Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors

This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23–0.99 U mg⁻¹ protein), butyrate kinase (Buk, < 0.03 U mg⁻¹ protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24–7.64 U mg⁻¹ protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH₃ and NH₄⁺-N), and a negative dependency can be postulated. Thus, high concentrations of NH₃ and NH₄⁺-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities.     

Gradients in microbial methanol uptake: productive coastal upwelling waters to oligotrophic gyres in the Atlantic Ocean

Methanol biogeochemistry and its importance as a carbon source in seawater is relatively unexplored. We report the first microbial methanol carbon assimilation rates (k) in productive coastal upwelling waters of up to 0.117±0.002 d⁻¹ (∼10 nmol l⁻¹d⁻¹). On average, coastal upwelling waters were 11 times greater than open ocean northern temperate (NT) waters, eight times greater than gyre waters and four times greater than equatorial upwelling (EU) waters; suggesting that all upwelling waters upon reaching the surface (⩽20 m), contain a microbial population that uses a relatively high amount of carbon (0.3–10 nmol l⁻¹d⁻¹), derived from methanol, to support their growth. In open ocean Atlantic regions, microbial uptake of methanol into biomass was significantly lower, ranging between 0.04–0.68 nmol l⁻¹d⁻¹. Microbes in the Mauritanian coastal upwelling used up to 57% of the total methanol for assimilation of the carbon into cells, compared with an average of 12% in the EU, and 1% in NT and gyre waters. Several methylotrophic bacterial species were identified from open ocean Atlantic waters using PCR amplification of mxaF encoding methanol dehydrogenase, the key enzyme in bacterial methanol oxidation. These included Methylophaga sp., Burkholderiales sp., Methylococcaceae sp., Ancylobacter aquaticus, Paracoccus denitrificans, Methylophilus methylotrophus, Methylobacterium oryzae, Hyphomicrobium sp. and Methylosulfonomonas methylovora. Statistically significant correlations for upwelling waters between methanol uptake into cells and both chlorophyll a concentrations and methanol oxidation rates suggest that remotely sensed chlorophyll a images, in these productive areas, could be used to derive total methanol biological loss rates, a useful tool for atmospheric and marine climatically active gas modellers, and air–sea exchange scientists.     

Microbial community structure in anaerobic co-digestion of grass silage and cow manure in a laboratory continuously stirred tank reactor

The impacts of feeding ratio and loading rate on the microbial community during co-digestion of grass silage with cow manure in an anaerobic laboratory continuously stirred tank reactor were investigated by 16S rRNA gene-based fingerprints. The microbial community remained stable when the reactor was fed with cow manure alone and with up to 20% of grass silage in feedstock at an organic loading rate (OLR) of 2 kg VS m⁻³ day⁻¹. Large changes in the bacterial community were observed when the loading ratio of grass was increased to 40%, while there was little change in the archaeal community. During the increase in OLR from 2 to 4 kg VS m⁻³ day⁻¹ the bacterial community structure showed few differences, whereas Archaea was undetectable. Sequencing of the major DGGE bands indicated that the phylum Bacteriodetes predominated in the bacterial community. Two unclassified bacteria with high abundance survived throughout the operation of the reactor.     

Carbon flow from volcanic CO2 into soil microbial communities of a wetland mofette

Effects of extremely high carbon dioxide (CO₂) concentrations on soil microbial communities and associated processes are largely unknown. We studied a wetland area affected by spots of subcrustal CO₂ degassing (mofettes) with focus on anaerobic autotrophic methanogenesis and acetogenesis because the pore gas phase was largely hypoxic. Compared with a reference soil, the mofette was more acidic (ΔpH ∼0.8), strongly enriched in organic carbon (up to 10 times), and exhibited lower prokaryotic diversity. It was dominated by methanogens and subdivision 1 Acidobacteria, which likely thrived under stable hypoxia and acidic pH. Anoxic incubations revealed enhanced formation of acetate and methane (CH₄) from hydrogen (H₂) and CO₂ consistent with elevated CH₄ and acetate levels in the mofette soil. ¹³CO₂ mofette soil incubations showed high label incorporations with ∼512 ng ¹³C g (dry weight (dw)) soil⁻¹ d⁻¹ into the bulk soil and up to 10.7 ng ¹³C g (dw) soil⁻¹ d⁻¹ into almost all analyzed bacterial lipids. Incorporation of CO₂-derived carbon into archaeal lipids was much lower and restricted to the first 10 cm of the soil. DNA-SIP analysis revealed that acidophilic methanogens affiliated with Methanoregulaceae and hitherto unknown acetogens appeared to be involved in the chemolithoautotrophic utilization of ¹³CO₂. Subdivision 1 Acidobacteriaceae assimilated ¹³CO₂ likely via anaplerotic reactions because Acidobacteriaceae are not known to harbor enzymatic pathways for autotrophic CO₂ assimilation. We conclude that CO₂-induced geochemical changes promoted anaerobic and acidophilic organisms and altered carbon turnover in affected soils.     

