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In this study, an engineered strain of Saccharomyces cerevisiae was used to produce taxadiene, a precursor in the biosynthetic pathway of the anticancer drug paclitaxel. Taxadiene was recovered in situ with the polymeric adsorbent Diaion © HP-20. Here we tested two bioreactor configurations and adsorbent concentrations to maximize the production and recovery of taxadiene. An external recovery configuration (ERC) was performed with the integration of an expanded bed adsorption column, whereas the internal recovery configuration (IRC) consisted in dispersed beads inside the bioreactor vessel. Taxadiene titers recovered in IRC were higher to ERC by 3.4 and 3.5 fold by using 3% and 12% (w/v) adsorbent concentration respectively. On the other hand, cell growth kinetics were faster in ERC which represents an advantage in productivity (mg of taxadiene/L*h). High resin bead concentration (12% w/v) improved the partition of taxadiene onto the beads up to 98%. This result represents an advantage over previous studies using a 3% resin concentration where the partition of taxadiene on the beads was around 50%. This work highlights the potential of in situ product recovery to improve product partition, reduce processing steps and promote cell growth. Nevertheless, a careful design of bioreactor configuration and process conditions is critical.

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Here, we compare the performance of various three-dimensional-printed Monolith Adsorption (PMA) columns designed from a triply periodic minimal surface geometry, the Schoen gyroid. The structures examined had designed hydraulic diameters between 203 and 458 µm and voidages of 40%-60%. We compare column efficiency, porosity, static binding capacity and dynamic binding capacity for various load volumes and flow rates. The results show that all structures allowed efficient passage of yeast cells (>97%) over a wide range of interstitial velocities (191 to 1911 cm/h) while maintaining a low pressure drop (<0.1 MPa). The structure with a voidage of 40% and a hydraulic diameter of 203 µm showed the best performance in all aspects evaluated. Bovine serum albumin (BSA) recoveries for all structures (27%-91% when the loaded volume was 180 mL) were significantly affected by hydraulic diameter, mean channel wall thickness, velocity and voidage. Moreover, biomass addition resulted in a decrease in BSA recovery, which became more obvious at high velocities. However, this did not lead to a dramatic reduction in saturated binding capacity, significant changes in axial dispersion, or blockage of channels and could be compensated for by recirculation of the feed, even at high velocity. PMA thus potentially provides an appealing alternative to Expanded Bed Adsorption, retaining the latter's advantages, while eliminating fluidization issues and minimizing both processing time and buffer consumption.

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Metallurgical wastewaters contain high concentrations of sulfate, up to 15 g L-1. Sulfate-reducing bioreactors are employed to treat these wastewaters, reducing sulfates to sulfides which subsequently co-precipitate metals. Sulfate loading and reduction rates are typically restricted by the total H2S concentration. Sulfide stripping, sulfide precipitation and dilution are the main strategies employed to minimize inhibition by H2S, but can be adversely compromised by suboptimal sulfate reduction, clogging and additional energy costs. Here, metallurgical wastewater was treated for over 250 days using two hydrogenotrophic granular activated carbon expanded bed bioreactors without additional removal of sulfides. H2S toxicity was minimized by operating at pH 8 ± 0.15, resulting in an average sulfate removal of 7.08 ± 0.08 g L-1, sulfide concentrations of 2.1 ± 0.2 g L-1 and peaks up to 2.3 ± 0.2 g L-1. A sulfate reduction rate of 20.6 ± 0.9 g L-1 d-1 was achieved, with maxima up to 27.2 g L-1 d-1, which is among the highest reported considering a literature review of 39 studies. The rates reported here are 6-8 times higher than those reported for other reactors without active sulfide removal and the only reported for expanded bed sulfate-reducing bioreactors using H2. By increasing the influent sulfate concentration and maintaining high sulfide concentrations, sulfate reducers were promoted while fermenters and methanogens were suppressed. Industrial wastewater containing 4.4 g L-1 sulfate, 0.036 g L-1 nitrate and various metals (As, Fe, Tl, Zn, Ni, Sb, Co and Cd) was successfully treated with all metal(loid)s, nitrates and sulfates removed below discharge limits.

