Lawrence Berkeley National Laboratory

The Protein Complex Analysis Project (PCAP, http://pcap.lbl.gov/), focuses on high-throughput analysis of microbial protein complexes in the anaerobic, sulfate-reducing organism, DesulfovibriovulgarisHildenborough(DvH).Interest in DvHas a model organism for bioremediation of contaminated groundwater sites arises from its ability to reduce heavy metals. D. vulgarishas been isolated from contaminated groundwater of sites in the DOE complex. To understand the effect of environmental changes on the organism, midlog-phase cultures are exposed to nitrate and salt stresses (at the minimum inhibitory concentration, which reduces growth rates by 50percent), and compared to controls of cultures at midlogand stationary phases. Large volumes of culture of consistent quality (up to 100 liters) are needed because of the relatively low cell density of DvHcultures (one order of magnitude lower than E. coli, for example) and PCAP's challenge to characterize low-abundance membrane proteins. Cultures are grown in continuous flow stirred tank reactors (CFSTRs) to produce consistent cell densities. Stressor is added to the outflow from the CFSTR, and the mixture is pumped through a plug flow reactor (PFR), to provide a stress exposure time of 2 hours. Effluent is chilled and held in large carboys until it is centrifuged. A variety of analyses -- including metabolites, total proteins, cell density and phospholipidfatty-acids -- track culture consistency within a production run, and differences due to stress exposure and growth phase for the different conditions used. With our system we are able to produce the requisite 100 L of culture for a given condition within a week.