Biochemical Engineering Journal (v.54, #2)

BEJ Keywords (IV).

► Optimization of alkali soaking and extrusion pretreatment of prairie cord grass for maximum sugar recovery by enzymatic hydrolysis. ► Biomass pretreatment is an essential step in the conversion of biomass to Bioethanol and extrusion is one of the promising continuous methods. ► In order to improve sugar recovery from prairie cord grass, influence of alkali soaking, extruder barrel temperature, screw speed, and particle size were studied. ► Statistical analyses revealed that all the independent variable had a strong influence on sugar recovery. ► The proposed quadratic model to predict sugar recoveries had high F and R 2 values with low p value adequately representing the relationship. ► The optimal pretreatment condition 114 °C barrel temperature, 122 rpm screw speed, 1.70% alkali concentration, and 8 mm particle size resulted a maximum glucose, xylose and combined sugar recoveries of 86.8, 84.5, and 82%, respectively, by enzymatic hydrolysis.Apart from corn stover and switchgrass, there are several other types of biomass that have comparable yield and can be grown across the country, and prairie cord grass (PCG) is one among them. Biomass pretreatment is an essential step in the conversion of biomass to bioethanol. Till date, no perfect conversion technology has been established for biofuel production from biomass on commercial scale. Extrusion pretreatment of PCG showed significant improvement on sugar recovery (63.5%). However, there is still room to improve sugar recovery from PCG by combining with alkali soaking. The current study was undertaken to investigate the influence of alkali (NaOH) concentration on sugar recovery and optimize extruder barrel temperature, screw speed, and particle size for maximum sugar recovery. In order to evaluate the sequential effect of alkali soaking and extrusion, PCG (2–10 mm) was soaked at different alkali concentrations (0.5–2.5%, w/v NaOH) for 30 min at room temperature and then extruded using a lab scale single screw extruder at various barrel temperatures (45–225 °C) and screw speeds (20–200 rpm). All the independent variable had a strong influence on sugar recovery and it was confirmed through statistical analyses. The proposed quadratic model to predict sugar recoveries had high F and R 2 values with low p value adequately representing the relationship. The optimal pretreatment condition 114 °C barrel temperature, 122 rpm screw speed, 1.70% alkali concentration, and 8 mm particle size resulted a maximum glucose, xylose and combined sugar recoveries of 86.8, 84.5, and 82%, respectively, by enzymatic hydrolysis.
Keywords: Biomass; Pretreatment; Enzymatic hydrolysis; Extrusion; Screw speed; Barrel temperature;

Utilization of different agro-industrial wastes for sustainable bioproduction of citric acid by Aspergillus niger by Gurpreet Singh Dhillon; Satinder Kaur Brar; Mausam Verma; Rajeshwar Dayal Tyagi (83-92).
► Economical and sustainable citric acid (CA) production using agro-industrial wastes. ► Economy use of negative cost agro-industrial wastes. ► To met the rising demand of CA in many advanced applications in food and biomedicines. ► To lessen the environmental hazards caused by the disposal of agro-industrial wastes. ► Environmental capture of waste carbon leading to mitigation of climate change.In view of ever growing demand of citric acid, there is an urgent need to look for inexpensive and novel substrates for feasible production of citric acid. In this context, the present study was carried out to evaluate the potential of different agro-industrial wastes for hyper production of citric acid through solid-state and submerged fermentation by Aspergillus niger NRRL 567 and NRRL 2001. It was found that among all the solid substrates utilized, apple pomace with 66.0 ± 1.9 g/kg of dry substrate proved to be an excellent substrate for citric acid production by A. niger NRRL 567 at 72 h of incubation. A. niger NRRL 2001 resulted in slightly lower citric acid concentration of 61.0 ± 1.9 g/kg of dry substrate at the same incubation time. APS-1 (apple pomace ultrafiltration sludge-1) gave highest citric acid production rate of 9.0 ± 0.3 g/l and 8.9 ± 0.3 g/l of substrate by A. niger NRRL 567 and NRRL 2001 by submerged fermentation, respectively. Further study with apple pomace and apple pomace ultrafiltration sludge-1 by A. niger NRRL 567 was carried out. Addition of 3% (v/w) ethanol and 4% (v/w) methanol to apple pomace gave significantly higher citric acid values of 127.9 ± 4.3 g/kg and 115.8 ± 3.8 g/kg of dry substrate by A. niger NRRL 567 by solid-state fermentation. Higher citric acid values of 18.2 ± 0.4 g/l and 13.9 ± 0.4 g/l of apple pomace ultrafiltration sludge-1 were attained after addition of 3% (v/v) ethanol and 4% (v/v) methanol, respectively by A. niger NRRL 567. Apple pomace solid waste and apple pomace ultrafiltration sludge-1 thus proved to be an excellent source for citric acid production, of the different substrates chosen.
Keywords: Agro-industrial waste; Citric acid; Solid-state fermentation; Submerged fermentation; Aspergillus niger;