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In this study, an expanded bed anion exchange in a countercurrent chromatography column (EB-CCC) technique for separation and purification of heparin, an anticoagulant and antithrombotic glycosaminoglycan, is reported for the first time. A comparative evaluation of the EB-CCC technique with the conventional fixed bed column chromatography (FBCC) revealed its effectiveness in improving adsorption at high flow rates and reducing separation time. A significantly higher maximum adsorption (91.66%) was exhibited by EB-CCC in comparison with FBCC (45.16%) at the eluent flow rate of 1 mL·min-1. Similarly, the experimental adsorption capacity of heparin was enhanced by 1.69, 2.06 and 2.58 times in the case of EB-CCC at the flow rates of 1, 2 and 5 mL·min-1, respectively. Moreover, the directly proportional amplification of double loaded resin and double column volume was demonstrated at an EB-CCC rotational speed of 300 rpm and a flow rate of 2 mL·min-1, and the experimental adsorption capacity was observed to increase from 66.42 to 136.48 mg·g-1 after amplification. Heparin purified by EB-CCC displayed higher potency (216.09 ± 11.89 IU·mg-1) as compared to FBCC (205.51 ± 7.90 IU·mg-1) and the initial crude heparin 134.17 ± 4.12 IU·mg-1. Furthermore, comparing to the purified heparin by FBCC, heparin purified by EB-CCC had low molecular weight, high FXa/FIIa, superior anticoagulation effect and enhanced suitability as an exogenous anticoagulant.

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The vortex flow reactor (VFR) can be used in many chemical engineering applications. This paper assesses its novel use in the purification of monoclonal antibodies from cell broth. To this end, the IgG2a antibody was purified from the unclarified fermentation broth of transgenic mouse 55/6 hybridoma cells. Visual experiments showed that the VFR worked in the laminar vortices flow regime and the vortices displaced slightly faster than the axial flow. The VFR has the advantage of creating two sorts of flows: axial flow to produce the expanded bed and an extra vortex flow to avoid channeling and stabilize the expanded bed, the hydrodynamic behavior of which is plug flow with an experimental Pèclet number higher than 20. The pH was adjusted in the untreated fermentation broth, which was directly introduced into the reactor thus reducing the number of stages. The IgG2a purification was carried out in a single device via two steps: antibody adsorption in the expanded bed and antibody elution in the settled bed using Streamline rProtein A. A thirty-fold increase in the high-purity antibody concentration was achieved at the top of the pH5 elution peak with a total recovery of 93.1% (w/w) between elution peaks pH 5 and 3.

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An ordered 3D printed chromatography stationary phase was used to purify M13 bacteriophage (M13) directly from crude cell culture. This new approach, which offers the same advantages as expanded bed adsorption (EBA) with regard to tolerating solids-laden feed streams but without the corresponding issues associated with fluidized bed stability that affect the latter, can be described as "printed monolith adsorption (PMA)". PMA columns (5, 10 and 15 cm length by 1 cm diameter) were made via a wax templating method from cross-linked cellulose hydrogel and functionalized with a quaternary amine ligand. The recovery of M13 was found to be strongly linked to load flow rate, with the highest recovery 89.7% ± 6% for 1.4 × 1011 pfu/mL of resin occurring at 76 cm/h with a 10 cm column length. A recovery of 87.7% ± 5% for 1.49 × 1011 pfu/mL of media was achieved with a 15 cm column length under conditions comparable to a reported EBA process. The PMA process was completed three times faster than EBA because PMA flow rates can readily be adjusted during operation, with high flow rates and low back pressure, which is unique to the ordered monolithic media geometry used. Equilibration, wash, and cleaning steps were carried out at high flow rates (611 cm/h), minimizing process time and were limited only by the volumetric flow rate capacity of the pumps used, rather than column back pressure (<0.1 MPa at 611 cm/hr). Initial capture of M13 appears to occur on the surface of the monolith solid phase (i.e. the mobile phase channel walls) and subsequently, at a slower rate, within the internal pores of the solid phase media. The difference in binding rate between these two sites is likely caused by slow pore diffusion of the large M13 particles into the pores, with similar slow diffusion out of the pores resulting in tailing of the elution peak. The results indicate that PMA is a promising technology for the efficient purification of viruses directly from crude cell culture.

