Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale.

Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization.

Oleaginous fungus Mortierella isabellina showed excellent lipid conversion on non-detoxified lignocellulosic hydrolysate. This study investigated the effects of inhibitory compounds (furfural, hydroxymethylfurfural, and ferulic and coumaric acids) in lignocellulosic hydrolysate on M. isabellina growth and lipid production. M. isabellina can tolerate furfural (∼1 g/L), hydroxymethylfurfural (∼2.5 g/L), ferulic (∼0.5 g/L) and coumaric acid (∼0.5 g/L) with normal growth rates. Synergistic effect of these inhibitors (2 g/L furfural, 0.4 g/L hydroxymethylfurfural, 0.02 g/L ferulic acid and 0.02 g/L coumaric acid) moderately reduces total fungal growth (by 28%), while the presence of these inhibitors has minor impact on cell lipid contents and lipid profiles. In the presence of inhibitory compounds, (13)C-tracing has revealed that M. isabellina can simultaneously utilize glucose and acetate, and acetate is mainly assimilated for synthesis of lipid and TCA cycle amino acids. The results also demonstrate that glucose has strong catabolite repression for xylose utilization for biomass and lipid production in the presence of inhibitors.

A self-sustaining advanced lignocellulosic biofuel production by integration of anaerobic digestion and aerobic fungal fermentation.

High energy demand hinders the development and application of aerobic microbial biofuel production from lignocellulosic materials. In order to address this issue, this study focused on developing an integrated system including anaerobic digestion and aerobic fungal fermentation to convert corn stover, animal manure and food wastes into microbial lipids for biodiesel production. Dairy manure and food waste were first anaerobically digested to produce energy and solid digestate (AD fiber). AD fiber and corn stover were then processed by a combined alkali and acid hydrolysis, followed by fungal lipid accumulation. The integrated process can generate 1L biodiesel and 1.9 kg methane from 12.8 kg dry dairy manure, 3.1 kg dry food wastes and 12.2 kg dry corn stover with a positive net energy of 57 MJ, which concludes a self-sustaining lignocellulosic biodiesel process and provides a new route to co-utilize corn stover and organic wastes for advanced biofuel production.

Lipid profiling and corresponding biodiesel quality of Mortierella isabellina using different drying and extraction methods.

Four lipid extraction methods (Bligh & Dyer, hexane & isopropanol, dichloromethane & methanol, and hexane) were evaluated to extract lipid from freeze- and oven-dried fungus Mortierella isabellina ATCC42613. The highest lipid yield (41.8%) was obtained from Bligh & Dyer extraction on the oven-dried fungal biomass with a methanol:chloroform:water ratio of 2:1:0.8. Other lipid extraction methods on both freeze- and oven-dried samples had lipid yields ranging from 20.7% to 35.9%. Non-polar lipid was the main lipid class (more than 90% of total lipid) in M. isabellina. Regarding fatty acid profile, there was no significant difference on fatty acid concentration between different drying and extraction methods. Estimation of biodiesel fuel properties using correlative models further demonstrated that the fungal biodiesel is a good alternative to fossil diesel.

Oleaginous fungal lipid fermentation on combined acid- and alkali-pretreated corn stover hydrolysate for advanced biofuel production.

A combined hydrolysis process, which first mixed dilute acid- and alkali-pretreated corn stover at a 1:1 (w/w) ratio, directly followed by enzymatic saccharification without pH adjustment, has been developed in this study in order to minimize the need of neutralization, detoxification, and washing during the process of lignocellulosic biofuel production. The oleaginous fungus Mortierella isabellina was selected and applied to the combined hydrolysate as well as a synthetic medium to compare fungal lipid accumulation and biodiesel production in both shake flask and 7.5L fermentor. Fungal cultivation on combined hydrolysate exhibited comparable cell mass and lipid yield with those from synthetic medium, indicating that the integration of combined hydrolysis with oleaginous fungal lipid fermentation has great potential to improve performance of advanced lignocellulosic biofuel production.

Fungal cellulase/xylanase production and corresponding hydrolysis using pretreated corn stover as substrates.

Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey's statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.

Integration of sewage sludge digestion with advanced biofuel synthesis.

Sewage sludge rich in carbohydrates and other nutrients could be a good feedstock for fuel/chemical production. In this study, fungal and engineered bacterial cultivations were integrated with a modified anaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was first adjusted to enable acetogenic bacteria to accumulate acetate. A fungus (Mortierella isabellina) and an engineered bacterium (Escherichia coli created by optimizing acetate utilization and fatty acid biosynthesis as well as overexpressing a regulatory transcription factor fadR) were then cultured on the acetate solution to accumulate fatty acids. The engineered bacterium had higher fatty acid yield and titer than the fungus. Both medium- and long-chain fatty acids (C12:0-C18:0) were produced by the engineered bacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0-C18:3). This study demonstrated a potential path that combines fungus or engineered bacterium with anaerobic digestion to achieve simultaneous organic waste treatment and advanced biofuel production.

Co-hydrolysis of lignocellulosic biomass for microbial lipid accumulation.

The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co-hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid-solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co-hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g(-1) dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g(-1) dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g(-1) dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g(-1) dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co-hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co-hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co-hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co-hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production.

Evaluation of lipid accumulation from lignocellulosic sugars by Mortierella isabellina for biodiesel production.

The filamentous fungus Mortierella isabellina ATCC42613 was used to assess the conversion of different carbon sources (glucose, xylose, mixed glucose/xylose, acid and alkali treated corn stover hydrolysate) in submerged media to lipid. Glucose and xylose cultures composed of varying initial sugar concentrations (28.1-91.7gL(-1), and 26.6-90.9gL(-1) respectively) showed a positive correlation to lipid accumulation, with significant quantities occurring at the upper limit of the substrate range (10.2, and 8.8gL(-1) lipid respectively). While lipid concentrations increased with each incremental glucose and xylose level, the lipid yield (0.41-0.44, and 0.39-0.43gg(-1) cell mass respectively), and intracellular fatty acid composition remained relatively constant. Additionally, sulfuric acid hydrolysate, without detoxification, exhibited greater cell mass, and equivalent lipid production compared to synthetic medium with similar initial glucose and xylose concentrations. These results elucidate the potential of utilizing filamentous fungal fermentation to accumulate lipids from lignocellulosic biomass for biodiesel production.