Microbial methanol uptake in northeast Atlantic waters

Methanol is the predominant oxygenated volatile organic compound in the troposphere, where it can significantly influence the oxidising capacity of the atmosphere. However, we do not understand which processes control oceanic concentrations, and hence, whether the oceans are a source or a sink to the atmosphere. We report the first methanol loss rates in seawater by demonstrating that ¹⁴C-labelled methanol can be used to determine microbial uptake into particulate biomass, and oxidation to ¹⁴CO₂. We have found that methanol is used predominantly as a microbial energy source, but also demonstrated its use as a carbon source. We report biological methanol oxidation rates between 2.1 and 8.4 nmol l⁻¹ day⁻¹ in surface seawater of the northeast Atlantic. Kinetic experiments predict a V_max of up to 29 nmol l⁻¹ day⁻¹, with a high affinity K_m constant of 9.3 n in more productive coastal waters. We report surface concentrations of methanol in the western English channel of 97±8 n (n=4) between May and June 2010, and for the wider temperate North Atlantic waters of 70±13 n (n=6). The biological turnover time of methanol has been estimated between 7 and 33 days, although kinetic experiments suggest a 7-day turnover in more productive shelf waters. Methanol uptake rates into microbial particles significantly correlated with bacterial and phytoplankton parameters, suggesting that it could be used as a carbon source by some bacteria and possibly some mixotrophic eukaryotes. Our results provide the first methanol loss rates from seawater, which will improve the understanding of the global methanol budget.     

Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean

Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world''s largest continuous rhodolith bed (of ∼21 000 km²) and has one of the largest marine CaCO₃ deposits (producing 25 megatons of CaCO₃ per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 μmol carbon m⁻²s⁻¹), allowing the entire Abrolhos rhodolith bed to produce 5.65 × 10⁵ tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles

Cereal distillers grains, a by-product from bioethanol industry, proved to be a suitable feedstock for biogas production in laboratory scale anaerobic digesters. Five continuously stirred tank reactors were run under constant conditions and monitored for biogas production and composition along with other process parameters. Iron additives for sulfide precipitation significantly improved the process stability and efficiency, whereas aerobic pretreatment of the grains had no effect. The microbial communities in the reactors were investigated for their phylogenetic composition by terminal restriction fragment length polymorphism analysis and sequencing of 16S rRNA genes. The bacterial subcommunities were highly diverse, and their composition did not show any correlation with reactor performance. The dominant phylotypes were affiliated to the Bacteroidetes. The archaeal subcommunities were less diverse and correlated with the reactor performance. The well-performing reactors operated at lower organic loading rates and amended with iron chloride were dominated by aceticlastic methanogens of the genus Methanosaeta. The well-performing reactor operated at a high organic loading rate and supplemented with iron hydroxide was dominated by Methanosarcina ssp. The reactor without iron additives was characterized by propionate and acetate accumulation and high hydrogen sulfide content and was dominated by hydrogenotrophic methanogens of the genus Methanoculleus.     

Characterization of Microbial Communities Removing Nitrogen Oxides from Flue Gas: the BioDeNOx Process

BioDeNOx is an integrated physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gases. In this process, the flue gas is purged through a scrubber containing a solution of Fe(II)EDTA²⁻, which binds the NOx to form an Fe(II)EDTA·NO²⁻ complex. Subsequently, this complex is reduced in the bioreactor to dinitrogen by microbial denitrification. Fe(II)EDTA²⁻, which is oxidized to Fe(III)EDTA⁻ by oxygen in the flue gas, is regenerated by microbial iron reduction. In this study, the microbial communities of both lab- and pilot-scale reactors were studied using culture-dependent and -independent approaches. A pure bacterial strain, KT-1, closely affiliated by 16S rRNA analysis to the gram-positive denitrifying bacterium Bacillus azotoformans, was obtained. DNA-DNA homology of the isolate with the type strain was 89%, indicating that strain KT-1 belongs to the species B. azotoformans. Strain KT-1 reduces Fe(II)EDTA·NO²⁻ complex to N₂ using ethanol, acetate, and Fe(II)EDTA²⁻ as electron donors. It does not reduce Fe(III)EDTA⁻. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene fragments showed the presence of bacteria closely affiliated with members of the phylum Deferribacteres, an Fe(III)-reducing group of bacteria. Fluorescent in situ hybridization with oligonucleotide probes designed for strain KT-1 and members of the phylum Deferribacteres showed that the latter were more dominant in both reactors.     