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In the present work, spherical carboxymethyl cellulose-cellulose-nickel (CMC-C-Ni) composite beads as novel adsorbent was synthesized to make a stable expanded bed adsorption (EBA) column for the treatment of the oily wastewater collected from the downstream of rapeseed industry. The morphology and structure of the CMC-C-Ni composite beads were studied by scanning electron microscopy (SEM) and optical microscope. The SEM images revealed that the synthesized composite beads were spherical with porous structure. The pore size of the beads was in the range of 90-200 nm. The physical characteristics of the CMC-C-Ni composite beads including wet density, porosity, and water content were respectively in the ranges of 1.23-1.63 g/cm3, 82.29-90.75%, and 52-76%. The factor of bed expansion in the range of 2-3 was corresponded with Richardson-Zaki equation. The results showed that by increasing the fluid viscosity, the terminal settling velocity (Ut) was reduced. The expansion index values were between 2.77 and 3.14 that were close to 4.8 (commonly utilized index in the laminar flow regimes). CMC-C-Ni composite beads were tested when the velocity of fluid was ˂ 700 cm/h, and the Daxl was found to be ˂ 1 × 10-5 m2/s (steady state).

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Jarosite precipitates formed in iron oxidising bioreactors have been shown to harbour iron-oxidisers. The aim of this study was to develop an iron oxidising bioprocess where microorganisms are retained solely on biogenic jarosite particles. Based on preliminary experiments using a fluidised-bed bioreactor (FBR), the formed jarosite particles started to disintegrate and wash out at upflow velocities of ≥0.21 cm/s. Therefore, the generation and use of biogenic jarosite carrier was studied in an expanded-bed bioreactor (J-EBR) with an upflow velocity of 0.19 cm/s. Inside J-EBR, the jarosite particles formed granules of 0.5-3 mm containing 200-460 mg/g of attached biomass. The performance of J-EBR was compared with an activated carbon biofilm FBR at 0.82 cm/s upflow velocity (AC-FBR). At 35 ± 2 °C with a feed ferrous iron concentration of 10 g/l, the highest obtained iron oxidation rate of J-EBR (6.8 g/l/h) was 33% lower than that of AC-FBR (10.1 g/l/h). This was likely due to the 80% lower recirculation rate and subsequently higher oxygen mass transfer limitation in J-EBR compared to AC-FBR. The present study demonstrates that biogenic jarosite can be used for retainment of iron oxidising biofilms in expanded-bed bioreactors that oxidise iron at high rates.

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BACKGROUND: An efficient biodegradation-strengthening approach was developed to improve penicillin G degradation from industrial bacterial residue in an expanded bed adsorption bioreactor (EBAB) is reported in this paper. RESULTS: Paracoccus sp. strain KDSPL-02 was isolated based on its ability to use penicillin G as the sole carbon and nitrogen source. Strain identification was based on analyses of morphology, physio-biochemical characteristics, and 16S rDNA sequences. The effects of temperature, pH, PVA-sodium alginate concentration, calcium chloride concentration and initial penicillin G concentration were investigated. Repeated operations of immobilized cells with EBAB, At initial penicillin concentrations below 2.0 g L- 1, the continuous mode could reach more than 20 times, and the degradation rate reached 100%. CONCLUSIONS: The present study suggests that the EBAB system can be utilized for the simple and economical biodegradation of penicillin G from industrial bacterial residue.

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Vitamin B12 and propionic acid that were simultaneous produced by Propionibacterium freudenreichii are both favorable chemicals widely used in food preservatives, medicine, and nutrition. While the carbon source and propionic acid accumulation reflected fermentation efficiency. In this study, using corn stalk as a carbon source and fed-batch fermentation process in an expanded bed adsorption bioreactor was studied for efficient and economic biosynthesis of acid vitamin B12 and propionic. With liquid hot water pretreated corn stalk hydrolysates as carbon source, 28.65 mg L-1 of vitamin B12 and 17.05 g L-1 of propionic acid were attained at 168 h in batch fermentation. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed corn stalk in expanded bed adsorption bioreactor (EBAB), giving 47.6 mg L-1 vitamin B12 and 91.4 g L-1 of propionic acid at 258 h, which correspond to product yields of 0.37 mg g-1 and 0.75 g g-1, respectively. The present study provided a promising strategy for economically sustainable production of vitamin B12 and propionic acid by P. freudenreichii fermentation using biomass cornstalk as carbon source and expanded bed adsorption bioreactor.

