Frank William Rowley Chaplen

Modern agriculture is sustained by application of inorganic nitrogen (N) fertilizer in the form of ammonium (NH ₄ ⁺ ). Up to 60% of NH ₄ ⁺ -based fertilizer can be lost through leaching of nitrifier-derived nitrate (NO ₃ ⁻ ), and through the emission of N oxide gases (i.e., nitric oxide [NO], N dioxide [NO ₂ ], and nitrous oxide [N ₂ O] gases), the latter being a potent greenhouse gas. Our approach to modeling of nitrification suggests that both biotic and abiotic mechanisms function as important sources and sinks of N oxides during microaerobic conditions and that previous models might have underestimated gross NO production during nitrification.

Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH ₃ ) to obtain energy for growth on carbon dioxide (CO ₂ ) and can also produce nitrous oxide (N ₂ O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na ₂ CO ₃ ) supplemented with atmospheric CO ₂ . IC-limited cultures oxidized 25 to 58% of available NH ₃ to nitrite, depending on the dilution rate and Na ₂ CO ₃ concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH ₃ -limited cultures. Rates of N ₂ O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH ₃ -N that was transformed to N ₂ O-N from 0.5% (replete) up to 4.4% (0.2 mM Na ₂ CO ₃ ). Transcriptome analysis showed differential expression ( P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C ₁ metabolism, including the genes for RuBisCO ( cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C ₁ moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N ₂ O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH ₃ oxidation from growth and increased N ₂ O production. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO ₂ ) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea , a model ammonia-oxidizing bacterium, and report on increased production of N ₂ O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N ₂ O emissions from nitrification in soils and wastewater treatment.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO₂⁻) as a source of energy and nitrogen. In most natural situations, NO₂⁻ oxidation is coupled closely to ammonium (NH₄⁺) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH₄⁺ to meet its N-biosynthetic requirements. Interestingly, NH₄⁺ delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH₄⁺ while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH₄⁺ concentration. Genes in assimilatory NO₂⁻ metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH₄⁺. Nitrobacter winogradskyi grown in medium with 25 mM NH₄⁺ upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH₄⁺ and is likely to be modified during coupled nitrification with NH₃ oxidizers.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH₄ ⁺ resulted in a cell density (0.20–0.29 OD₆₀₀) greater than the sum of the densities in single chemostat cultures, i.e., 0.09–0.14 OD₆₀₀ for N. europaea with 60 mM NH₄ ⁺and 0.04–0.06 OD₆₀₀ for N. winogradskyi with 60 mM NO₂ ⁻. The NO₂ ⁻- and NH₄ ⁺-dependent O₂ uptake rates, qRT–PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD₆₀₀ (~80 %) and N. winogradskyi ~0.05 OD₆₀₀ (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

This project used the cyanobacterial species Synechocystis PCC 6803 to pursue two lines of inquiry, with each line addressing one of the two main factors affecting hydrogen (H2) production in Synechocystis PCC 6803: NADPH availability and O2 sensitivity. H2 production in Synechocystis PCC 6803 requires a very high NADPH:NADP+ ratio, that is, the NADP pool must be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-hydrogenase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild type (WT) Synechocystis PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we used H2 production profiling and metabolicmore » flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work provided rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we used site-directed mutagenesis to create mutants with hydrogenase enzymes exhibiting greater O2 tolerance. The research addressed the following four tasks: 1. Evaluate the effects of various culture conditions (N, S, or P limitation; light/dark; pH; exogenous organic carbon) on H2 production profiles of WT cells and an NDH-1 mutant; 2. Conduct metabolic flux analyses for enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production. « less

Anode properties are critical for the performance of microbial electrolysis cells (MECs). In the present study, Fe nanoparticle-modified graphite disks were used as anodes to investigate the effects of nanoparticles on the performance of Shewanella oneidensis MR-1 in MECs. Results demonstrated that the average current densities produced with Fe nanoparticle-decorated anodes up to 5.89-fold higher than plain graphite anodes. Whole genome microarray analysis of the gene expression showed that genes encoding biofilm formation were significantly up-regulated as a response to nanoparticle-decorated anodes. Increased expression of genes related to nanowires, flavins, and c-type cytochromes indicates that enhanced mechanisms of electron transfer to the anode may also have contributed to the observed increases in current density. The majority of the remaining differentially expressed genes associated with electron transport and anaerobic metabolism demonstrate a systemic response to increased power loads.