Projected shortages of global phosphate have prompted investigation of methods that could be employed to capture and recycle phosphate, rather than continue to allow the resource to be essentially irreversibly lost through dilution in surface waters. Hydrothermal carbonization of animal manures from large farms was investigated as a scenario for the reclamation of phosphate for agricultural use and mitigation of the negative environmental impact of phosphate pollution. Hydrothermal reaction conditions were identified for poultry, swine, and cattle manures that resulted in hydrochar yields of 50-60% for all three manures, and >90% of the total phosphorus present in these systems was contained in the hydrochars as precipitated phosphate salts. Phosphate recovery was achieved in yields of 80-90% by subsequent acid treatment of the hydrochars, addition of base to acid extracts to achieve a pH of 9, and filtration of principally calcium phosphate. Phosphate recovery was achieved in yields of 81-87% based on starting manures by subsequent acid treatment of the hydrochars, addition of base to acid extracts to achieve a pH of 9, and filtration of principally calcium phosphate. Swine and cattle manures produced hydrochars with combustion energy contents comparable to those of high-end sub-bituminous coals.

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.

The conversion of branched-chain amino acids to branched-chain acids or alcohols is an important aspect of flavor in the food industry and is dependent on the Ehrlich pathway found in certain lactic acid bacteria. A key enzyme in the pathway, the 2-keto acid decarboxylase (KDC), is also of interest in biotechnology applications to produce small branched-chain alcohols that might serve as improved biofuels or other commodity feedstocks. This enzyme has been extensively studied in the model bacterium Lactococcus lactis, but is also found in other bacteria and higher organisms. In this report, distinct homologs of the L. lactis KDC originally annotated as pyruvate decarboxylases from Psychrobacter cryohalolentis K5 and P. arcticus 273-4 were cloned and characterized, confirming a related activity toward specific branched-chain 2-keto acids derived from branched-chain amino acids. Further, KDC activity was confirmed in intact cells and cell-free extracts of P. cryohalolentis K5 grown on both rich and defined media, indicating that the Ehrlich pathway may also be utilized in some psychrotrophs and psychrophiles. A comparison of the similarities and differences in the P. cryohalolentis K5 and P. arcticus 273-4 KDC activities to other bacterial KDCs is presented.