Biochemical Engineering Journal (v.37, #2)

BEJ Keywords (IV).

This work studied the effect of hot pressurized water extraction of Acanthopanax senticosus (AS) roots on the activity, phagocytosis and nitric oxide (NO) molecules releasing of rat peripheral blood macrophages. Experimental results indicated that a kind of phenyl-propanoid glycosides, i.e., syringin plays a role in stimulating rat-blood macrophages to perform phagocytosis. Suitable concentrations of the syringin standard in the aqueous solution, ranging from 4 to 16 mg/L, notably sustain the activity of macrophages, but speed up the releasing of NO molecules from macrophages after incubation from 24 to 72 h. A two-factor central composite experimental designed hot pressurized water extractions of A. senticosus indicated that the enhancement of phagocytosis after the 24, 48 and 72 h incubations may be induced by a co-solute effect of syringin and other ingredients in the extracts.
Keywords: Hot pressurized water extraction; Syringin; Acanthopanax senticosus; Rat-blood macrophages; Activation; Phagocytosis;

Process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer production by Nostoc muscorum by Nirupama Mallick; Suneel Gupta; Bhabatarini Panda; Ramkrishna Sen (125-130).
This study aims at process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalarate) [P(3HB-co-3HV)] co-polymer production by Nostoc muscorum with respect to variation in different parameters, viz. carbon concentration, time of incubation and pH using response surface methodology (RSM). Under pre-optimized condition, P(3HB-co-3HV) accumulation in N. muscorum reached up to 28.2% (w/w) of dry cells (dcw) in presence of 0.2% acetate + 0.4% propionate, when incubated for 14 days at pH 8.5. A five-level four-factorial central composite design was employed to find out the interactions of four variables, viz. concentrations of acetate and propionate, pH and days of incubation. Using RSM, a second order polynomial equation was obtained by regression analysis. An optimum co-polymer yield of 31.4% (dcw) was achieved at a reduced level of carbons, i.e. 0.11% acetate and 0.08% propionate at pH 8.1 and an incubation period of 16 days. Thus after optimization, though the product yield was increased only by 11%, acetate and propionate requirements were reduced by 45 and 80%, respectively.
Keywords: Acetate; P(3HB-co-3HV); Propionate; Response surface methodology (RSM);

Treatment of diesel fuel contaminated soil in jet–fluidized bed by Jazia Arrar; Nadia Chekir; Fatiha Bentahar (131-138).
Bioremediation of diesel-oil contaminated soil (4 wt%) was studied in a jet–fluidized reactor by the stimulation of indigenous oil micro-organisms. The experiments were carried out using different rates of aeration and/or jet air flow and consequently various mixing rates to investigate their influence on biodegradation and removal ratios. The diesel-oil content decreased rapidly in all experiments in the first 7 days, with different removal ratios. The presence of the jet favoured inter-particle exchanges, transfers between the various phases involved, and accelerated the diesel-oil removal process. Also, the influence of aeration seemed negligible compared with that of the jet. Removal and biodegradation ratios ranged from 69% to 99%, and 54% to 84%, respectively after 15 days of treatment. The best biodegradation ratio of 84% occurred in the case of an expanded bed with minimum fluidization, operating at a jet velocity of 37 m/s. The diesel-oil biodegradation was governed by first-order kinetics. Important air flows enhanced the efficiency of diesel-oil removal, and abiotic loss and hence decreased the biodegradation ratio.
Keywords: Bioremediation; Biostimulation; Biodegradation; Jet–fluidized bed; Aeration; Diesel fuel;

Grifola umbellata is one of the most famous traditional Chinese medicinal mushrooms and has also been termed as Polyporus umbellatus or Chuling in scientific reports. A key problem in cultivating G. umbellata is that its mycelia growth is much slower than that of other mushroom species. In this study, a brownish G. umbellata broth concentrate (GBC) harvested at the beginning of the growth phase was collected and used as an additive in the medium to investigate its effect on facilitating mycelium germination. The results showed that the addition of GBC in solid-state culture promoted mycelia growth. Moreover, the GBC addition in submerged culture significantly shortened the lag phase and the maximum mycelia production was enhanced by 18%, from 10.8 to 12.78 g/1. These results demonstrate that GBC is capable of stimulating G. umbellata growth in all respects.
Keywords: Grifola umbellata; Conditioned medium; Stimulated effect; Solid-state culture; Submerged culture;