High rates of denitrification and nitrate removal in cold seep sediments

We measured denitrification and nitrate removal rates in cold seep sediments from the Gulf of Mexico. Heterotrophic potential denitrification rates were assayed in time-series incubations. Surficial sediments inhabited by Beggiatoa exhibited higher heterotrophic potential denitrification rates (32 μ N reduced day⁻¹) than did deeper sediments (11 μ N reduced day⁻¹). Nitrate removal rates were high in both sediment horizons. These nitrate removal rates translate into rapid turnover times (<1 day) for the nitrate pool, resulting in a faster turnover for the nitrate pool than for the sulfate pool. Together, these data underscore the rigorous nature of internal nitrogen cycling at cold seeps and the requirement for novel mechanisms to provide nitrate to the sediment microbial community.     

Linking activity,composition and seasonal dynamics of atmospheric methane oxidizers in a meadow soil

Microbial oxidation is the only biological sink for atmospheric methane. We assessed seasonal changes in atmospheric methane oxidation and the underlying methanotrophic communities in grassland near Giessen (Germany), along a soil moisture gradient. Soil samples were taken from the surface layer (0–10 cm) of three sites in August 2007, November 2007, February 2008 and May 2008. The sites showed seasonal differences in hydrological parameters. Net uptake rates varied seasonally between 0 and 70 μg CH₄ m⁻² h⁻¹. Greatest uptake rates coincided with lowest soil moisture in spring and summer. Over all sites and seasons, the methanotrophic communities were dominated by uncultivated methanotrophs. These formed a monophyletic cluster defined by the RA14, MHP and JR1 clades, referred to as upland soil cluster alphaproteobacteria (USCα)-like group. The copy numbers of pmoA genes ranged between 3.8 × 10⁵–1.9 × 10⁶ copies g⁻¹ of soil. Temperature was positively correlated with CH₄ uptake rates (P<0.001), but had no effect on methanotrophic population dynamics. The soil moisture was negatively correlated with CH₄ uptake rates (P<0.001), but showed a positive correlation with changes in USCα-like diversity (P<0.001) and pmoA gene abundance (P<0.05). These were greatest at low net CH₄ uptake rates during winter times and coincided with an overall increase in bacterial 16S rRNA gene abundances (P<0.05). Taken together, soil moisture had a significant but opposed effect on CH₄ uptake rates and methanotrophic population dynamics, the latter being increasingly stimulated by soil moisture contents >50 vol% and primarily related to members of the MHP clade.     

Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario,Canada

Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000 copies ml⁻¹ and were considered ‘high abundance'', whereas the other two Prasinovirus-related genes peaked at abundances <1000 copies ml⁻¹ and were considered ‘low abundance''. Of the genes related to chloroviruses, one peaked at ca 1600 copies ml⁻¹, whereas the other reached only ca 300 copies ml⁻¹. Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100–1000 copies ml⁻¹ while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100 copies ml⁻¹. Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.     

Impact of crop species on bacterial community structure during anaerobic co-digestion of crops and cow manure

The bacterial communities in three continuously stirred tank reactors co-digesting cow manure with grass silage, oat straw, and sugar beet tops, respectively, were investigated by 16S rRNA gene-based fingerprints and clone libraries. The analyses revealed both clearly distinct and similar phylotypes in the bacterial communities between the reactors. The major groups represented in the three reactors were Clostridia, unclassified Bacteria, and Bacteroidetes. Phylotypes affiliated with Bacilli or Deltaproteobacteria were unique to the sugar beet and straw reactor, respectively. Unclassified Bacteria dominated in sugar beet reactor while in the straw and grass reactor Clostridia was the dominant group. An increase in organic loading rate from 2 to 3 kg volatile solids m^?3 d^?1 resulted in larger changes in the bacterial community in the straw compared to grass reactor. The study shed more light on the evolution of bacterial community during anaerobic co-digestion of different crops and manure to methane.     

