Guhan Jayaraman

Lactococcus lactis is a promising host for pathway-engineered production of high molecular weight hyaluronic acid (HA). This work investigates the effect of hemin-supplemented respiratory metabolism in an engineered L. lactis ldh mutant strain (Δldh L. lactis) as a strategy to enhance HA titer in the batch fermentation process. L. lactis lacks a functional electron transport chain (ETC) and ultimately relies on substrate-level phosphorylation for energy production. We conducted detailed investigations under different dissolved oxygen (DO) conditions and found a steady increase in HA titer with increased DO levels. We found a strong correlation between cofactor availability (NAD+ and acetyl-CoA) and the intracellular concentration of HA precursors, UDP-GlcUA and UDP-GlcNAc. We correlated the intracellular precursor levels with the HA titer obtained for the four conditions. We found that these were higher in hemin-supplemented cultures, with the best HA titer of 4.6 g/l. Finally, we observed that the molecular weight of HA (MWHA) correlates strongly with the ratio of HA precursors concentrations. We also found an inverse correlation between MWHA and HA titer across these experiments. These results can be used in optimizing the trade-off between molecular weight and HA production in bioprocesses involving recombinant L. lactis cultures.

Background: Simultaneous saccharification and fermentation (SSF) of pre-treated lignocellulosics to biofuels and other platform chemicals has long been a promising alternative to separate hydrolysis and fermentation processes. However, the disparity between the optimum conditions (temperature, pH) for fermentation and enzyme hydrolysis leads to execution of the SSF process at sub-optimal conditions, which can affect the rate of hydrolysis and cellulose conversion. The fermentation conditions could be synchronized with hydrolysis optima by carrying out the SSF at a higher temperature, but this would require a thermo-tolerant organism. Economically viable production of platform chemicals from lignocellulosic biomass (LCB) has long been stymied because of the significantly higher cost of hydrolytic enzymes. The major objective of this work is to develop an SSF strategy for D-lactic acid (D-LA) production by a thermo-tolerant organism, in which the enzyme loading could significantly be reduced without compromising on the overall conversion. Results: A thermo-tolerant strain of Lactobacillus bulgaricus was developed by adaptive laboratory evolution (ALE) which enabled the SSF to be performed at 45 °C with reduced enzyme usage. Despite the reduction of enzyme loading from 15 Filter Paper Unit/gLCB (FPU/gLCB) to 5 FPU/gLCB, we could still achieve ~ 8% higher cellulose to D-LA conversion in batch SSF, in comparison to the conversion by separate enzymatic hydrolysis and fermentation processes at 45 °C and pH 5.5. Extending the batch SSF to SSF with pulse-feeding of 5% pre-treated biomass and 5 FPU/gLCB, at 12-h intervals (36th–96th h), resulted in a titer of 108 g/L D-LA and 60% conversion of cellulose to D-LA. This is one among the highest reported D-LA titers achieved from LCB. Conclusions: We have demonstrated that the SSF strategy, in conjunction with evolutionary engineering, could drastically reduce enzyme requirement and be the way forward for economical production of platform chemicals from lignocellulosics. We have shown that fed-batch SSF processes, designed with multiple pulse-feedings of the pre-treated biomass and enzyme, can be an effective way of enhancing the product concentrations.

This study investigates the behavior and intracellular changes in Escherichia coli (model organism) during electro-oxidation with Ti/Sb–SnO2/PbO2 anode in a chlorine free electrochemical system. Preliminary studies were conducted to understand the effect of initial E. coli concentration and applied current density on disinfection. At an applied current density 30 mA cm−2, 7 log reduction of E. coli was achieved in 75 min. The role of reactive oxygen species’ (ROS) in E.coli disinfection was evaluated, which confirmed hydroxyl (•OH) radical as the predominant ROS in electro-oxidation. Observations were carried out at cell and molecular level to understand E.coli inactivation mechanism. Scanning electron microscopy images confirmed oxidative damage of the cell wall and irreversible cell death. Intracellular and extracellular protein quantification and genetic material release further confirmed cell component leakage due to cell wall rupture and degradation due to •OH radical interaction. Change in cell membrane potential suggests the colloidal nature of E. coli cells under applied current density. Plasmid deoxyribonucleic acid degradation study confirmed fragmentation and degradation of released genetic material. Overall, effective disinfection could be achieved by electro-oxidation, which ensures effective inactivation and prevents regrowth of E. coli. Disinfection of real wastewater was achieved in 12 min at an applied current density 30 mA cm−2. Real wastewater study further confirmed that effective disinfection is possible with a low cost electrode material such as Ti/Sb–SnO2/PbO2. Energy consumed during disinfection was determined to be 4.978 kWh m−3 for real wastewater disinfection at applied current density 30 mA cm−2. Cost of operation was estimated and stability of the electrode was studied to evaluate the feasibility of large scale operation. Relatively low energy and less disinfection time makes this technology suitable for field scale applications.