A polyhydroxyalkanoate (PHA) synthase negative mutant termed Aeromonas hydrophila CQ4 was constructed from its wild type strain A. hydrophila 4AK4. Heterologous expression of a low-substrate-specificity PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 in A. hydrophila CQ4 could copolymerize short-chain-length (SCL) 3-hydroxybutyrate (3HB) and medium-chain-length (MCL) 3-hydroxyalkanoates (3HA). Co-expressing (R)-specific enoyl-CoA hydratase and PHA synthase PhaC2Ps in A. hydrophila CQ4 (phaJ Ah , phaC2 Ps ) led to accumulation of 20.86 wt% PHA copolyesters containing 59 mol% 3HB, 37 mol% 3-hydroxyhexanoate (3HHx) and 4 mol% of other MCL 3HA in shake flask culture. When grown in fermentor, cellular dry weight (CDW) and PHA content were 22.75 g L−1 and 20.08 wt%, respectively, with 72.38 mol% 3HB, 25.18 mol% 3HHx and 2.45 mol% other MCL 3HA in the copolymer. If beta-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha were co-expressed with PhaC2Ps, A. hydrophila CQ4 (phaC2 Ps , phbA Re , phbB Re ) accumulated PHA copolyesters containing enhanced 3HB content up to 79.95 mol% and reduced 3HHx fraction of 8.55 mol%. When grown in fermentor it produced 16.28 g L−1 CDW containing 11.64 wt% PHA consisting of 87.38 mol% 3HB, 9.75 mol% 3HHx and 2.87 mol% other MCL 3HA. These results further demonstrate that A. hydrophila is a good candidate for metabolic engineering for diverse PHA production.
Keywords: Polyhydroxyalkanoates (PHA); Aeromonas hydrophila; Fermentation; Metabolic engineering; PHA synthase;

Biosorption of Pb(II) and Ni(II) from aqueous solution by lichen (Cladonia furcata) biomass by Ahmet Sarı; Mustafa Tuzen; Özgür Doğan Uluözlü; Mustafa Soylak (151-158).
Equilibrium, thermodynamic and kinetic studies were carried out for the biosorption of Pb(II) and Ni(II) ions from aqueous solution using the lichen (Cladonia furcata) biomass. Langmuir, Freundlich and Dubinin–Radushkevich (D–R) isotherm models were applied to describe the biosorption of the metal ions onto C. furcata biomass. The influences of pH, biomass dosage, contact time and temperature of solution on the biosorption were studied. Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The monolayer biosorption capacity of the biomass was found to be 12.3 and 7.9 mg/g for Pb(II) and Ni(II) ions, respectively. From the D–R model, the mean free energy was calculated as 9.1 kJ/mol for Pb(II) biosorption and 9.8 kJ/mol for Ni(II) biosorption, indicating that the biosorption of both metal ions was taken place by chemical ion-exchange. Thermodynamic parameters, the change of free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) of the biosorption were also calculated. These parameters showed that the biosorption process of Pb(II) and Ni(II) ions onto C. furcata biomass was feasible, spontaneous and exothermic under studied conditions. Experimental data were also tested in terms of kinetic characteristics and it was found that biosorption processes of both metal ions followed well pseudo-second-order kinetics.
Keywords: Lichen biomass; Cladonia furcata; Pb(II); Ni(II); Biosorption; Isotherm; Thermodynamics; Kinetics;

Design of polymer brushes for immobilizing enzymes onto hollow fiber micropores in organic media reaction by Muneharu Goto; Tokie Okubo; Hidetaka Kawakita; Kazuya Uezu; Satoshi Tsuneda; Kyoichi Saito; Masahiro Goto; Masao Tamada; Takanobu Sugo (159-165).
To immobilize lipase for enzymatic reactions in organic solvent, various functional [epoxy (GMA-fiber), hydroxyl (OH-fiber) or diethyl amino (DEA-fiber)] groups were introduced onto porous hollow-fiber membranes by radiation-induced graft polymerization of glycidyl methacrylate and chemical modification. Lipase from Candida rugosa was immobilized on polymer brushes by permeation of lipase. The activities of immobilized lipase were measured by esterification reactions between lauric acid and benzyl alcohol in isooctane. The activity of immobilized lipase on GMA-fibers, DEA-fibers and OH-fibers was 0.70 mol/(h kg-lipase), 0.50 mol/(h kg-lipase), and 2.45 mol/(h kg-lipase), respectively. Immobilized lipase on DEA-fibers or OH-fibers was reused three times after it was used in the batch reactor for 24 h. It was found that lipase activity showed no signs of denaturation. However, when native lipase was used, lipase activity remarkably decreased after reusing.
Keywords: Enzyme activity; Enzyme technology; Esterification reaction; Immobilized enzymes; Lipase; Polymer brush;

