Hydrogen Oxidation Influences Glycogen Accumulation in a Verrucomicrobial Methanotroph.

Metabolic flexibility in aerobic methane oxidizing bacteria (methanotrophs) enhances cell growth and survival in instances where resources are variable or limiting. Examples include the production of intracellular compounds (such as glycogen or polyhydroxyalkanoates) in response to unbalanced growth conditions and the use of some energy substrates, besides methane, when available. Indeed, recent studies show that verrucomicrobial methanotrophs can grow mixotrophically through oxidation of hydrogen and methane gases via respiratory membrane-bound group 1d [NiFe] hydrogenases and methane monooxygenases, respectively. Hydrogen metabolism is particularly important for adaptation to methane and oxygen limitation, suggesting this metabolic flexibility may confer growth and survival advantages. In this work, we provide evidence that, in adopting a mixotrophic growth strategy, the thermoacidophilic methanotroph, Methylacidiphilum sp. RTK17.1 changes its growth rate, biomass yields and the production of intracellular glycogen reservoirs. Under nitrogen-fixing conditions, removal of hydrogen from the feed-gas resulted in a 14% reduction in observed growth rates and a 144% increase in cellular glycogen content. Concomitant with increases in glycogen content, the total protein content of biomass decreased following the removal of hydrogen. Transcriptome analysis of Methylacidiphilum sp. RTK17.1 revealed a 3.5-fold upregulation of the Group 1d [NiFe] hydrogenase in response to oxygen limitation and a 4-fold upregulation of nitrogenase encoding genes (nifHDKENX) in response to nitrogen limitation. Genes associated with glycogen synthesis and degradation were expressed constitutively and did not display evidence of transcriptional regulation. Collectively these data further challenge the belief that hydrogen metabolism in methanotrophic bacteria is primarily associated with energy conservation during nitrogen fixation and suggests its utilization provides a competitive growth advantage within hypoxic habitats.

Mixotrophy drives niche expansion of verrucomicrobial methanotrophs.

Aerobic methanotrophic bacteria have evolved a specialist lifestyle dependent on consumption of methane and other short-chain carbon compounds. However, their apparent substrate specialism runs contrary to the high relative abundance of these microorganisms in dynamic environments, where the availability of methane and oxygen fluctuates. In this work, we provide in situ and ex situ evidence that verrucomicrobial methanotrophs are mixotrophs. Verrucomicrobia-dominated soil communities from an acidic geothermal field in Rotokawa, New Zealand rapidly oxidised methane and hydrogen simultaneously. We isolated and characterised a verrucomicrobial strain from these soils, Methylacidiphilum sp. RTK17.1, and showed that it constitutively oxidises molecular hydrogen. Genomic analysis confirmed that this strain encoded two [NiFe]-hydrogenases (group 1d and 3b), and biochemical assays revealed that it used hydrogen as an electron donor for aerobic respiration and carbon fixation. While the strain could grow heterotrophically on methane or autotrophically on hydrogen, it grew optimally by combining these metabolic strategies. Hydrogen oxidation was particularly important for adaptation to methane and oxygen limitation. Complementary to recent findings of hydrogenotrophic growth by Methylacidiphilum fumariolicum SolV, our findings illustrate that verrucomicrobial methanotrophs have evolved to simultaneously utilise hydrogen and methane from geothermal sources to meet energy and carbon demands where nutrient flux is dynamic. This mixotrophic lifestyle is likely to have facilitated expansion of the niche space occupied by these microorganisms, allowing them to become dominant in geothermally influenced surface soils. Genes encoding putative oxygen-tolerant uptake [NiFe]-hydrogenases were identified in all publicly available methanotroph genomes, suggesting hydrogen oxidation is a general metabolic strategy in this guild.

The outcome of pedicle screw instrumentation removal for ongoing low back pain following posterolateral lumbar fusion.

BACKGROUND: Our aim was to determine whether patients derived benefit from removal of pedicle screw instrumentation for axial pain without other cause using our surgical technique and patient selection. A secondary aim was to investigate factors that were associated with poorer outcomes for this procedure as well as complication rate in this cohort. METHODS: Theater records from a single spinal surgeon's practice were reviewed to identify patients that had undergone lumbar fusion for discogenic back pain with subsequent pedicle screw instrumentation removal (Expedium, DePuy Synthes) in the preceding 3 years with a minimum of 18 months follow-up. Inclusion criteria were persisting midline axial back pain with computed tomography (CT)-confirmed solid fusion with non-radicular symptoms and nil other potential causes found, e.g., infection. Case note review along with pre- and post-operative Oswestry disability index (ODI) questionnaires and visual analog scores (VAS) were assessed for all patients. Surgical technique included re-use of previous midline posterior incision and the Wiltse approach with removal of implants, confirmation of a solid fusion mass, washout and bone grafting of removal sites. RESULTS: From 50 consecutive patients who underwent removal of posterolateral instrumentation for an index elective lumbar fusion for discogenic back pain, 34 patients were identified that met the criteria with a mean follow-up of 25 months (range, 18-36 months). The VAS and ODI improved in 22/34 (65%) of participants. The mean cohort VAS score was 6.6 pre-surgery and 4.3 post-surgery (P=0.04). Preoperative and postoperative mean Oswestry disability scores were 64 and 41, respectively (P=0.05). There was a statistically significant difference in the proportion of patients with poorer compared to satisfactory outcomes with regards to compensable status, preoperative grade II opioid use and shorter time between fusion and removal procedure. Complications were one postoperative hematoma and one superficial wound infection, both of which settled without re-operation. CONCLUSIONS: Approximately two thirds of patients were satisfied with removal of instrumentation for treatment of residual low back pain (LBP) following elective lumbar fusion and recorded reduced VAS and grade II opioid use. A subset of patients remained that did not derive benefit and were associated with compensable status, preoperative grade II opioid use and a shorter time between fusion and removal procedure. A prospective cohort study with preoperative diagnostic injections and standardized imaging and microscopic techniques would strengthen future studies. However, this study suggests that removal of instrumentation is safe and provides modest benefit as a palliative procedure for a subset of patients with significant disability from chronic LBP without an underlying cause following lumbar fusion.

Application of hydrothermal treatment to affect the fermentability of Pinus radiata pulp mill effluent sludge.

A hybrid technique incorporating a wet oxidation stage and secondary fermentation step was used to process Pinus radiata pulp mill effluent sludge. The effect of hydrothermal oxidation at high temperature and pressure on the hydrolysis of constituents of the waste stream was studied. Biochemical acidogenic potential assays were conducted to assess acid production resulting from anaerobic hydrolysis of the wet oxidised hydrolysate under acidogenic conditions. Significant degradation of the lignin, hemicellulose, suspended solids, carbohydrates and extractives were observed with wet oxidation. In contrast, cellulose showed resistance to degradation under the experimental conditions. Extensive degradation of biologically inhibitory compounds by wet oxidation did not show a beneficial impact on the acidogenic or methanogenic potential compared to untreated samples.