Expression, purification of a novel alkaline Staphylococcus xylosus lipase acting at high temperature by Ahlem Bouaziz; Habib Horchani; Nadia Ben Salem; Youssef Gargouri; Adel Sayari (93-102).
Display Omitted► A newly isolated Staphylococcus xylosus produce a thermostable and alkaline lipase. ► The Staphylococcus xylosus lipase is the most active lipase in the staphylococcal family. ► The immobilized SXL2 was used as biocatalyst to synthesise a high added value molecules.A new Staphylococcus xylosus strain was isolated. The extracellular lipase of S. xylosus (wt-SXL2) was purified to homogeneity from the culture medium. The specific activity of the purified enzyme, measured at pH 8.5 and 55 °C using tributyrin or olive oil emulsion, reached, respectively, 6300 U/mg or 2850 U/mg. The sequenced 18 N-terminal amino acid showed a high degree of identity with known staphylococcal lipase sequences.The gene encoding the mature lipase was cloned and sequenced. The deduced amino acid sequence showed a significant similarity with various staphylococcal lipases. The highest overall identity (98.74%) was found with S. xylosus lipase (SXL1). The mature part of the lipase was expressed in Escherichia coli. The recombinant lipase was purified by affinity chromatography. The specific activity of the recombinant lipase was 4100 or 1500 U/mg using tributyrin or olive oil emulsion as substrate, respectively, at pH 8.5 and 55 °C.The wild type and recombinant lipases presented a quite interesting thermal stability, after an incubation of 60 min at 55 °C and they are found to be highly stable at a pH ranging from 4 to 11. Due to its stability at high temperature and in organic solvent, the wt-SXL2 was used as biocatalyst to synthesise a high added value molecules.
Keywords: Staphylococcus xylosus lipase; Purification; Sequencing; Thermo-alkaline; Expression; Synthesis;

Recovery and isolation of recombinant human monoclonal antibody from transgenic tobacco plants by Mukesh Mayani; Michael D. McLean; J. Christopher Hall; Carlos D.M. Filipe; Raja Ghosh (103-108).
► Transgenic plants offer an alternative to cell culture based production of protein biopharmaceuticals ► Recovery and purification of biopharmaceuticals from transgenic plants is significantly more challenging than their purification from cell culture supernatant primarily due to the extreme complexity of plant extracts ► The early steps in the purification process for obtaining recombinant antibody from transgenic plants can significantly affect the overall purity and recovery. ► Monoclonal antibody recovery could be enhanced by minimizing its interactions with native tobacco proteins and other components of tobacco tissue. ► Parameters such as salt concentration, tissue grinding time and pH affected both recovery and purity of monoclonal antibody.Early steps in the purification process for obtaining recombinant antibody from transgenic plants can significantly affect the overall purity and recovery. We investigate the effects of salt concentration, tissue grinding time and lowering pH on recovery and purification of a recombinant human monoclonal antibody (anti-Pseudomonas aeruginosa serotype O6ad) from transgenic tobacco plants. The presence of 450 mM sodium chloride in the extraction buffer resulted in an 8-fold increase in antibody recovery when compared to using a sodium chloride-free buffer. The grinding time and the pH at which the extract was maintained were also found to have significant effects on antibody recovery. Generally, monoclonal antibody recovery could be enhanced by minimizing its interactions with native tobacco proteins and other components of the tobacco tissue. The overall yield of monoclonal antibody after optimization of the grinding and extraction steps was 30.3 mg/kg of tobacco tissue, this being 12.5 times higher than that at a non-optimized extraction condition.
Keywords: Monoclonal antibody; Transgenic; Tobacco; Recovery; Purification;