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While anammox is a cost-effective nitrogen treatment process for wastewater with high nutrient strength, phosphorus remains untouched during this process and needs further treatment. In this study, the nitrogen removal and phosphorus recovery were achieved simultaneously at 25 °C using an anammox expanded bed. A stable high nitrogen removal efficiency of 83.7 ± 4.8% at a 1500 mgN/L influent total nitrogen concentration and a phosphorus removal efficiency of 94.2 ± 1.2% at 100 mg P/L influent total phosphorus were obtained during continuous operation. The effects of the nitrogen loading rate, hydraulic retention time (HRT), pH, Ca2+ and PO43- concentration on the phosphorus removal was verified in the long-term operation of the reactor. The sludge produced contained a high content of phosphorus mainly in the form of hydroxyapatite (HAP), and the sludge composition strongly reflected the nitrogen and phosphorus loading. The structure of the anammox-HAP composite granules was illustrated by the use of fluorescence in situ hybridization (FISH) and Raman spectroscopic mapping analysis. The results in this study indicate that by controlling the operation parameters, it is possible to integrate a high efficiency phosphorus recovery with the anammox process, and significantly reduce the nutrient loading for further wastewater treatment.

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The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men's fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid-liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid-liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L-1 and productivity of 3.2 mg L-1 h-1. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules.

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Phosphorus recovery from wastewater is an important approach for sustainable phosphorus use. In this work, a process combining anammox and hydroxyapatite (HAP) precipitation in an expanded bed reactor for simultaneous nitrogen removal and phosphorus recovery was developed by applying specific Ca/P ratio and pH control. A high phosphorus removal rate (0.14 ±â€¯0.01 kg-P/m3/d) was obtained while a stable nitrogen removal efficiency (87.4 ±â€¯2.9%) maintained with an effluent recirculation system applied. Average 13.4% phosphorus (30.7% in P2O5) accumulation in the dry sludge and a Ca/P ratio very close to HAP was observed by quantitative elemental analysis. In this work, different analysis revealed the two layers structure with anammox biofilm attached to inorganic core of the granules. Different spectral analysis determined the major phase of the inorganic content as hydroxyapatite. With proper Ca/P ratio and pH control, anammox expanded bed reactor was transformed into an efficient process to simultaneously remove nitrogen and recover phosphorus.

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Bio-based industries need efficient downstream solutions to process complex streams. This was addressed through a technology integration approach, where expanded-bed adsorption (EBA) is integrated with simulated moving-bed (SMB) technology. Current work involved adaptation of an SMB apparatus and control principle to implement expanded-bed level control. As an outcome, EBA-SMB technology was successfully applied for purification of γ-aminobutyric acid (GABA). This resulted in two-fold increase in productivity and a GABA purity ≥ 92 % in one step from unclarified fermentation broth, compared to ≥ 93 % purity in case of clarified broth and packed-bed SMB. These results proved that EBA-SMB technology is able to enhance process efficiency and economics of bioprocesses.

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Expanded-bed adsorption (EBA) is an efficient downstream technology that enhances the techno-economic potential of bio-based industries. However, application of EBA for bulk biochemicals requires the use of industrial resins. Therefore, two cation exchangers, namely, gel-type CS16GC and porous IRC747, were tested to purify γ-aminobutyric acid (GABA) from unclarified E. coli fermentation broth. Experiments compared the impact of gel-type and macroporous resin properties on the EBA process performance. As an outcome, the gel-type resin exhibited higher GABA binding capacity of compared to that of macroporous resin. This was due to improved hydrodynamics and uniform flow distribution in the case of gel-type resin. Further, CS16GC effectively removed ≥ 99 % of impurities and achieved ≥ 97 % GABA yield.

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In this study, a general rate model was applied to the entire process of expanded bed adsorption chromatography (EBAC) for the chitosanases purification protocol from unclarified fermentation broth produced by Paenibacillus ehimensis using the anionic adsorbent Streamline® DEAE. For the experiments performed using the expanded bed, a homemade column (2.6cm×30.0cm) was specially designed. The proposed model predicted the entire EBA process adequately, giving R2 values higher than 0.85 and χ2 as low as 0.351 for the elution step. Using the validated model, a 33 factorial design was used to investigate other non-tested conditions as input. It was observed that the superficial velocity during loading and washing steps, as well as the settled bed height, has a strong positive effect on the F objective function used to evaluate the production of the purified chitosanases.

