Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l⁻¹ was the most suitable. Sole nitrate nitrogen of 30 mmol l⁻¹ was the best for PLBs growth. Metal ions (Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) showed different influences on PLBs growth. The optimal concentration of Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was 4.5 mmol l⁻¹, 0.1 mmol l⁻¹, 0.5 mmol l⁻¹ and 0.06 mmol l⁻¹, respectively. In the optimized medium (sucrose, nitrate, Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l⁻¹ PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l⁻¹) was obtained in the case of 50 g l⁻¹ sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.     

Production of a new exopolysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682 grown on glycerol

Pure glycerol and glycerol-rich product (GRP) obtained from the biodiesel industries were used as carbon source for the production of a new extracellular polysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682. The influence of temperature (20–40 °C) and pH (6.0–8.0) was studied. A temperature of 30 °C and pH control at 6.8 gave the maximum cell growth and EPS production. The culture attained a maximum cell dry weight (CDW) of 9.55 g l⁻¹ and an EPS concentration of 11.82 g l⁻¹ when cultivated with pure glycerol. GRP was a suitable carbon source, as shown by the slightly higher EPS concentration (12.18 g l⁻¹). The EPS productivity obtained with GRP (3.85 g l⁻¹ d⁻¹) was almost twice that obtained with pure glycerol (2.00 g l⁻¹ d⁻¹). Also, the yield on glycerol was higher for the cultivation with GRP (0.36 g g⁻¹) than for pure glycerol (0.28 g g⁻¹). The EPS was a high molecular weight heteropolysaccharide, composed by neutral sugars (37–80 wt% galactose, 2–30 wt% glucose, 0.5–25 wt% mannose and 0.5–20 wt% rhamnose) and containing acyl group substituents (pyruvil, acetyl and succinyl were identified). The EPS forms highly viscous aqueous dispersions with many potential commercial applications.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

Assessment of Lemna gibba L. (duckweed) as a potential ecological indicator for contaminated aquatic ecosystem by boron mine effluent

Duckweeds, as a group, are important early warning indicators for the assessment of contaminated ecosystems due to their propensity to accumulate pollutants. In the present study, we investigated the potential use of Lemna gibba L. (Lemnaceae) as an ecological indicator for boron (B) mine effluent containing B concentration above 10 mg l⁻¹. For this purpose, L. gibba fronds were grown for 7 days in simulated water contaminated with B mine effluent. The important note is that this study was carried out in Kırka (Eskişehir, Turkey) B reserve area, which is the largest borax reserve in all over the world, under natural climatic conditions in the field. The results demonstrated that accumulations of B by L. gibba gradually increased based on the initial B concentrations (10, 25, 50, 100, and 150 mg l⁻¹) of the mine effluent. B concentration in the dry weight of the plant reached 639 mg kg⁻¹ when the minimum initial dosage (10 mg l⁻¹) was applied and 2711 mg kg⁻¹ when the maximum initial dosage (150 mg l⁻¹) was applied during the study. However, significant reductions in their relative growth rates occurred in 50, 100 and 150 mg l⁻¹ initial B concentrations. Results suggest that 25 mg l⁻¹ B concentration in water seemed to be a sensitive endpoint for L. gibba that could be used as a critical bioindicator level of B contaminated water. Following our data, we also constructed a simple growth model under the climatic conditions in this region of Turkey, but in instructive as a worldwide model. L. gibba is, therefore, suggested to be able to use as both an indicator and a phytoremediation tool because of its high accumulation capacity for B contaminated water.     

Signal transducer and oxidative stress mediated modulation of phenylpropanoid pathway to enhance rosmarinic acid biosynthesis in fungi elicited whole plant culture of Solenostemon scutellarioides

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation with phytopathogenic fungi. Amongst selected fungi, Aternaria alternata caused significant elevation (p < 0.05–0.01) in RA accumulation (∼1.3–1.6-fold) between 25 and 100 μg l⁻¹. However, elicitation at the dose of 50 μg l⁻¹ has been found to be most effective and intracellular RA content reached almost ∼1.6-fold (p < 0.01) higher in day 7. Therefore, A. alternata (50 μg l⁻¹) was selected for mechanism evaluation. A significant elevation of intercellular jasmonic acid was observed up to day 6 after elicitation with A. alternata (50 μg l⁻¹). A significant increase in tissue H₂O₂ and lipid peroxidation coupled with depletion of antioxidant enzymes superoxide dismutase and catalase indicated augmented oxidative stress associated with biotic interaction. Preceding the elicitor-induced RA accumulation, a notable alteration in the specific activities of biosynthetic enzymes namely PAL and TAT was recorded, while, no significant change in the activities of RAS was observed. HPPR activity was slightly improved in elicited plant. Therefore, it could be concluded that A. alternata elicited the biosynthesis of rosmarinic acid via signal transduction through jasmonic acid coupled with elicitor induced oxidative stress and associated mechanism.     

