Gordon V. Wolfe

Grazing-activated chemical defence in a unicellular marine alga

Marine plankton use a variety of defences against predators, some of which affect trophic structure and biogeochemistry. We have previously shown that, during grazing by the protozoan Oxyrrhis marina on the alga Emiliania huxleyi, dimethylsulphoniopropionate (DMSP) from the prey is converted to dimethyl sulphide (DMS) when lysis of ingested prey cells initiates mixing of algal DMSP and the enzyme DMSP lyase. Such a mechanism is similar to macrophyte defence reactions,. Here we show that this reaction deters protozoan herbivores, presumably through the production of highly concentrated acrylate, which has antimicrobial activity. Protozoan predators differ in their ability to ingest and survive on prey with high-activity DMSP lyase, but all grazers preferentially select strains with low enzyme activity when offered prey mixtures. This defence system involves investment in a chemical precursor, DMSP, which is not self-toxic and has other useful metabolic functions. We believe this is the first report of grazing-activated chemical defence in unicellular microorganisms.

The cycling of sulfur in surface seawater of the northeast Pacific

Oceanic dimethylsulfide (DMS) emissions to the atmosphere are potentially important to the Earths radiative balance. Since these emissions are driven by the surface seawater concentration of DMS, it is important to understand the processes controlling the cycling of sulfur in surface seawater. During the third Pacific Sulfur/Stratus Investigation (PSI-3, April 1991) we measured the major sulfur reservoirs (total organic sulfur, total low molecular weight organic sulfur, ester sulfate, protein sulfur, dimethylsulfoniopropionate (DMSP), DMS, dimethylsulfoxide) and quantified many of the processes that cycle sulfur through the upper water column (sulfate assimilation, DMSP consumption, DMS production and consumption, air-sea exchange of DMS, loss of organic sulfur by particulate sinking). Under conditions of low plankton biomass ( 8 μM nitrate), 250 km off the Washington State coast, DMSP and DMS were 22% and 0.9%, respectively, of the total particulate organic sulfur pool. DMS production from the enzymatic cleavage of DMSP accounted for 29% of the total sulfate assimilation. However, only 0.3% of sulfate-S assimilated was released to the atmosphere. From these data it is evident that air-sea exchange is currently only a minor sink in the seawater sulfur cycle and thus there is the potential for much higher DMS emissions under different climatic conditions.

PRODUCTION AND CELLULAR LOCALIZATION OF NEUTRAL LONG-CHAIN LIPIDS IN THE HAPTOPHYTE ALGAE ISOCHRYSIS GALBANA AND EMILIANIA HUXLEYI1

Isochrysis galbana Parke, Emiliania huxleyi (Lohm.) Hay and Mohler, and some related prymnesiophyte algae produce as neutral lipids a set of polyunsaturated long‐chain (C37–39) alkenones, alkenoates, and alkenes (PULCA). These biomarkers are widely used for paleothermometry, but the biosynthesis and cellular location of these unique lipids remain largely unknown. By staining with the fluorescent lipophilic dye Nile Red, we found that I. galbana and E. huxleyi, like many other algae, package their neutral lipid into cytoplasmic vesicles or lipid bodies. We found that these lipid bodies increase in abundance under nutrient limitation and disappear under prolonged darkness and show that this pattern correlates well with the concentration of PULCA as measured by TLC. In addition, we show that lipid vesicles purified by sucrose density gradient centrifugation consist predominantly of PULCA. We also found significant pools of neutral lipid associated with chloroplasts, and PULCA component profiles in lipid vesicles and chloroplasts are similar. Examination of cell ultrastructure shows conspicuous cytoplasmic and chloroplast lipid bodies, and we suggest that PULCA may be synthesized in chloroplasts and then exported to cytoplasmic lipid bodies for storage and eventual metabolism. Our results connect and extend prior observations of lipid bodies and membrane‐unbound PULCA in I. galbana and E. huxleyi, as well as the behavior of PULCA during nutrient and light stress.

Physiological impacts on alkenone paleothermometry

We conducted isothermal (15°C) batch culture experiments with the coccolithophorid Emiliania huxleyi (strain NEPCC 55a) to evaluate the extent to which nutrient and light stress contribute to variability in the alkenone unsaturation index U37K′. Alkenone content and composition were constant throughout exponential growth in both experiments when nutrients (nitrate and orthophosphate) were replete. Stationary phase (nutrient-starved) cells continued to produce alkenones, amassing concentrations (ΣAlk) ≥ 3 times higher than those dividing exponentially (1.5–2 pg cell−1), and the U37K′ of “excess” alkenone dropped by 0.11 units. In contrast, 5 days of continuous darkness resulted in a 75% decrease in cellular ΣAlk and a significant U37K′ increase (+0.11 units). Given an established 0.034 unit/°C response for exponentially growing cells of this strain, the observed range of U37K′ variability at 15°C corresponds to an uncertainty of ±3.2°C in predicted growth temperature. This level of variability matches that of the global U37K′ annual mean sea surface temperature calibration for surface marine sediments, begging the question: What is the physiological condition of alkenone-producing cells exported to marine sediments? Comparison of our laboratory results for a strain of E. huxleyi isolated from the subarctic Pacific Ocean with depth profiles for alkenones in surface waters from two contrasting sites in the northeast Pacific Ocean suggests that the answer to this question depends on the ocean regime considered, a possibility with significant bearing on how stratigraphic U37K′ records in marine sediments are to be interpreted paleoceanographically.

