Biochemical Engineering Journal (v.86, #C)

IFC (CO2).

BEJ Keywords (IV).

Disposable shaking bioreactors are a promising alternative to other disposable bioreactors owing to their ease of operation, flexibility, defined hydrodynamics and characterization. Shaken bioreactors of sizes 20 L and 50 L are characterized in terms of heat transfer characteristics in this research work. Water and an 80% glycerol–water system were used as fluid. Results indicated large heat generation due to shake mixing which was observed by temperature difference between the fluid inside the vessel and the surrounding air outside the vessel. Maximum temperature difference of ca. 30 K was encountered for a 50 L vessel, at 300 rpm and 20 L filling volume. Outside heat transfer rate was governing the overall heat transfer process. Lateral air flow did increase heat transfer rates to large extent. An empirical correlation of overall heat transfer coefficient was obtained in terms of filling volume, rotational speed and lateral air flow rate. However, as the vessel thickness increased, the overall heat transfer process was limited by vessel wall resistance.
Keywords: Disposable; Bioreactors; Orbitally-shaken; Characterization; Heat transfer; Newtonian fluids;

Enhancement of hydrolytic activity of thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 through optimization of amino acid residues surrounding the substrate binding site by Naoya Tamamura; Wataru Saburi; Atsushi Mukai; Naoki Morimoto; Toshihiko Takehana; Seiji Koike; Hirokazu Matsui; Haruhide Mori (8-15).
The hydrolytic activity of a thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A, I509A, and K549A showed notably higher activity than the wild type at 162–254% of wild-type activity. Tyr426, His431, and Ile509 were predicted to be located near subsite −2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M.
Keywords: Amylase; Starch; Enzyme activity; Enzyme production; Mutagenesis; Glycoside hydrolase family 13;

Structural insights on laccase biografting of ferulic acid onto lignocellulosic fibers by Jorge Rencoret; Elisabetta Aracri; Ana Gutiérrez; José C. del Río; Antonio L. Torres; Teresa Vidal; Angel T. Martínez (16-23).
Treatment of high-kappa sisal pulp with Trametes villosa laccase and ferulic acid resulted in strong increases of kappa-number and acid-group content due to biografting of this phenolic acid, as shown by pyrolysis in the presence of tetramethylammonium hydroxide. The coupling linkages were investigated by 2D NMR of the lignin isolated from pulps. The aromatic region of the spectra showed incorporation of the cinnamic molecule, representing ∼4% of the lignin content, that according to the displacement of its olefinic 13Cβ1Hβ signal to 117.0/6.40 ppm would be C4-etherified. The aliphatic region of the spectra showed that ferulic acid also incorporates as the corresponding β–β′ dilactone (another ∼4% of the total lignin) with characteristic 13Cα1Hα and 13Cβ1Hβ correlations at 81.8/5.69 and 47.9/4.19 ppm, respectively. The sisal lignin in the treated pulps was only slightly modified (including a small increase of Cα-oxidized units) revealing that the main effect of the treatment was ferulic acid biografting.
Keywords: Laccase; Ferulic acid; Biografting; Dilactone; Pyrolysis; 2D NMR;

Maximum stable drop size measurements indicate turbulence attenuation by aeration in a 3 m3 aerated stirred tank by A. Daub; M. Böhm; S. Delueg; M. Mühlmann; G. Schneider; J. Büchs (24-32).
In this study, break-up controlled drop dispersion experiments in a 3 m3 pilot scale reactor with 1.2 m inner diameter are presented that allow a correlation of the maximum local energy dissipation rate in stirred reactors with intense aeration. Experiments with two different setups of 6-bladed Rushton turbine impellers with diameters of d  = 0.41 m (d/D R  = 0.34) and d  = 0.51 m (d/D R  = 0.43) in a 3 impeller configuration were conducted. The results from experiments without aeration are well in agreement with the existing literature on drop dispersion. The results from experiments with aeration indicate a strong attenuation of turbulence intensity in stirred tank reactors by the presence of air. The ratio between maximum and volume-averaged energy dissipation rate was reduced by aeration by 64% for the d/D R  = 0.34 impeller and by 52% for the d/D R  = 0.43 impeller when compared with unaerated operating conditions on the basis of equal volumetric power input. The value of the aeration rate had no measurable effect in the range of aeration rates applied, which was between 0.1 vvm (volume of gas/volume liquid/minute) and 1 vvm.
Keywords: Multiphase bioreactors; Fluid mechanics; Aeration; Agitation; Hydromechanical stress; Drop size;

Simultaneous enhancement of CO2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy by You-Ping Xie; Shih-Hsin Ho; Chun-Yen Chen; Ching-Nen Nathan Chen; Chen-Chun Liu; I.-Son Ng; Ke-Ju Jing; Sheng-Chung Yang; Chi-Hui Chen; Jo-Shu Chang; Ying-Hua Lu (33-40).
This study aimed to improve lutein production using a thermo-tolerant lutein-rich microalga Desmodesmus sp. F51. To achieve this goal, four fed-batch cultivation strategies were investigated for CO2 fixation and lutein production of Desmodesmus sp. F51. Among them, Fed-batch IV showed the best performance, giving the highest CO2 fixation rate and lutein productivity of 1582.4 mg/L/d and 3.91 mg/L/d, respectively. Both increasing the light intensity and limiting the nutrients led to a lower carotenoids content in the microalga, with a higher proportion of lutein and lower proportion of β-carotene being obtained in the carotenoids. The carotenoid present in the biomass was mainly lutein, accounting for 50–66% of total carotenoids. Repeated operations of the Fed-batch IV strategy could effectively improve CO2 fixation and lutein production of Desmodesmus sp. F51, giving the best results of 1826.0 mg/L/d, and 4.61 mg/L/d, respectively. This performance is better than most of the previously reported values.
Keywords: Microalgae, Lutein; Fed-batch culture; Desmodesmus sp.; Bioreactors; Bioprocess design;