Hydrogen sulphide removal from air by biotrickling filter using open-pore polyurethane foam as a carrier

A study was conducted on H₂S removal in a biotrickling filter packed with open-pore polyurethane foam. Thiobacillus denitrificans was used as inoculum and a mixed culture population was developed during the process. The inhibitory effect of sulphate concentration (1.8–16.8 g L⁻¹), pH (6.9–8.6), trickling liquid velocity (TLV, 9.1–22.8 m h⁻¹), H₂S inlet concentration (20–157 ppmv) and the empty bed residence time (EBRT, 9–57 s) on the H₂S removal efficiency (RE) were thoroughly investigated. An increase in pH from 6.9 to 8.5 led to a corresponding increase in H₂S removal. In addition, an inhibitory effect of sulphate concentration was observed from 16.8 g L⁻¹ and the maximum elimination capacity was found to be 22 gS m⁻³ h⁻¹ (RE 98%). The RE was constant (98.8 ± 0.30%) for EBRT ≥ 16 s, but a decrease in the EBRT from 16 to 9 s led to a corresponding decrease in RE from 98.2 to 89.6% for a TLV of 9.1 m h⁻¹ and from 97.9 to 94.9% for a TLV of 22.8 m h⁻¹ (inlet load of 11.0 ± 0.2 gS m⁻³ h⁻¹). The sulphur oxidation capacity in the biotrickling filter was not diminished by the presence of other bacteria.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Characterization of a recombinant Escherichia coli TOP10 [pQR239] whole-cell biocatalyst for stereoselective Baeyer–Villiger oxidations

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min⁻¹ g⁻¹ dry weight) than that of sonicated cells (500 μmol min⁻¹ g⁻¹ dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l⁻¹ and at product concentrations above 4.5–5 g l⁻¹ the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.     

Comparison of two matrices for selective recovery of C595 diabody fragment (dbFv) from Escherichia coli lysates

A comparative study of the performance of two types of adsorbent (Streamline Quartz Base and Upfront Matrices), derivatized with the same affinity ligand (RPAP) to recover C595 diabody fragment (dbFv) from Escherichia coli lysates has been undertaken. Both streamline and Upfront Matrices are characterized by a particle size range of 100–300 μm. Streamline has a density of 1.20 g cm⁻³ and ligand concentration of 0.85 μmol ml⁻¹. Upfront has a density of 1.35 g cm⁻³ and ligand concentration of 0.83 μmol ml⁻¹. The release of C595 dbFv from E. coli cells was achieved by a chemical lysis method. The recovery performance of both adsorbents was evaluated in terms of operational productivity and elution yield of C595 dbFv in packed bed (clarified feedstock) and expanded bed (unclarified and clarified feedstock) chromatography systems. Streamline and Upfront adsorbents exhibited diabody operational productivities of 131 and 202 mg l⁻¹, respectively, with an elution yield of 92 and 94%, respectively, in packed bed operation. Streamline and Upfront adsorbents exhibited diabody operational productivities of 54.5 and 123.7 mg l⁻¹, respectively, with an elution yield of 89 and 92%, respectively, in expanded bed operation.     

Mathematical analysis of trickling filter response to pentachlorophenol shock load

The response of a laboratory trickling filter to a step increase in pentachlorophenol (PCP) feed concentration was analyzed using continuous stirred tank (CSTR) and plug flow reactor (PFR) models. The CSTR model provided a slightly better fit to experimental data than the plug flow model when specific growth rate, μ, and PCP-degrading biomass concentration before the shock load, X₀, were variable parameters but was clearly superior when the mean residence time, τ, was added as a third parameter. The three-parameter CSTR model accurately represented six of seven concentration response curves corresponding to step increases in PCP feed concentration of 12–165 mg l⁻¹ and 20–150 mg l⁻¹. The continuing improvement in system response to repetitive 20–150 mg l⁻¹ shock loads was reflected by a monotonic increase in the optimal estimates of initial rate of biomass production.     

Biodegradation of organochlorine pesticides by bacteria grown in microniches of the porous structure of green bean coffee

In this paper, the authors propose a model for DDT biodegradation by bacteria grown in microniches created in the porous structure of green bean coffee. Five bacteria isolated from coffee beans, identified as Pseudomonas aeruginosa, P. putida, Stenotrophomonas maltophilia, Flavimonas oryzihabitans, and Morganella morganii. P. aeruginosa and F. oryzihabitans, were selected for pesticide degradation. Bacteria were selected according to their ability to grow on mineral media amended with: (a) glucose (10 g l⁻¹), (b) peptone (2 g l⁻¹), and (c) ground coffee beans (2 g l⁻¹). These three media were supplemented with 50 mg l⁻¹ of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) and endosulfan. GC/MS analysis demonstrated that the greatest DDT removal was obtained in the medium supplemented with coffee beans, where 1,1-dichloro-2,2′-bis (4-chlorophenyl)ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethane (DDMU) and 2,2′-bis (p-chlorophenyl)ethanol (DDOH) were detected. DDMU is a product of the reductive dechlorination of DDE, which in this system could be carried out under the anaerobic conditions in microniches present in the porous structure of the coffee bean. This was supported by scanning electron microscopy. Green bean coffee could be used as a nutrient source and as a support for bacterial growth in pesticide degradation.