B) in this species.
Keywords: Delila; Rosea1; Phenylpropanoid metabolism; Salvianolic acid B; Rosmarinic acid; Salvia miltiorrhiza;

► Solid state enzymolysis, fermentation and separation were integrated into one system. ► The steam explosion vessel of 5 m3 was applied to the industrial production of ethanol. ► The NSSSFS equipment had been scaled up to 110 m3 and operated successfully.To alleviate the problems of low substrate loading, nonisothermal, end-product inhibition of ethanol during the simultaneous saccharification and fermentation, a nonisothermal simultaneous solid state saccharification, fermentation, and separation (NSSSFS) process was investigated; one novel pilot scale nonisothermal simultaneous solid state enzymatic saccharification and fermentation coupled with CO2 gas stripping loop system was invented and tested. The optimal pretreatment condition of steam-explosion was 1.5 MPa for 5 min in industrial level. In the NSSSFS, enzymatic saccharification and fermentation proceeded at around 50 °C and 37 °C, respectively, and were coupled together by the hydrolyzate loop; glucose from enzymatic saccharification was timely consumed by yeast, and the formed ethanol was separated online by CO2 gas stripping coupled with adsorption of activated carbon; the solids substrate loading reached 25%; ethanol yields from 18.96% to 30.29% were obtained in fermentation depending on the materials tested. Based on the pilot level of 300 L fermenter, a novel industrial-level of 110 m3 solid state enzymatic saccharification, fermentation and ethanol separation plant had been successfully established and operated. The NSSSFS was a novel and feasible engineering solution to the inherent problems of simultaneous saccharification and fermentation, which would be used in large scale and in industrial production of ethanol.
Keywords: Bioconversion; Bioreactors; Ethanol; Noisothermal; Solid state enzymatic saccharification; Scale-up;

► Developed a new data representation structure to capture the experimental data systematically. ► Created ontology for modelling the relationship of data properties. ► Established a computational system to search relevant data based to predict required process conditions and to suggest a new set of experiments for process development. ► Demonstrated centrifugation data and knowledge system and its evaluation.Bioprocess design requires substantial resources during the experimental investigation of the options for each bioprocess step. This is both time-consuming and expensive. The amount of data available has increased exponentially since the expansion of new biological drug development. Data are heterogeneous, sometimes inconsistent and incomplete, making them difficult to be systematically utilised for analysis for any new bioprocess design. In this paper, we report a novel computational method that harnesses the bioprocess experimental data to assist design decision making, and perhaps identify further needed experiments. First, we develop a new data representation structure to capture the experimental data systematically. Then the ontology for modelling the relationship of data properties is created. A computational system has been developed to search relevant data, or to predict required process conditions, or to suggest a new set of experiments for process development. A prototype for harnessing centrifugation experimental data has been built, and is then used to illustrate the method and demonstrate the type of results that can be obtained. Evaluations show that such a system has significant potential to mine the relevant experimental data to assist new drug bioprocess development, which should reduce process development time and cost.
Keywords: Data representation; Ontology; Reasoning system; Centrifugation; Data mining;