► The paper provides a study of downstream operations for laccase recovery. ► This is probably the first report on laccase separation using TPP. ► TPP was optimized to concentrate and purify laccase effectively. ► Laccase purity using TPP was better than ion exchange chromatography. ► This work helps select recovery protocols with a trade between purity and yield.Three-phase partitioning (TPP), an efficient bioseparation technique, was used to purify laccase from fermentation broths of Ganoderma sp. WR-1. In a two-step procedure, the physiochemical parameters of ammonium sulfate concentration, the aqueous phase-to-t-butanol ratio, temperature and pH were optimized for laccase separation. In step 1 with 20% ammonium sulfate and a 1:0.5 (v/v) aqueous phase-to-t-butanol ratio at 35 °C and pH 7, most of the laccase remained in the lower aqueous phase. Step 2, with 90% ammonium sulfate and the other variables the same as in step 1, resulted in laccase concentrated at the interface. The two-step TPP led to 60% recovery of laccase with 13.2-fold purification. The TPP protocol developed in our study for the concentration and purification of laccase is rapid, simple and highly efficient.
Keywords: Laccase; Precipitation; Purification; Three phase partitioning; Ultrafiltration; White rot fungi;

Solvent-free polyglycerol polyricinoleate synthesis mediated by lipase from Rhizopus arrhizus by J.L. Gómez; J. Bastida; M.F. Máximo; M.C. Montiel; M.D. Murcia; S. Ortega (111-116).
► We have obtained for the first time the food emulsifier E-476 enzymatically. ► Obtaining E-476 has been carried out in the absence of solvents and immobilised enzyme, fulfilling the main premises of green chemistry. ► The improved quality of the final product and the energy savings, makes this process a serous alternative for the production of PGPR (E-476).The enzymatic biosynthesis of polyglycerol polyricinoleate (PGPR) (E-476) is described in detail for the first time. Starting from polyglycerol and polyricinoleic acid, Rhizopus arrhizus lipase was used as catalyst. The reaction, which is really a reversal of hydrolysis, takes place in the presence of a very limited amount of aqueous phase. No organic solvent is necessary to solubilise the substrates, which allows a reaction medium solely composed of the necessary substrates to be used.Immobilisation of the lipase by physical adsorption onto an anion exchange resin provided good results in terms of activity, enzyme stability and the reuse of immobilised derivative. Using this immobilised derivative, PGPR with an acid value of 16 mg KOH/g was obtained, far above the requirements of the European Commission Directive 2008/84/EC (<6 mg KOH/g). In an attempt to force the reaction equilibrium towards the synthetic pathway, polyglycerol polyricinoleate was synthesised under controlled atmosphere in a vacuum reactor with dry nitrogen intake. This equipment allowed us to synthesise PGPR with an acid value of 4.9 mg KOH/g, which complies with the European Commission Directive and the results were entirely reproducible. This investigation represents a good starting point for using the enzymatic procedure in the industrial biosynthesis of PGPR.
Keywords: Polyglycerol polyricinoleate; Solvent-free; Lipase; Immobilised enzyme; Biosynthesis;