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor.

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with 14C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to 14CO2 by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.

Protist genetic diversity in the acidic hydrothermal environments of Lassen Volcanic National Park, USA

ABSTRACT. We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devils Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85°C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68°C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%–99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68°C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassens acidic hydrothermal features may harbor novel protists.

Comparison of Rhizosphere Bacterial Communities in Arabidopsis thaliana Mutants for Systemic Acquired Resistance

Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a β-glucoronidase (GUS) reporter construct driven by the β-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon–Weiner, and Simpson’s diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.

Cation distribution, cycling, and removal from mineral soil in Douglas-fir and red alder forests

Overstory species influence the distribution and dynamics of nutrients in forest ecosystems. Ecosystem-level estimates of Ca, Mg, and K pools and cycles in 50-year old Douglas-fir and red alder stands were used to determine the effect of overstory composition on net cation removal from the mineral soil, i.e. cation export from the soil in excess of additions. Net cation removal from Douglas-fir soil was 8 kg Ca ha−1 yr−1, 1 kg Mg ha−1 yr−1, and 0.3 kg K ha−1 yr−1. Annual cation export from soil by uptake and accumulation in live woody tissue and O horizon was of similar magnitude to leaching in soil solution. Atmospheric deposition partially off-set export by adding cations equivalent to 28–88% of cation export. Net cation removal from red alder soil was 58 kg Ca ha−1 yr−1, 9 kg Mg ha−1 yr−1, and 11 kg K ha−1 yr−1. Annual cation accumulation in live woody tissue and O horizon was three times greater than in Douglas-fir, while cation leaching in soil solution was five to eight times greater. The lack of excessive depletion of exchangeable cations in the red alder soil suggests that mineral weathering, rather than exchangeable cations, was the source of most of the removed cations. Nitric acid generated during nitrification in red alder soil led to high rates of weathering and NO3-driven cation leaching.

Accumulation of Dissolved DMSP by Marine Bacteria and its Degradation via Bacterivory

Several bacterial isolates enriched from seawater using complex media were able to accumulate dimethylsulfoniopropionate (DMSP) from media into cells over several hours without degrading it. Uptake only occurred in metabolically active cells, and was repressed in some strains by the presence of additional carbon sources. Accumulation was also more rapid in osmotically-stressed cells, suggesting DMSP is used as an osmotic solute. Uptake could be blocked by inhibitors of active transport systems (2,4-dinitrophenol, azide, arsenate) and of protein synthesis (chloramphenicol). Some structural analogs such as glycine betaine and S-methyl methionine also blocked DMSP uptake, suggesting that the availability of alternate organic osmolytes may influence DMSP uptake. Stresses such as freezing, heating, or osmotic down shock resulted in partial release of DMSP back to the medium. One strain which contained a DMSP-lyase was also able to accumulate DMSP, and DMS was only produced in the absence of alternate carbon sources. Bacteria containing DMSP were prepared as prey for bacterivorous ciliates and flagellates, to examine the fate of the DMSP during grazing. In all cases, predators metabolized the DMSP in bacteria. In some cases, DMS was produced, but it is not clear if this was due to the predators or to associated bacteria in the non-axenic grazer cultures. Bacterivores may influence DMSP cycling by either modulating populations of DMSP-metabolizing bacteria, or by metabolizing DMSP accumulated by bacterial prey.

16 Human Modification of Global Biogeochemical Cycles

Publisher Summary This chapter discusses the human modification of global biogeochemical cycles. Many biogeochemical and physical processes are involved in determining the climate of the Earth, and some of these are being significantly perturbed by human activity. The physical and chemical composition of the atmosphere determines the transmission, absorption, and reflection of incoming solar radiation and outgoing terrestrial radiation, and the resulting energy balance determines surface temperature. The biogeochemical cycles of sulfur—a crucial component of clouds and most aerosols—and of carbon and nitrogen, which form radiatively important trace gases, are central to the radiative properties of the atmosphere. The combustion of fossil fuels, and the consequent oxidation of nitrogen in combustion air has greatly modified the natural atmospheric cycles of C, N, and S. Although the change of atmospheric CO 2 has little effect on the chemical composition of precipitation, sulfur- and nitrogen-containing acids have a major impact on the chemical composition of rain and snow. These acids in turn perturb the cycles of important minor elements, such as aluminum, through the weathering process in rocks and soils.