The marine microalgal strain Isochrysis galbana CCMP 1324 was cultured in Walne medium to examine the changes in lipid bodies and fatty acid composition, with special emphasis on different iron, nitrogen, and phosphorus sources associated changes in the growth kinetic parameters and fatty acid composition. All the experiments were performed at 20 °C, with the culture medium at pH 9.0, a specific rate of air supply of 1.0 vv−1  m−1 and a continuous illumination of 68 μEm−2  s−1. Lipid bodies in I. galbana CCMP 1324 were observed at different growth phase in Walne medium by light and transmission electron microscopy. Lipid granules from 0.4 to 2.0 μm were observed from exponential growth stage to stationary stage. The I. galbana CCMP 1324 had the highest contents of saturated and monounsaturated fatty acids (SFA + MUFA) as well as polyunsaturated fatty acids (PUFA) in the early stationary phase. The n-3/n-6 value reached a maximal value of 4.9 in the late stationary phase. The Walne medium could be a good iron, nitrogen and phosphorus sources to culture I. galbana to maximize PUFA production and harvesting the biomass at stationary phase may enable better yields in lipid and PUFA composition. The ratio of SFA + MUFA to PUFA was highest in NH4NO3 modified Walne culture and minimal in Walne culture. The n-3 PUFA was predominant in Walne culture. A compromise between the docosahexaenoic acid (DHA) production and growth kinetics can be achieved by using NH4NO3 as N-source in modified Walne medium, which provides the greatest value of DHA production (15.6% of total fatty acids) with the highest maximum specific growth rate (2.34 per day) even though the microalgal cell productivity (43.1 mg l−1 per day) is not the highly attained. The approaches presented in this study could be employed for the design of pilot or full-scale photoreactor for PUFA commercial productions by I. galbana CCMP 1324.
Keywords: Isochrysis galbana; Growth phase; Nutrient source; Polyunsaturated fatty acids; Photoreactor;

Study of levan production by Zymomonas mobilis using regional low-cost carbohydrate sources by Marcos Roberto de Oliveira; Rui Sérgio Santos Ferreira da Silva; João Batista Buzato; Maria Antonia Pedrine Colabone Celligoi (177-183).
The use of alternative regional low-cost substrates has become very interesting because in addition to the ease of acquisition it presents a relatively low cost. In this study using statistical instruments, the exopolysaccharide levan production by the Zymomonas mobilis microorganism was analyzed varying the carbon source (commercial sucrose, molasses and sugar cane syrups) and the fermentation medium constituents. There was a decrease in levan production in the molasses medium (2.533 g L−1) when compared to the commercial sucrose (21.685 g L−1) and syrup (15.456 g L−1) media. Yeast extract and KH2PO4 were significant in the commercial sucrose medium for levan production and in the syrup medium the yeast extract and MgSO4 were significant. Although sugar cane syrup up produced about 28.724% less levan compared to commercial sucrose, biomass production in the syrup was 2.76 times greater than in commercial sucrose (0.857 and 2.366 g L−1) that could justify joint levan and Z. mobilis ATCC 31821 biomass production. Studies are needed on the use of alternative substrates and complexes in biotechnology to assess the composition of these media, avoiding unnecessary supplementation with vitamins and minerals salts, when using the formulations of the mediums defined for the complex mediums.
Keywords: Zymomonas mobilis; Levan; Molasses sugar cane; Sugar cane syrups;

The influences of different environmental stimuli on the soluble expression of anti-human ovarian carcinoma × anti-human CD3 single-chain bispecific antibody in Escherichia coli were examined to improve its yield. Often, it was found that the amount ratio of the inclusion body fraction to the soluble fraction of the recombinant protein varied greatly with the changes of environmental conditions in fermentation process. Wherefore, an improved strategy based on various undercritical stimuli, including such environmental factors as temperature-shift, pH-shift and repetitious pH-shift, was employed to enhance the soluble expression of the target protein. As a result, an optimal factor-shift process was determined and the soluble yield of the target protein increased by 82.9% (from 68.9 to 126.6 mg protein/g DCW) in flask fermentation compared with the result cultured at 30 °C. And the high soluble expression level was also obtained in a 3.7 L fermentor based on this strategy. This improved strategy that factor-shifts under undercritical environmental stimuli improve the soluble expression of exogenous proteins in recombinant E. coli may become a new alternative besides the genetic engineering considerations.
Keywords: Anti-human ovarian carcinoma × anti-human CD3; Environmental stimuli; Factor-shift; Inclusion body; Recombinant E. coli; Soluble expression;