Microbial community structures in anoxic freshwater lake sediment along a metal contamination gradient

Contamination, such as by heavy metals, has frequently been implicated in altering microbial community structure. However, this association has not been extensively studied for anaerobic communities, or in freshwater lake sediments. We investigated microbial community structure in the metal-contaminated anoxic sediments of a eutrophic lake that were impacted over the course of 80 years by nearby zinc-smelting activities. Microbial community structure was inferred for bacterial, archaeal and eukaryotic populations by evaluating terminal restriction fragment length polymorphism (TRFLP) patterns in near-surface sediments collected in triplicate from five areas of the lake that had differing levels of metal contamination. The majority of the fragments in the bacterial and eukaryotic profiles showed no evidence of variation in association with metal contamination levels, and diversity revealed by these profiles remained consistent even as metal concentrations varied from 3000 to 27 000 mg kg⁻¹ total Zn, 0.125 to 11.2 μ pore water Zn and 0.023 to 5.40 μ pore water As. Although most archaeal fragments also showed no evidence of variation, the prevalence of a fragment associated with mesophilic Crenarchaeota showed significant positive correlation with total Zn concentrations. This Crenarchaeota fragment dominated the archaeal TRFLP profiles, representing between 35% and 79% of the total measured peak areas. Lake DePue 16S rRNA gene sequences corresponding to this TRFLP fragment clustered with anaerobic and soil mesophilic Crenarchaeota sequences. Although Crenarchaeota have been associated with metal-contaminated groundwater and soils, this is a first report (to our knowledge) documenting potential increased prevalence of Crenarchaeota associated with elevated levels of metal contamination.     

Wildland fire as an atmospheric source of viable microbial aerosols and biological ice nucleating particles

The environmental sources of microbial aerosols and processes by which they are emitted into the atmosphere are not well characterized. In this study we analyzed microbial cells and biological ice nucleating particles (INPs) in smoke emitted from eight prescribed wildland fires in North Florida. When compared to air sampled prior to ignition, samples of the air–smoke mixtures contained fivefold higher concentrations of microbial cells (6.7 ± 1.3 × 10⁴ cells m⁻³) and biological INPs (2.4 ± 0.91 × 10³ INPs m⁻³ active at temperatures ≥ −15 °C), and these data significantly positively correlated with PM₁₀. Various bacteria could be cultured from the smoke samples, and the nearest neighbors of many of the isolates are plant epi- and endophytes, suggesting vegetation was a source. Controlled laboratory combustion experiments indicated that smoke emitted from dead vegetation contained significantly higher numbers of cells, INPs, and culturable bacteria relative to the green shrubs tested. Microbial viability of smoke aerosols based on formazan production and epifluorescent microscopy revealed no significant difference in the viable fraction (~80%) when compared to samples of ambient air. From these data, we estimate each fire aerosolized an average of 7 ± 4 × 10⁹ cells and 2 ± 1 × 10⁸ biological INPs per m² burned and conclude that emissions from wildland fire are sources of viable microbial aerosols to the atmosphere.Subject terms: Air microbiology, Microbial ecology, Microbial ecology, Forest ecology     

Aerobic nitrous oxide production through N-nitrosating hybrid formation in ammonia-oxidizing archaea

Soil emissions are largely responsible for the increase of the potent greenhouse gas nitrous oxide (N₂O) in the atmosphere and are generally attributed to the activity of nitrifying and denitrifying bacteria. However, the contribution of the recently discovered ammonia-oxidizing archaea (AOA) to N₂O production from soil is unclear as is the mechanism by which they produce it. Here we investigate the potential of Nitrososphaera viennensis, the first pure culture of AOA from soil, to produce N₂O and compare its activity with that of a marine AOA and an ammonia-oxidizing bacterium (AOB) from soil. N. viennensis produced N₂O at a maximum yield of 0.09% N₂O per molecule of nitrite under oxic growth conditions. N₂O production rates of 4.6±0.6 amol N₂O cell⁻¹ h⁻¹ and nitrification rates of 2.6±0.5 fmol NO₂⁻ cell⁻¹ h⁻¹ were in the same range as those of the AOB Nitrosospira multiformis and the marine AOA Nitrosopumilus maritimus grown under comparable conditions. In contrast to AOB, however, N₂O production of the two archaeal strains did not increase when the oxygen concentration was reduced, suggesting that they are not capable of denitrification. In ¹⁵N-labeling experiments we provide evidence that both ammonium and nitrite contribute equally via hybrid N₂O formation to the N₂O produced by N. viennensis under all conditions tested. Our results suggest that archaea may contribute to N₂O production in terrestrial ecosystems, however, they are not capable of nitrifier-denitrification and thus do not produce increasing amounts of the greenhouse gas when oxygen becomes limiting.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL^−/−) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3^−/− and MLKL^−/− mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3^−/−, but not MLKL^−/− mice, were protected against postinjury motor and cognitive deficits at 1–4 weeks (RIPK3^−/− vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3^−/− mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24–48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.Subject terms: Molecular neuroscience, Brain injuries