Recent advances in methanol production from methanotrophs.

Methanol, the simplest aliphatic molecule of the alcohol family, finds diverse range of applications as an industrial solvent, a precursor for producing other chemicals (e.g., dimethyl ether, acetic acid and formaldehyde), and a potential fuel. There are conventional chemical routes for methanol production such as, steam reforming of natural gas to form syngas, followed by catalytic conversion into methanol; direct catalytic oxidation of methane, or hydrogenation of carbon dioxide. However, these chemical routes are limited by the requirement for expensive catalysts and extreme process conditions, and plausible environmental implications. Alternatively, methanotrophic microorganisms are being explored as biological alternative for methanol production, under milder process conditions, bypassing the requirement for chemical catalysts, and without imposing any adverse environmental impact. Methanotrophs possess inherent metabolic pathways for methanol production via biological methane oxidation or carbon dioxide reduction, thus offering a surplus advantage pertaining to the sequestration of two major greenhouse gases. This review sheds light on the recent advances in methanotrophic methanol production including metabolic pathways, feedstocks, metabolic engineering, and bioprocess engineering approaches. Furthermore, various reactor configurations are discussed in view of the challenges associated with solubility and mass transfer limitations in methanotrophic gas fermentation systems.

Process engineering strategy for improved methanol production in Methylosinus trichosporium through enhanced mass transfer and solubility of methane and carbon dioxide.

Methanol was produced in a two-stage integrated process using Methylosinus trichosporium NCIMB 11131. The first stage involved sequestration of methane to produce methanotrophic biomass, which was utilized as biocatalyst in the second stage to convert CO2 into methanol. A combinatorial process engineering approach of design of micro-sparger, engagement of draft tube, addition of mass transfer vector and elevation of reactor operating pressure was employed to enhance production of biomass and methanol. Maximum biomass titer of 7.68 g/L and productivity of 1.46 g/L d-1 were achieved in an airlift reactor equipped with a micro-sparger of 5 µm pore size, in the presence of draft tube and 10 % v/v silicone oil, as mass transfer vector. Maximum methane fixation rate was estimated to be 0.80 g/L d-1. Maximum methanol titer of 1.98 g/L was achieved under an elevated operating pressure of 4 bar in a high-pressure stirred tank reactor.

Enhanced production of Bacopa saponins by repeated batch strategy in bioreactor.

Cultivation of cell suspension culture of Bacopa monnieri targeting the production of bacosides was explored in a 5-l stirred tank reactor using statistically optimized conditions. The bioreactor cultivation conditions were modified and this led to profuse biomass growth (2.81 ± 0.20 g/l) and total bacosides (1.26 ± 0.23 mg/g in cells and 0.60 ± 0.11 mg/l in fermenter broth) production in 9 days. The values of static volumetric mass transfer coefficient (kLa), dimensionless mixing time (Nm) were measured in the bioreactor. The culture grew efficiently and produced enhanced amount of bacoside A (5.59 ± 0.41 mg/g total bacosides in cells and 3.12 ± 0.13 mg/l in the fermenter broth) using one cycle of repeated batch strategy adopted in the bioreactor for 15 days. The intracellular concentration of bacoside A3 (1.18 ± 0.11 mg/g), bacopaside II (2.09 ± 0.35 mg/g), bacopaside X (0.79 ± 0.17 mg/g) and bacopasaponin C (2.24 ± 0.23 mg/g) were significantly higher in repeated batch as compared to batch bioreactor cultivation. The yield of total bacosides in the fermenter broth was 5-times higher in repeated batch as compared to batch cultivation. This strategy can be helpful for the enhanced production of other valuable triterpenoid saponins.

Two-stage integrated process for bio-methanol production coupled with methane and carbon dioxide sequestration: Kinetic modelling and experimental validation.

The study demonstrates a two-stage integrated process for bio-methanol production using Methylosinus trichosporium NCIMB 11131, coupled with sequestration of methane and carbon dioxide. The first stage involved generation of methanotrophic biomass via sequestration of methane; which was used as biocatalyst to reduce carbon dioxide into methanol in the second stage. Maximum biomass titer of 3.39 g L-1 and productivity of 0.60 g L-1 d-1 were achieved in semi-batch stirred tank reactor with methane concentration in the inlet gas mixture of 2.5% v/v and gas flow rate of 0.5 vvm. Methane fixation rate was estimated to be 0.32 g L-1 d-1. Maximum methanol titer of 0.58 g L-1 was achieved at headspace carbon dioxide concentration of 50% v/v and liquid to headspace volume ratio 10:90. Subsequently, a kinetic model was developed to predict and understand the system behaviour in terms of dynamic profile of growth, methanol formation, concentration of dissolved methane or carbon dioxide in the aqueous phase and headspace carbon dioxide concentration, in response to varying process parameters. The model can serve as a tool for estimation of process parameters and aid in overall production optimization.

Biotransformation of Methane and Carbon Dioxide Into High-Value Products by Methanotrophs: Current State of Art and Future Prospects.

Conventional chemical methods to transform methane and carbon dioxide into useful chemicals are plagued by the requirement for extreme operating conditions and expensive catalysts. Exploitation of microorganisms as biocatalysts is an attractive alternative to sequester these C1 compounds and convert them into value-added chemicals through their inherent metabolic pathways. Microbial biocatalysts are advantageous over chemical processes as they require mild-operating conditions and do not release any toxic by-products. Methanotrophs are potential cell-factories for synthesizing a wide range of high-value products via utilizing methane as the sole source of carbon and energy, and hence, serve as excellent candidate for methane sequestration. Besides, methanotrophs are capable of capturing carbon dioxide and enzymatically hydrogenating it into methanol, and hence qualify to be suitable candidates for carbon dioxide sequestration. However, large-scale production of value-added products from methanotrophs still presents an overwhelming challenge, due to gas-liquid mass transfer limitations, low solubility of gases in liquid medium and low titer of products. This requires design and engineering of efficient reactors for scale-up of the process. The present review offers an overview of the metabolic architecture of methanotrophs and the range of product portfolio they can offer. Special emphasis is given on methanol biosynthesis as a potential biofuel molecule, through utilization of methane and alternate pathway of carbon dioxide sequestration. In view of the gas-liquid mass transfer and low solubility of gases, the key rate-limiting step in gas fermentation, emphasis is given toward reactor design consideration essential to achieve better process performance.

Statistical optimization of bacoside A biosynthesis in plant cell suspension cultures using response surface methodology.

The in vitro cultures of Bacopa monnieri show poor production of the anti-Alzheimer's drug, bacoside A. Therefore, suitable bioprocess optimization strategy was developed for callus induction from leaf explants (30 days), followed by callus proliferation (15 days). Central Composite Design was implemented to analyze the effect of pH, photoperiod, naphthalene acetic acid (NAA), and benzylaminopurine (BAP) concentration for maximum biosynthesis of bacoside A using leaf explants as well as callus explants as the inoculum. Using the CCD responses, it was predicted that the best biomass concentration of 4.56 ± 0.53 g/l DW and bacoside A production of 14.04 ± 1.31 mg/g DW can be obtained using 5.4 pH, 18 h/6 h L/D photoperiod regime, and 1.2 mg/l BAP in combination with 0.2 mg/l NAA. The kinetic parameter values for maximum specific growth rate (0.16/day), saturation constant (7.35 g/l), inhibition constant (120 g/l), biomass yield (0.011 g/g), maintenance coefficient (0.02 g/g/day), and growth-associated (0.627 mg/g) and non-growth-associated (1.096 mg/g/day) bacoside A formation constants were determined experimentally in batch cultures using optimized conditions.