Biochemical Engineering Journal (v.107, #C)


Biofilters are popular for the removal of odours from gaseous emissions in wastewater treatment plants because of their low capital costs and low energy requirements. In an aerobic environment, the microbes in biofilter oxidize odorous gases like hydrogen sulphide (H2S) and ammonia (NH3) to non-odorous sulphate and nitrate. This paper describes a pilot plant biofilter setup at a local waste water treatment plant (WWTP) which has been in continuous operation for more than 150 days, removes both H2S and NH3 at an average removal efficiency of 91.96% and 100%, respectively. Unlike a conventional biofilter, the pH of this biofilter was not adjusted by addition of chemicals or buffers and the H2SO4 produced from the biological conversion of H2S is periodically washed down and allowed to accumulate in a concentrated form at the base of the biofilter. NH3 entering at the base is removed, not by biological oxidation, but by the chemical reaction of ammonium with sulphate to form ammonium sulphate. The ammonium sulphate produced in biofilter is washed down and the volume of leachate produced is less than 0.2 mL of leachate/L of reactor/day. Estimated cost savings of converting the current chemical scrubber used at the WWTP to a similar biofilter described in this study is included with this paper.
Keywords: Biofilter; Chemical NH3 removal; Biological H2S removal; Wastewater treatment plant; Odour removal;

The evolution of cell line and bioprocess development for biopharmaceutical manufacturing using mammalian cell culture over the past few decades has been striking. Despite this success, the future presents new challenges that include productivity increase, product quality modulation for comparability and biosimilarity, and efficient manufacturing of novel modalities. While empirical process development techniques will continue to play an important role in addressing these challenges, approaches based on mechanistic understanding are likely to be more impactful for solving the more complex multidimensional problems by providing insights into the interplays between the cell line, bioprocess, and product quality. Systems biology is one such approach that provides information on cellular physiology at the molecular level, which, when rigorously interpreted, can provide targets for cell line and/or process modifications. We present a general framework for applying systems biology to biotherapeutic-producing mammalian cells and summarize published work that exemplifies successful applications of this technique. We highlight gaps in our current understanding that limit widespread application of systems biology to mammalian cell-based bioprocess development and propose remediation methods that can encourage increased adoption. More nuanced understanding of cellular physiology and the interplay between expressing novel proteins and product quality attributes is possible through systems biology and this understanding will better position the field to successfully engage with the challenges of the future.
Keywords: Systems biology; Cell line development; Bioprocess design; Modeling; Bioreactors; Mammalian cell culture;

Affinity chromatography of human IgG with octapeptide ligands identified from eleven peptide-ligand candidates by Aiying Xue; Wei-Wei Zhao; Xiaoguang (Margaret) Liu; Yan Sun (18-25).
We have reported earlier on the characterization of four peptides from a library of 15 peptide ligand candidates for human IgG (hIgG) obtained by a biomimetic design strategy and identified three high-affinity octapeptides. In this work, the left 11 peptides were evaluated and we found two more octapeptides (FYCHWQDE and FYCHNQDE) that showed high affinity for hIgG. The binding pH ranges for the two ligands were different, but the optimum pH values were the same for each other (pH 6.0). Both the ligands showed high specificity and bound hIgG mainly by electrostatic interactions. Ligand binding competition experiments revealed that the binding sites on hIgG for the two octapeptides were similar to those for Protein A. Finally, hIgG was purified from human serum with high purities and recovery yields with the two peptide affinity columns. Thus, among the 15 peptide candidates, a total of five octapeptides were identified as high-affinity ligands of hIgG. The five ligands were all derived from the same peptide model FYxHxxxE (where x denotes any amino acid) and contained four common hot spots F132, Y133, H137, and E143 of the affinity motif of Protein A. Analyses and evaluation of the peptide library would help deepen our understanding of the affinity binding of Protein A to IgG, and promote application of the biomimetic strategy in the design of affinity ligands for different proteins.
Keywords: Protein; Affinity; Adsorption; Chromatography; Purification; Human immunoglobulin G;

