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1.  Sources and resources: importance of nutrients, resource allocation, and ecology in microalgal cultivation for lipid accumulation 
Applied Microbiology and Biotechnology  2014;98(11):4805-4816.
Regardless of current market conditions and availability of conventional petroleum sources, alternatives are needed to circumvent future economic and environmental impacts from continued exploration and harvesting of conventional hydrocarbons. Diatoms and green algae (microalgae) are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed stocks, the use of autotrophic microorganisms can further contribute to reduced CO2 emissions through utilization of atmospheric CO2. Because of the inherent connection between carbon, nitrogen, and phosphorus in biological systems, macronutrient deprivation has been proven to significantly enhance lipid accumulation in different diatom and algae species. However, much work is needed to understand the link between carbon, nitrogen, and phosphorus in controlling resource allocation at different levels of biological resolution (cellular versus ecological). An improved understanding of the relationship between the effects of N, P, and micronutrient availability on carbon resource allocation (cell growth versus lipid storage) in microalgae is needed in conjunction with life cycle analysis. This mini-review will briefly discuss the current literature on the use of nutrient deprivation and other conditions to control and optimize microalgal growth in the context of cell and lipid accumulation for scale-up processes.
PMCID: PMC4024127  PMID: 24695829
Biofuel; Recycle; Algal biofilm; Biofuel ecology
2.  Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park 
The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park (YNP), Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatures (44°C, 63°C, 75°C). The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi) at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus) at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44 and 63°C springs and a thermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus Nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus Caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different) to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts.
PMCID: PMC3824361  PMID: 24282404
16S rRNA pyrosequencing; alkaline hot spring; Heart Lake Geyser Basin; methanogenic community; phylogeny; Thermus; Yellowstone National Park; thermoalkaline
3.  Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum 
Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination.
The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases).
The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.
PMCID: PMC3457861  PMID: 22672912
Algae; Diatom; Lipid-accumulation; Transcriptomics; Biofuel; Carbon fixation; RNA-seq; Bio-oil
4.  Resolution of volatile fuel compound profiles from Ascocoryne sarcoides: a comparison by proton transfer reaction-mass spectrometry and solid phase microextraction gas chromatography-mass spectrometry 
AMB Express  2012;2:23.
Volatile hydrocarbon production by Ascocoryne sacroides was studied over its growth cycle. Gas-phase compounds were measured continuously with a proton transfer reaction-mass spectrometry (PTR-MS) and at distinct time points with gas chromatography-mass spectrometry (GC-MS) using head space solid phase microextraction (SPME). The PTR-MS ion signal permitted temporal resolution of the volatile production while the SPME results revealed distinct compound identities. The quantitative PTR-MS results showed the volatile production was dominated by ethanol and acetaldehyde, while the concentration of the remainder of volatiles consistently reached 2,000 ppbv. The measurement of alcohols from the fungal culture by the two techniques correlated well. Notable compounds of fuel interest included nonanal, 1-octen-3-ol, 1-butanol, 3-methyl- and benzaldehyde. Abiotic comparison of the two techniques demonstrated SPME fiber bias toward higher molecular weight compounds, making quantitative efforts with SPME impractical. Together, PTR-MS and SPME GC-MS were shown as valuable tools for characterizing volatile fuel compound production from microbiological sources.
PMCID: PMC3402149  PMID: 22480438
Biofuel; Solid phase microextraction; Proton transfer reaction-mass spectrometry; Volatile organic compounds; Fungal hydrocarbons; Gas chromatography-mass spectrometry
5.  Application of Molecular Techniques To Elucidate the Influence of Cellulosic Waste on the Bacterial Community Structure at a Simulated Low-Level-Radioactive-Waste Site▿ † 
Applied and Environmental Microbiology  2010;76(10):3106-3115.
Low-level-radioactive-waste (low-level-waste) sites, including those at various U.S. Department of Energy sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a nonradioactive model low-level-waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more operational taxonomic units, and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the fill and fill-waste interface (FW) layers and greater in the wood waste and waste-clay interface layers. Principal-coordinate analysis and lineage-specific analysis determined that the Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose-degrading microorganisms suggest that the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.
PMCID: PMC2869122  PMID: 20305022
6.  Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State▿ †  
Applied and Environmental Microbiology  2008;74(15):4877-4888.
The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.
PMCID: PMC2519324  PMID: 18552187
7.  Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer 
Applied and Environmental Microbiology  2004;70(10):6037-6046.
Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.
PMCID: PMC522124  PMID: 15466548
8.  Copper-Induced Inhibition of Growth of Desulfovibrio desulfuricans G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead 
Applied and Environmental Microbiology  2001;67(10):4765-4772.
The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using Desulfovibrio desulfuricans G20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of D. desulfuricans membrane integrity. The results show that D. desulfuricans in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.
PMCID: PMC93230  PMID: 11571183

Results 1-8 (8)