► Recombinant lipase has higher specific activity than native one. ► More than one form of lipase was found in both extracts. ► An esterase activity was found in the native extract. ► Different specificity towards p-nitrophenol esters. ► Determination of the influence of ionic strength on lipolytic activity.Extracts containing mature Rhizopus oryzae lipase overexpressed in Pichia pastoris (rROL) and commercial ones from the native microorganism (nROL) have been characterized. The specific activity of rROL extract was more than 40-fold higher than nROL. The presence of multiple bands of lipases around 34 kDa was detected by western blot and zymogram analysis in both extracts. Nevertheless, rROL showed a slightly lower molecular weight than nROL. The presence of hydrolytic activity not recognised as derived from a lipase was also detected in nROL at higher molecular weights.The influence of the ionic strength on lipase activity was assayed and there was an effect both on pH and optimal temperature. Some differences were found between the two extracts. The specificity against triacylglycerol esters and p-nitrophenol substrates was also analyzed. Similar behaviour was noted towards triacylglycerol esters; however rROL showed opposite behaviour compared to nROL towards p-nitrophenol esters with preferences for long chain derivatives.The properties of rROL were partially different from those reported for nROL showing the influence of the pre-pro-sequence of ROL, the post-translational modifications of Pichia pastoris and the effect of an esterase in nROL powder.
Keywords: Pichia pastoris; Rhizopus oryzae; Recombinant lipase; Biochemical properties; Protein extract; Specificity;

Recovery of gold(III) from an aqueous solution onto a durio zibethinus husk by Mahani A.Z. Abidin; Aishah A. Jalil; Sugeng Triwahyono; S. Hazirah Adam; N.H. Nazirah Kamarudin (124-131).
Display Omitted► Durio zibethinus husk (DZH) as an alternative low-cost adsorbent for recovery of gold(III) ion. ► Adsorption fitted well to Langmuir isotherm and followed pseudo second-order kinetic equation. ► Adsorption was exothermic and chemisorptions process. ► DZH has a potential to be used in industrial wastewater treatment.The recovery of gold(III) ions from an aqueous solution onto a durio zibethinus husk (DZH) was examined after varying pH, contact time, adsorbent dosage, initial Au(III) concentration, and temperature. The functional groups of DZH were analyzed by FTIR and Au(III) recovery onto DZH was verified by FESEM–EDX and XRD analysis. Adsorption equilibrium isotherms and kinetics of the DZH were studied using Freundlich and Langmuir models, as well as pseudo first-order, second-order kinetic and intraparticle diffusion equations. The experimental data obtained with DZH fitted best to the Langmuir isotherm model and exhibited a maximum adsorption capacity (q max) of 1724 μmol g−1. The data followed the pseudo second-order equation. The activation energy of the adsorption (E a) was estimated to be 38.5 kJ mol−1. Thermodynamic parameters, such as changes in enthalpy, entropy and Gibbs free energy, showed that the adsorption is exothermic, spontaneous at low temperature, and is a chemisorption process. These results indicate that DZH adsorbs efficiently and could be used as a low-cost alternative for the adsorption of Au(III) in wastewater treatment.
Keywords: Recovery; Gold(III); Durio zibethinus husk;

► A novel foam fractionation column has been developed for biosurfactant recovery. ► Fermentation and foam column operating conditions can be independently optimised. ► Integrated foam fractionation reduced uncontrolled overflow by a factor of 17. ► Optimising the foam separation increased biomass retention from 88% to 95%. ► An HFBII recovery of 70% was achieved at an enrichment of 6.6.A novel foam fractionation design has been developed for continuously recovering extracellular biosurfactants from fermenters. The apparatus design allows for the operating conditions of the foam fractionation process, feed rate and airflow rate, to be chosen independently of the fermentation parameters. Optimal conditions can then be established for each process, such as the aeration rate required to meet the biological oxygen demand of the cell population. The recirculating foam fractionation process was tested on fed batch fermentations producing the hydrophobin protein HFBII. It is shown that by using foam fractionation to strip HFBII from fermentation broth in situ the amount of uncontrolled overflow from the fermenter was greatly reduced from 770 g to 44.8 g, compared to previous fermentations without foam fractionation. Through optimisation of the foam column operating conditions the proportion of dry matter retained in the fermenter was increased from 88% to 95%, in contrast to dry matter retention of 66% for fermentation without the new design. With the integrated foam fractionation process an HFBII recovery of 70% was achieved at an enrichment of 6.6. This study demonstrates the utility of integrated foam fractionation in minimising uncontrolled foaming in fermenters whilst recovering an enriched product.
Keywords: Bioseparations; Biosurfactant; Foam fractionation; Downstream processing; Process integration; Yeast;