Display Omitted S-Adenosyl-l-methionine (SAM), with diverse pharmaceutical applications, is biosynthesized from l-methionine and ATP. To enhance SAM accumulation in Saccharomyces cerevisiae CGMCC 2842 (2842), a new strategy based on yeast acetyl-CoA metabolism combined with introducing a methionine adenosyltransferase (metK1) from Leishmania infantum, was presented here. It was found that over-expressing acs2 (encoding acetyl-CoA synthase) and deleting mls1 (encoding malate synthase) increased SAM by 0.86- and 1.30-fold, respectively. To eliminate feedback inhibition of SAM synthase, a codon-optimized metK1 was introduced into 2842, and an increase of 1.45-fold of SAM was observed. Subsequently, metK1 and acs2 were co-expressed in the mls1 deleted strain, obtained the highly SAM-productive strain Ymls1 △GAPmK, and 2.22 g/L of SAM accumulated, which was 3.36-fold that in 2842. Moreover, the Ymls1 △GAPmK strain yielded 6.06 g/L SAM, which was 9.18-fold that in 2842, by fed-batch fermentation in a 10-L fermenter. Finally, the isolation and purification of SAM from yeast cell and preparation of SAM sulfate were preliminarily investigated. This study demonstrated that up-regulating acs2 and deleting mls1, which elevated intracellular acetyl-CoA levels, effectively enhanced the intracellular methionine biosynthesis. The elevated intracellular acetyl-CoA levels ultimately enhanced SAM accumulation, whereas the introduction of metK1 enhanced the redirection of acetyl-CoA to SAM biosynthesis in Ymls1 △GAPmK strain.
Keywords: S-Adenosyl-l-methionine; Biosynthesis; Intracellular acetyl-CoA levels; Recombinant DNA; Yeast; Metabolite over production;

Use of physical and biological process models to understand the performance of tubular anaerobic digesters by Maureen N. Kinyua; Jie Zhang; Fabricio Camacho-Céspedes; Andres Tejada-Martinez; Sarina J. Ergas (35-44).
Tubular anaerobic digesters are used in developing countries to produce biogas from livestock waste. In this research, field measurements and physical and biological process modeling studies were used to investigate transport and transformation mechanisms for particulate and soluble organic matter in household-scale tubular digesters in the Monteverde region of Costa Rica. Greater than 75% removal of volatile solids and biochemical oxygen demand (BOD5) were observed. The high effluent quality was attributed to the formation of a biologically active floccular sludge layer, which allowed for separation of hydraulic and mean cell residence times (HRT and MCRT). A reduced order transport model was developed and validated using field tracer study data. Key assumptions of the reduced order model were verified via computational fluid dynamics (CFD) analysis. The mean HRT predicted by the reduced order model was 23 days and was in good agreement with the tracer experiment. A simplified floccular sludge biological process model was developed and used to estimate an average MCRT of 115 days. The results showed that household-scale tubular anaerobic digesters can provide enough biogas to meet households’ cooking energy needs, which was consistent with field results. This is the first study to combine mathematical modeling with field studies of tubular anaerobic digester performance.
Keywords: Anaerobic processes; Bioreactors; Computational fluid dynamics; Modeling; Tubular anaerobic digester; Waste treatment;

Display OmittedWe recently described the development of a versatile and efficient esterase (rPPE01W187H) for the preparation of enantiopure 2-hydroxy acids. Herein, a primary amino-functionalized resin (ESR-1) was selected for immobilizing rPPE01W187H, and the resulting immobilized enzyme, rPPE01W187H@ESR-1, exhibited notably enhanced stability. No obvious inactivation was observed for rPPE01W187H@ESR-1 following its incubation at 30 °C for 1440 h, whereas the half-life of the free enzyme was only 50.2 h under the same conditions. A continuous process was subsequently developed using rPPE01W187H@ESR-1 in a packed bed reactor, which allowed for the immobilized enzyme to be conveniently recycled. The space-time yield for biocatalytic resolution of 2-acetoxy-2-(2′-chlorophenyl)acetate in this packed bed reactor reached as high as 3.34 kg L−1  d−1 following the optimization of the critical process parameters, including the initial pH, H:D ratio and flow direction. Further investigation of the operational stability indicated that the enzymatic process could be continuously operated for at least 42 d with approximately 50% conversion and nearly perfect optical purity, giving an outstanding total turnover number of 1.13 × 107 for rPPE01W187H. The continuous, long-term and high-performance manufacturing process developed in this study therefore highlights the potential feasibility of using rPPE01W187H for the large-scale production of optically pure chiral 2-hydroxy acids.
Keywords: Biocatalysis; Enantioseparation; Immobilized enzyme; Packed bed bioreactor; Chiral hydroxy acids; (R)-2-hydroxy-2-(2′-chlorophenyl)acetate;

