Freshwater diatoms as a source of lipids for biofuels.

Until recently, biodiesel production has been derived from terrestrial plants such as soybean and canola, leading to competition between biodiesel production and agricultural production for source materials. Microalgae have the potential to synthesize 30 times more oil per hectare than terrestrial plants without competing for agricultural land. We examined four genera (Cyclotella, Aulacoseira, Fragilaria, Synedra) of common freshwater diatoms (Bacillariophyceae) for growth and lipid content in defined medium (sD11) that replicates hypereutrophic conditions in lakes and wastewater treatment plant effluents and optimized the medium for silicon content. Cyclotella and Aulacoseira produced the highest levels of total lipids, 60 and 43 µg total lipids/ml, respectively. Both diatoms are rich in fatty acids C14, C16, C16:1, C16:2,7,10, and C22:5n3. Of the diatoms examined, Cyclotella reached the highest population density (>2.5 × 10(6) cells/ml) in stationary phase when many of the cells appeared to be filled entirely with oil. Silicon enrichment studies indicated that for optimal utilization of phosphorus and nitrogen by diatoms growing in wastewater effluent, the amount of silicon present or added to the effluent should be 17.5 times the mass of phosphorus in the effluent. With high growth rates, high lipid contents, and rapid settling rates, Cyclotella and Aulacoseira are candidates for biodiesel production.

Membrane stresses induced by overproduction of free fatty acids in Escherichia coli.

Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.

Bacterial production of free fatty acids from freshwater macroalgal cellulose.

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 µM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 µg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.

Isolation of improved free fatty acid overproducing strains of Escherichia coli via Nile red based high-throughput screening.

Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially-produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose. Strains with increased titers can be obtained through both rational and random approaches. While powerful, random approaches require a genetic selection or facile screen that is amenable to high throughput platforms. Here, we present the use of a high throughput screen for fatty acids based on the hydrophobic dye Nile red. The method was applied to screening a transposon library of a free fatty acid overproducing strain of Escherichia coli in search of high producing mutants. Ten gene targets were identified via primary and secondary screening. A strain comprising a clean knockout of one of the identified genes led to a 20% increase in titer over the baseline strain. A selection strategy that combines these findings and can act in an iterative fashion has been developed and can be used for future strain optimization in hydrocarbon producing strains.