Engineering of Nitrosomonas europaea to express Vitreoscilla hemoglobin enhances oxygen uptake and conversion of ammonia to nitrite.

Nitrosomonas europaea was transformed with a recombinant plasmid bearing the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla under control of the N. europaea amoC P1 promoter. Vgb was maintained stably and appeared to be expressed in the transformants at VHb levels of about 0.75 nmol/g wet weight. Expression of VHb in the N. europaea transformants was correlated with an approximately 2 fold increase in oxygen uptake rate by whole cells at oxygen concentrations in the range of 75-100% saturation, but no change in oxygen uptake rate at oxygen concentrations below 25% saturation. VHb expression was also correlated with an increase of as much as about 30% in conversion of ammonia to nitrite by growing cells. The results suggest that engineering of key aerobic wastewater bacteria to express bacterial hemoglobins may be a useful strategy to produce species with enhanced respiratory abilities.

Directed evolution to produce sludge communities with improved oxygen uptake abilities.

Two activated sludge cultures, seeded with activated sludge from the same source, were cultivated for 370 days in synthetic wastewater. Both cultures were transferred weekly to fresh medium; one culture was operated at high dissolved oxygen (DO) (near saturation) and the other at low DO (0.25 mg O2/L). There were significant changes in the abundances of bacterial species and phyla present in each culture throughout the 370-day operational period. In the low DO culture, over time, there was a continuously increasing proportion of cells of species known to encode truncated hemoglobins (Hbs). These are the types of Hbs which may enhance delivery of oxygen to the respiratory chain, to enhance ATP production, especially under low aeration conditions. The levels of heme b, the heme found in Vitreoscilla hemoglobin, increased in parallel to the increase in Hb-encoding species, to much higher levels in the low DO culture than in the high DO culture. Specific oxygen uptake rates increased by 3 % for the high DO culture near the end of the 370-day period, while those for the low DO culture increased steadily to a level 28 % higher than that of the starting culture. Thus, imposition of low DO conditions may, due to selection for Hb-expressing species, be useful in developing bacterial communities with enhanced ability to function efficiently in aerobic wastewater treatment, especially under low aeration conditions.

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions.

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O2/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.

Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions.

This study has investigated the acclimation of ammonia-oxidizing communities (AOC) to low dissolved oxygen (DO) concentrations. Under controlled laboratory conditions, two sequencing batch reactors seeded with activated sludge from the same source were operated at high DO (near saturation) and low DO (0.1 mg O2/L) concentrations for a period of 220 days. The results demonstrated stable and complete nitrification at low DO conditions after an acclimation period of approximately 140 days. Acclimation brought about increased specific oxygen uptake rates and enhanced expression of a particular heme protein in the soluble fraction of the cells in the low DO reactor as compared to the high DO reactor. The induced protein was determined not to be any of the enzymes or electron carriers present in the conventional account of ammonia oxidation in ammonia-oxidizing bacteria (AOB). Further research is required to determine the specific nature of the heme protein detected; a preliminary assessment suggests either a type of hemoglobin protein or a lesser-known component of the energy-transducing pathways of AOB. The effect of DO on AOC dynamics was evaluated using the 16S rRNA gene as the basis for phylogenetic comparisons and organism quantification. Ammonium consumption by ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria was ruled out by fluorescent in situ hybridization in both reactors. Even though Nitrosomonas europaea was the dominant AOB lineage in both high and low DO sequencing batch reactors at the end of operation, this enrichment could not be linked in the low DO reactor to acclimation to oxygen-limited conditions.

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose.

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.