A novel bioreactor to study the dynamics of co-culture systems by M.H. Kim; M. Liang; Q.P. He; J. Wang (52-60).
Co-culture strategy has drawn increasing interest in the last a few years to ferment the mixture of glucose and xylose or lignocellulosic hydrolysate. However, existing research has been mostly qualitative which mainly examined the ethanol production performance of the co-culture system such as final ethanol yield and productivity, with little or no attempt made to understand the dynamic interactions between the two microbes. This is partially due to the difficulties associated with monitoring and control of co-culture systems. In this work, we developed a bioreactor and associated protocols and control strategies to facilitate quantitative and systematic study of co-culture systems. In particular, the reported equipment, operation protocols and control strategies can deliver chemostat operation under controlled stable operation conditions by achieving stable oxygen utilization rates at various levels. In addition, the developed membrane-separated co-culture bioreactor enables independent control of the dissolved oxygen (DO) levels in each chamber, and easy tracking of individual biomass of each strain. The new protocol is a dual continuous/pseudo-continuous operation mode, which allows the control of constant biomass by adjusting the operation time of each mode, dilution rate and feed concentration. Experimental results on the co-culture system of Saccharomyces cerevisiae and Scheffersomyces stipitis are provided to demonstrate the capabilities of the developed co-culture bioreactor. With the developed equipment and protocol, simultaneous and complete utilization of both glucose and xylose was achieved around 70 h into the experiments we conducted, and was maintained as long as 800 h. Such complete utilization can be maintained even longer if desired. In addition, different OUR conditions (ranging 0.0036–0.0045 mmol O2/gDCW/h) were tested under controlled chemostat. Under the different operation conditions tested, ethanol yields and conversion rates varied in the range of 0.12–0.44 g/g and 0.22–1.95 g EtOH/L/h, respectively, which are in line with results reported in the literature.
Keywords: Co-culture; Bioreactors; Control; Bioprocess monitoring; Fermentation; Ethanol;

Hydrolysis of lignocellulose at high solids content is the prerequisite condition to obtain high ethanol titer broth and reduce the distillation cost. Design of large scale bioreactors requires the determination of rheological properties of the hydrolysate, but regular rheometers are no longer applicable to high solids content hydrolysate feedstocks. This study reported an on-site method using the torque meter equipped on hydrolysis reactors for its rheological property measurement. The measured torque data were transformed into the apparent viscosity and then the rheological parameters of the power law model after correlation. The determined parameters were applied to develop the computational fluid dynamics (CFD) model for simulating mixing efficiency and power consumption. This study provided a practical method for measurement of rheological parameters and design of optimal structure of large scale bioreactors of high solids content hydrolysis system.
Keywords: Cellulose; Rheology; Enzyme bioreactors; High solids content; On-site torque measurement; Computational fluid dynamics (CFD);

Batch experiments under different COD/NO3 -N ratios were carried out to investigate physicochemical characteristics and microbial community structure of granular sludge under the simultaneous denitrification and methanogenesis (SDM) process. COD/NO3 -N ratio of 8.0 was proved to be a critical point of the SDM process and sludge at this ratio was selected for analysis. BET, SEM, FTIR and zeta potential measurement were used to characterize the micro-structure, functional groups and surface charge of the granular sludge related to nitrate addition. SEM observation showed that rod-shaped bacteria were predominant at the surface of granules and FTIR spectrum (1745 cm−1) presented an evidence for the carboxyl group protonation upon reduction of the cytochrome c oxidase. Furthermore, high-throughput sequencing technology was used to analyze the microbial structure and diversity. Archaea was found to be accounted for 3.33% of the total microbial communities and Methanosaeta and Methanobacterium were the dominant archaeas. Otherwise, Proteobacteria (63.00%), Bacteroidetes (21.79%) and Firmicutes (9.73%) phyla were identified to be the three dominant bacterial communities. Enterobacteriaceae was detected with a content of 50.24% of the total bacterial sequences and might be the core bacterium contributed to the SDM process. The results would provide vital guidances for the design and stable operation of nitrate-containing wastewater treatment.
Keywords: Anaerobic processes; Biodegradation; Waste-water treatment; Microbial growth; Denitrification; Microbial community;

Pretreatment and upward liquid velocity effects over granulation in hydrogen producing EGSB reactors by Christian Daniela Bárcenas-Ruiz; Julián Carrillo-Reyes; Luis Arellano-García; Lourdes B. Celis; Felipe Alatriste-Mondragón; Elías Razo-Flores (75-84).
Display OmittedHydrogen production by fermentation with granular anaerobic biomass has been regarded as one alternative to obtain clean energy. However critical aspects such as process conditions to obtain hydrogenogenic granules have been scarcely investigated. In this work two inoculum pretreatments and different upward liquid velocities (ULV) in expanded granular sludge bed (EGSB) reactors were applied to determine the influence of these parameters on the formation, structure and specific hydrogenogenic activity (SHA) of the granules. Heat pretreated inoculum produced granules with greater manipulation resistance compared to those produced with wash-out pretreatment. Furthermore, the increase of ULV (2.5–4.5 m/h) generated bigger granules with a granule size distribution with a trend to a normal distribution and higher protein to carbohydrate ratios. Mass balances showed that propionate was the main metabolite, nonetheless, its production decreased substantially as the ULV applied increased, indicating a selective wash-out of propionate-producing bacteria. In the opposite way, SHA values were higher as the ULV applied increased as a result of selective enrichment and better mass transfer conditions. The whole results indicated that the inoculum pretreatment and hydrodynamic conditions play a key role in the formation, structure and in the biological properties of hydrogen-producing granules in EGSB reactors.
Keywords: Heat treatment; Hydrogen production; Hydrodynamics; Granular sludge; Upward liquid velocity; Wash-out;