The adsorption of lead onto formaldehyde or sulphuric acid treated acorn waste was studied using a batch sorber. The aim of this study was to understand the mechanisms that govern lead removal and find a suitable equilibrium isotherm and kinetic model for the lead removal in a batch reactor. The experimental isotherm data were analyzed using the Langmuir, Freundlich and Temkin equations. The equilibrium data fit well the Langmuir isotherm. The experimental data were analyzed using four adsorption kinetic models – the pseudo first- and second-order equations, intraparticle diffusion equation and the Elovich equation – to determine the best fit equation for the adsorption of lead ions onto formaldehyde or sulphuric acid treated acorn waste. The rate constants, equilibrium capacities and related correlation coefficients for each kinetic model were calculated and discussed. Also, predicted q t values from the kinetic equations were compared with the experimental data. Results show that the pseudo second-order equation provides the best correlation for the adsorption process, whereas the Elovich equation also fits the experimental data well.
Keywords: Acorn waste; Lead; Isotherm; Adsorption kinetics; Elovich equation; Pseudo second-order equation;

In this work, the biodegradation of mixed chlorophenols by a microbial consortium was studied in steady state continuous culture. Amplified ribosomal DNA restriction analysis (ARDRA) was used to evaluate the consortium's predominating bacterial species when chemostat was fed with pure or mixed chlorophenols. Removal efficiencies, cell growth yields, volumetric and specific biodegradation rates of phenol and chlorophenols were compared to those obtained with pure strains in the same culture conditions. Using phenol as the primary carbon and energy source, the microbial consortium cometabolically degraded mono-, di- and trichlorophenols, with overall removal efficiencies ranging from 95 to 99.8% and COD removal efficiencies from 85 to 97.8%. In all cultures, the microbial consortium showed better overall removal efficiencies than single strains. Binary mixtures of chlorophenols produced a remarkably increase in specific biodegradation rates of substrates q c, beyond those obtained when the consortium grew on single compounds. The results suggest that chlorophenols collapsed cell's proton motive force; thus, energy spilling, reflected by the q c increase, could be the cell's response to compensate the short-circuit in membrane electrochemical proton gradient.
Keywords: Cometabolism; Chlorophenols; Energy spilling; Phenol; Microbacterium phyllosphaerae; Uncoupling;

Two-layer continuous-process design for the biodesulfurization of diesel oils under bacterial growth conditions by Jianzhong Yang; Yongqi Hu; Dishun Zhao; Shaozhao Wang; Peter C.K. Lau; Ian W. Marison (212-218).
An aqueous–organic two-layer partitioning and continuous process was designed to make efficient use of growing cells for the biodesulfurization of diesel oils. This biphasic system was shown to have a significant advantage over batch and fed-batch processes in the maintenance of the biodesulfurization activity for an extended period of time. Specifically, it was demonstrated that in a 2-L bioreactor 1500 ppm total sulfur was removed from 250 ml diesel oil within a 5-day period.
Keywords: Biodesulfurization; Rhodococcus globerulus; Diesel; Two-layer and continuous bioreactor;

Kinetic analysis of anaerobic digestion of cattail by rumen microbes in a modified UASB reactor by Zhen-Hu Hu; Han-Qing Yu; Zheng-Bo Yue; Hideki Harada; Yu-You Li (219-225).
The kinetics of anaerobic digestion of cattail by rumen microbes in a modified upflow anaerobic sludge blanket (UASB) reactor was systematically analyzed in this study. The Monod and first-order equations were combined to develop kinetic models to describe the substrate degradation, microbial growth and product formation. At an influent cattail concentration (volatile solids) of 12.1 g/L, hydraulic retention time of 0.75 day and solids retention time of 1.0–4.0 days, the concentration of reducing sugars was negligible, compared to the hydrolyzed cattail, indicating that the lignocellulosic hydrolysis was the rate-limiting step in the fermentation. The rate constant (k) of cattail degradation, true microbial growth yield (Y EG) and the maintenance coefficient (m) were calculated as 1.50 day−1, 0.11 g/g and 0.103 g/(g h), respectively. The measured and estimated substrate degradation, microbial growth and product formation were in good agreement.
Keywords: Anaerobic digestion; Cattail; Kinetics; Lignocellulose; Rumen microbes; Upflow anaerobic sludge blanket (UASB);

Effects of high pressure on the accumulation of trehalose and glutathione in the Saccharomyces cerevisiae cells by Yongsheng Dong; Qinzheng Yang; Shiru Jia; Changsheng Qiao (226-230).
The metabolism of trehalose, a marker of general physiological stress, and glutathione (GSH), another marker of oxidative stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with high pressure air. The growth of yeast cells was dramatically decreased under high pressure and the duration of stable growth was significantly shortened. Scanning electron microscopy showed that high pressure led to the disruption of membrane structure. Yeast cells were wrinkled or broken under higher pressure, indicating high pressure was a strong stress factor. The maximum yields of trehalose and GSH were obtained when the cells were cultured under 1.0 MPa pressure for 3 and 6 h, respectively. The increase of the production of trehalose and GSH indicated that trehalose and glutathione had protection effects under the stress of high pressure.
Keywords: High-pressure; Trehalose; Glutathione; Saccharomyces cerevisiae;