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The current processing paradigm of large manufacturing facilities dedicated to single product production is no longer an effective approach for best manufacturing practices. Increasing competition for new indications and the launch of biosimilars for the monoclonal antibody market have put pressure on manufacturers to produce at lower cost. Single-use technologies and continuous upstream processes have proven to be cost-efficient options to increase biomass production but as of today the adoption has been only minimal for the purification operations, partly due to concerns related to cost and scale-up. This review summarizes how a single-use holistic process and facility strategy can overcome scale limitations and enable cost-efficient manufacturing to support the growing demand for affordable biologics. Technologies enabling high productivity, right-sized, small footprint, continuous, and automated upstream and downstream operations are evaluated in order to propose a concept for the flexible facility of the future.

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Panax ginseng has been applied in traditional Chinese medicine for over 2000 years. It is still one of the most popular herbs in recent decades. The prescribed ginseng-containing medicines consist of protopanaxadiol and protopanaxatriol ginsenosides, which are the major constituents of the herb. Minor ginsenosides at low levels in the herb, such as Rg3 and Rg5 , have attracted more rising attention than the major ones. The existing approaches to prepare Rg3 and Rg5 usually rely on either steamed red ginseng as the source or chemical/enzymatic conversion of protopanaxadiol to the targets. It is still highly desirable to effectively achieve such minor components. In this paper, a method integrated extraction of protopanaxadiol and conversion of it to Rg3 and Rg5 has been proposed. Protopanaxadiol was extracted and simultaneously converted to Rg3 and Rg5 by d,l-tartaric acid. The targets were absorbed by resins on expanded bed adsorption chromatography and were then separated from other ginsenosides in different stages. Compared with conventional methods, the developed process has advantages in shortening time consumption and improving the conversion ratio of protopanaxadiol, which is promising in directly achieving Rg3 and Rg5 from P. ginseng.

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The use of human mesenchymal stem cells (hMSC) in clinical applications has been increasing over the last decade. However, to be applied in a clinical setting hMSC need to comply with specific requirements in terms of identity, potency and purity. This study reports the improvement of established tangential flow filtration (TFF)-based washing strategies, further increasing hMSC purity, using negative mode expanded bed adsorption (EBA) chromatography with a new multimodal prototype matrix based on core-shell bead technology. The matrix was characterized and a stable, expanded bed could be obtained using standard equipment adapted from what is used for conventional packed bed chromatography processes. The effect of different expansion rates on cell recovery yield and protein removal capacity was assessed. The best trade-off between cell recovery (89%) and protein clearance (67%) was achieved using an intermediate expansion bed rate (1.4). Furthermore, we also showed that EBA chromatography can be efficiently integrated on the already established process for the downstream processing (DSP) of hMSC, where it improved the washing efficiency more than 10-fold, recovering approximately 70% of cells after global processing. This strategy showed not to impact cell viability (>95%), neither hMSC's characteristics in terms of morphology, immunophenotype, proliferation, adhesion capacity and multipotent differentiation potential.

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A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01. The purification and characterization of two chitosanases were studied. The purification assay was accomplished by ion exchange expanded-bed chromatography. Experiments were carried out in the presence and in the absence of cells through different expansion degree to evaluate the process performance. The adsorption experiments demonstrated that the biomass does not affect substantially the adsorption capacity of the matrix. The enzyme bound to the resin with the same extent using clarified and unclarified broth (0.32 and 0.30 U/g adsorbent, respectively). The fraction recovered exhibited 31% of the yield with a 1.26-fold increase on the specific activity concerned to the initial broth. Two chitosanases from different elution steps were recovery. Chit A and Chit B were stable at 30-60°C, pH 5.5-8.0 and 5.5-7.5, respectively. The highest activity was found at 55°C, pH 5.5 to Chit A and 50°C, pH 6.5 to Chit B. The ions Cu(2+), Fe(2+) and Zn(2+) indicated inhibitory effect on chitosanases activities that were significantly activated by Mn(2+). The methodology applied in this study enables the partial purification of a stable chitosanase using a feedstock without any pre-treatment using a single-step purification.

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