Thermal-aggregation suppression of proteins by a structured PEG analogue: Importance of denaturation temperature for effective aggregation suppression by Takahiro Muraoka; Nabanita Sadhukhan; Mihoko Ui; Shunichi Kawasaki; Enrikko Hazemi; Kota Adachi; Kazushi Kinbara (41-48).
Development of protein stabilizing reagents, that suppress aggregation and assist refolding, is an important issue in biochemical technology related with the synthesis and preservation of therapeutic or other functional proteins. In the precedent research, we have developed a structured poly(ethylene glycol) (PEG) analogue with triangular geometry, which turns into a dehydrated state above ca. 60 °C. Focusing on this rather lower dehydration temperature than that of conventional linear PEGs, a capability of the triangle-PEG to stabilize proteins under thermal stimuli was studied for citrate synthase, carbonic anhydrase, lysozyme and phospholipase. Variable temperature high-tension voltage and circular dichroism spectroscopic studies on the mixtures of these proteins and the triangle-PEG showed that the triangle-PEG stabilizes carbonic anhydrase, lysozyme and phospholipase that exhibit denaturation temperatures higher than 60 °C, while substantially no stabilization was observed for citrate synthase that denatures below 60 °C. Hence, the dehydrated triangle-PEG likely interacts with partially unfolded proteins through the hydrophobic interaction to suppress protein aggregation.
Keywords: Poly(ethylene glycol); Aggregation; Circular dichroism spectroscopy; High-tension voltage analysis; Protein denaturation; Refolding;

Interest in alkaline anaerobic fermentation of waste activated sludge (WAS) is on the rise recently due to its excellent result in sludge reduction and biomass reuse. However, there is still lack of ideal approaches to monitor the fermentation process. The authors present here a fast method for tracing status of microbes and soluble fluorescent organics during alkaline anaerobic fermentation by coupling flow cytometry (FCM) and three-dimensional excitation–emission matrix (3D-EEM) fluorescence spectroscopy. FCM primarily reveals the cell integrity and identifies sources of soluble organics, while 3D-EEM further characterizes them in different fermentation phases by correlation with fluorophores such as soluble protein. High correlation was obtained between fluorescence intensities of three peaks, especially those related to tryptophan-like (Ex/Em: 280/340 nm) and main soluble organics. The proposed method significantly reduced the time by over 90% compared with conventional methods. The method demonstrates great potential for application in efficient controlling and monitoring in practical alkaline fermentation process.
Keywords: Activated sludge; Waste treatment; Biomass reuse; Anaerobic alkaline fermentation; Process control; 3D-EEM;

Rheological characteristics of highly concentrated anaerobic digested sludge by Jiankai Jiang; Jing Wu; Souhila Poncin; Huai Z. Li (57-61).
Highly concentrated anaerobic sludge digestion is a favorable way to treat a considerable quantity of sewage sludge and recover biogas from both environmental and energetic point of view. For enhancing mass transfer and ensuring efficient anaerobic digestion, pumping and mixing are crucial operations which are directly conditioned by the rheology of sludge. The present work aims at studying the rheological characteristics of highly concentrated anaerobic digested sludge with TS (total solid) content more than 8%. The dependence on both TS content and temperature is investigated, respectively, in dynamic and flow measurements. The results show a shear-thinning behavior with a yield stress under flow measurements and a viscoelastic property in dynamic measurements, comparable to a pasty material. The Herschel–Bulkley model can describe these experimental results and a master curve is then proposed. In addition, the yield stress as well as cohesion energy increase with TS content following a power-law. The effect of temperature is relatively smaller, as a consequence of pronounced internal interactions strengthening the interior structure after the digestion, in the form of networks and steric linking.
Keywords: Rheology; Highly concentrated digested sludge; Shear-thinning; Yield stress; Viscoelasticity;

This study investigated the characteristics of nitrous oxide (N2O) emission from intermittently aerated sequencing batch reactors (IASBRs) treating high strength slaughterhouse wastewater at 11 °C, where partial nitrification followed by denitrification (PND) was achieved. N2O generation and emission was examined at three aeration rates of 0.4, 0.6, and 0.8 L air/min in three IASBRs (SBR1, SBR2, and SBR3, respectively). The slaughterhouse wastewater contained chemical oxygen demand (COD) of 6057 ± 172.6 mg/L, total nitrogen (TN) of 576 ± 15.1 mg/L, total phosphorus (TP) of 52 ± 2.7 mg/L and suspended solids (SS) of 1843 ± 280.5 g/L. In the pseudo-steady state, the amount of N2O emission was up to 5.7–11.0% of incoming TN. The aeration rate negatively affected N2O emission and the ratio of N2O emission to incoming TN was reduced by 48.2% when the aeration rate was increased from 0.4 to 0.8 L air/min. Results showed that more N2O was generated in non-aeration periods than in aeration periods. Lower DO concentrations enhanced N2O generation in the aeration periods (probably via nitrifier denitrification) while low DO concentrations (lower than 0.2 mg/L) did not affect N2O generation in the non-aeration periods (probably via heterotrophic denitrification). When PHB was utilized as the organic substrate for denitrification, there was a high N2O generation potential. It was estimated that 1.8 mg N2O-N was generated accompanying per mg PHB consumed.
Keywords: Intermittent aeration; Slaughterhouse wastewater; Aeration rate; Nitrite; Dissolved oxygen;