Environmental Technologies for Contaminated Solids, Soils and Sediments
4th cohort

Nirakar Pradhan

Hydrogen and lactic acid synthesis through capnophilic lactic fermentation by Thermotoga neapolitana

The environmental impact of excessive exploitation of fossil fuel reserves has inspired the innovation of several sustainable neo-carbon-neutral technologies. To that end, the biological processes like fermentation may be leveraged to bioconvert carbohydrate-rich feedstocks to fuels like hydrogen (H2) or commercially valuable organic acids like lactic acid. This research work investigated the engineering techniques for improving simultaneous synthesis of H2 and lactic acid under capnophilic (CO2-dependent) lactic fermentation (CLF) conditions by a lab strain of Thermotoga neapolitana.  Primarily, the genotypic comparison between the lab strain and the wild-type revealed DNA homology of 88.1 (± 2.4)%. Genotyping by RiboPrint® and matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses showed a genetic differentiation beyond subspecies level, hence the lab strain was proposed as a new subspecies, T. neapolitana subsp. lactica. The lab strain produced 10-90% more lactic acid, based on the phenotypic characterization, than the wild-type strain under similar operating conditions without impairing the H2 yield.  The lab strain was then studied to optimize the growth conditions as well as to estimate the growth kinetic parameters. A new kinetic model based on the dark fermentation (DF) principles and the Monod-like mathematical expressions were developed to enable the simulation of biomass growth, substrate consumption and product formation. The model failed to estimate acetic and lactic acid accurately, as the DF model did not consider the carboxylation of acetic acid to lactic acid by the pyruvate:ferredoxin oxidoreductase (PFOR) enzyme under CLF conditions. The model was then incorporated with the CLF mechanism and the kinetic parameters were recalibrated.  The kinetic parameters, i.e. maximum specific uptake rate (k), semi-saturation constant (kS), biomass yield coefficient (Y) and endogenous decay rate (kd) were 1.30 1/h, 1.42 g/L, 0.12 and 0.02 1/h, respectively, under CLF conditions. Interestingly, the new CLF-based model perfectly fitted the experimental results and estimated that about 40-80% of the lactic acid production is attributed to the recycling of acetic acid and CO2. In addition, the adsorption of lactic acid by activated carbon and anionic polymeric resins was successfully applied as a downstream processing technique for the recovery of lactic acid from a model T. neapolitana fermentation broth. This research work serves as a practical milestone in the field of microbial fermentation with a scope for wider scientific applications, including the development of bio-based renewable energy and industrial lactic acid production.