Microsphere hydroxyapatite (HAp) is widely used in various biomedical and chromatographic applications. The work described in this manuscript focuses on a dissolution precipitation method for production of HAp microspheres. This method overcomes certain drawbacks of conventional preparation methods used for HAp preparation, which produce polydisperse particles and are time-consuming and expensive. In the present work, the calcium carbonate (calcite) particles were directly and rapidly converted into HAp microspheres using an inexpensive dissolution precipitation method. The effects of the reaction temperature, time, and mechanical stirring rates were studied, and the reaction parameters were optimized. As confirmed by the XRD studies, the higher reaction temperature and time promote complete HAp conversion, while calcite residues were observed for lower temperatures and times. SEM images show the influence of reaction parameters on the surface microstructure of the microspheres produced. It was observed that the HAp microspheres undergo disintegration at a higher stirring rate. The reaction parameters optimized in this work were ideal for preparing HAp microspheres. The resultant HAp particles were utilized as matrices for chromatographic separation of protein mixtures.

The development of reliable kinetic models of bioprocesses from data is a challenging task. In this work, a systematic approach to develop a kinetic model of bioprocess involving single biomass from concentration data is proposed without imposing any kinetic model a priori. The proposed incremental model identification approach decomposes the model-building task into a set of sub-tasks such as determining the yield coefficients and maintenance coefficient, specific growth rate structure identification, and parameter estimation. It is shown that the proposed approach allows identifying the mechanism of product formation. The proposed approach is applied to the microbial production of Hyaluronic acid (HA), an important biopolymer, using a recombinant Lactococcus lactis MKG6. An unstructured kinetic model is developed for the HA production from data. It is shown that HA production is a growth-associated process. Further, the specific growth rate of HA production is identified from a set of rate candidates. It is revealed that the specific growth rate in the HA production follows the non-competitive HA inhibition model. The parameters obtained by the incremental identification are further refined to obtain statistically optimal estimates using the simultaneous model identification. Validation of the identified kinetic model of HA production on new experimental data shows that the proposed approach leads to a reliable kinetic model with the optimal parameter estimates.

Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110–130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175–190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

Hydroxyapatite ((Ca10(PO4)6(OH)2, HAp) based chromatography matrix has attracted great interest in the field of protein separation. However, researchers have been trying to combat the growing costs associated with the HAp matrix. In the present investigation, we utilized a cheap biological waste material, viz. eggshells, for the development of hydroxyapatite (HAp) resins and evaluated them for protein purification. Initially, the calcite of the eggshell carbonate was converted into metastable vaterite microspheres. The HAp microspheres (ECHAp) were then prepared from eggshell carbonate microspheres using a novel dissolution-precipitation process. Synthetic source calcium carbonate was also used to prepare HAp microspheres (CHAp) for comparison. The purity and morphology of the apatite microspheres were characterized using X-ray diffraction (XRD) method, Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and laser diffraction particle analysis. Although both the apatites have similar morphology, the ECHAp has a larger surface area of 33.8 m2 g−1 compared to CHAp of 17.27 m2 g−1 by surface area analysis method. A commercial HAp matrix (CHT) with similar properties was also studied for comparison. All the apatite microspheres were found to have a similar protein binding capacity for bovine serum albumin (BSA). But ECHAp showed better protein separation for BSA and lysozyme mixture compared to CHAp and CHT matrices. The ECHAp matrix was also found to be highly stable over 20 purification cycles. Hence, the eggshell waste seems to have the potential for HAp matrix by a novel carbonate route with ease of preparation and also an economical packing material for chromatographic purification of biomolecules.