The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg 2؉ or Mn 2؉ , strongly inhibited by Ca 2؉ , and independent of protein oligomerization. Linear dimeric GMP was shown to be 5pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.

This review describes the recent results in hydrothermal liquefaction (HTL) of biomass in continuous-flow processing systems. Although much has been published about batch reactor tests of biomass HTL, there is only limited information yet available on continuous-flow tests, which can provide a more reasonable basis for process design and scale-up for commercialization. High-moisture biomass feedstocks are the most likely to be used in HTL. These materials are described and results of their processing are discussed. Engineered systems for HTL are described; however, they are of limited size and do not yet approach a demonstration scale of operation. With the results available, process models have been developed, and mass and energy balances determined. From these models, process costs have been calculated and provide some optimism as to the commercial likelihood of the technology.

Off-gas 10% of total Carbon AHTL Oil 72% of total Carbon (77% algal carbon recovery) Natural Gas 3.5% of total Carbon in Water & Solids Recycle to Ponds 8% of total Carbon as dissolved CO2 (9% of algal carbon) Reformer & Heater Exhaust 23% of total Carbon (Includes 21% of algal carbon) Natural Gas Drier & Exhaust 3.5% of total Carbon in

Wet algae slurries can be converted into an upgradeable biocrude by hydrothermal liquefaction (HTL). High levels of carbon conversion to gravity separable biocrude product were accomplished at relatively low temperature (350°C) in a continuous-flow, pressurized (sub-critical liquid water) environment (20 MPa). As opposed to earlier work in batch reactors reported by others, direct oil recovery was achieved without the use of a solvent and biomass trace components were removed by processing steps so that they did not cause process difficulties. High conversions were obtained even with high slurry concentrations of up to 35 wt.% of dry solids. Catalytic hydrotreating was effectively applied for hydrodeoxygenation, hydrodenitrogenation, and hydrodesulfurization of the biocrude to form liquid hydrocarbon fuel. Catalytic hydrothermal gasification was effectively applied for HTL byproduct water cleanup and fuel gas production from water soluble organics, allowing the water to be considered for recycle of nutrients to the algae growth ponds. As a result, high conversion of algae to liquid hydrocarbon and gas products was found with low levels of organic contamination in the byproduct water. All three process steps were accomplished in bench-scale, continuous-flow reactor systems such that design data for process scale-up was generated.

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