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Methanotrophs: Multifunctional bacteria with promising applications in environmental bioengineering
Abstract
Methane is an important greenhouse gas which is produced from many natural and anthropogenic sources. It plays an important role in overall global warming. A significant amount of methane is removed through microbiological oxidation by methanotrophic bacteria, which are widespread in the environment, including many extreme environments. The key enzyme of these microorganisms, methane monooxygenase (MMO), especially the soluble MMO, is remarkable in its broad substrate specificity. This unique capability, i.e. catalyzing reactions of environmental importance, has attracted great attention for applied microbiologists and biochemical engineers. In this review, recent advances in the application of methanotrophs to environmental bioengineering are summarized, including biodiversity, catalytic properties, and cultivation, etc. We have focused on two aspects of the application and potential value of methanotrophs in environmental bioengineering, namely methane removal and biodegradation of toxic compounds. The removal of methane produced from landfills has been widely studied, and much of this work can be used as a source of reference for coal mine gas removal. Many bioreactors using methanotrophs in bioremediation have been developed in recent years. These reactors have two forms of configuration, single-stage and multi-stage. Current limitations which may affect the engineering applications of methanotrophs are discussed, such as the lack of suitable methanotrophic isolate, gas transfer limitation, competitive inhibition of MMO, regeneration of reducing equivalents for MMO and product toxicity.
... Among these biotechnologies, Methylococcus capsulatus has attracted widespread interest due to its ability to use methane as both a carbon and energy source. Additionally, the bacteria contain methane monooxygenase (MMO), an enzyme that facilitates the conversion of methane into methanol precursor for biofuel production [14,15]. Its distinct attributes include a high-affinity methane uptake system and the presence of particulate methane monooxygenase (pMMO), which positions the bacteria as a promising candidate for methane capture and utilization [16,17]. ...
... It comprises several copper ions incorporated in the active site of the enzyme, which has central importance for its enzymatic function. In this pathway, methane (CH 4 ) is oxidized in a copper-dependent process [14,38]. During this oxidation process, there is oxidation of ferrous iron (Fe 2+ ) which is incorporated within the enzyme into ferric iron (Fe 3+ ) which is also involved in the reaction cycle of the enzyme [39]. ...
... Transesterification principally employs catalysts including sodium hydroxide (NaOH), potassium hydroxide (KOH), or sulfuric acid (H 2 SO 4 ). These catalysts assist in increasing the rate of reaction, thus enhancing the production of biodiesel and glycerol [14]. Biodiesel is in high demand, as it has been shown to lower the emissions of carbon monoxide, particulate matter, and unburned hydrocarbons compared to fossil fuels. ...
Methane is the second largest contributor to global warming after carbon dioxide. Once it is released into the atmosphere, methane lingers for over 10 years, during which it traps heat, contributes to the formation of ground-level ozone, and affects air quality adversely. Conversely, methane has some benefits that could be harnessed to address its impact on the environment while utilizing it for good. Methane’s significant role in global warming and potential for energy production and other beneficial applications necessitate the adoption of innovative solutions to remediate the gas from the atmosphere and harness some of its benefits. This article explores Methylococcus capsulatus, a methanotrophic bacterium, and its potential for revolutionizing sustainable methane capture and utilization. With its unique metabolic abilities, M. capsulatus efficiently oxidizes methane, making it a promising candidate for biotechnological applications. We review current research in its current and potential applications in methane capture and utilization, emphasizing key characteristics, implementation challenges, benefits, and limitations in methane capture and conversion. We also highlight the importance of interdisciplinary collaborations and technological advancements in synthetic biology to maximize its energy production potential. Our article analyzes M. capsulatus’ role in addressing methane-related environmental concerns and advancing sustainable energy solutions.
... Methanotrophs contain the enzyme methane monooxygenase (MMO), responsible for the oxidation of methane to methanol. MMO occurs in two forms, a particulate membrane-associated form (pMMO) and a soluble cytoplasmic form (sMMO) and (Jiang et al. 2011). The particulate form has been found in all methanotrophs, except for the genera Methyloferula (Vorobev et al. 2011) and Methylocella (Theisen et al. 2005), in contrast sMMO has been found in fewer strains (Murrell et al. 2000). ...
... The sMMO genes are found in an operon (mmoXYBZDC) which encodes the α, β, and γ subunits of the hydroxylase (mmoXYZ), the reductase (mmoC) and a regulatory or coupling protein (mmoB) (Jiang et al. 2011). MMOD is a regulatory element to repress expression of sMMO (Kim et al. 2019;Koo and Rosenzweig 2021;Merkx and Lippard 2002;Sazinsky et al. 2004;Semrau et al. 2013). ...
... The substrate range for sMMO includes alicyclic hydrocarbons, alkanes, alkenes, monoaromatics, diaromatics and substituted methane derivatives (Jiang et al. 2011). ...
Soil and groundwater were investigated for the genes encoding soluble and particulate methane monooxygenase/ammonia monooxygenase (sMMO, pMMO/AMO), toluene 4-monooxygenase (T4MO), propane monooxygenase (PMO) and phenol hydroxylase (PH). The objectives were (1) to determine which subunits were present, (2) to examine the diversity of the phylotypes associated with the biomarkers and (3) to identify which metagenome associated genomes (MAGs) contained these subunits. All T4MO and PH subunits were annotated in the groundwater metagenomes, while few were annotated in the soil metagenomes. The majority of the soil metagenomes included only four sMMO subunits. Only two groundwater metagenomes contained five sMMO subunits. Gene counts for the pMMO subunits varied between samples. The majority of the soil metagenomes were annotated for all four PMO subunits, while three out of eight groundwater metagenomes contained all four PMO subunits. A comparison of the blast alignments for the sMMO alpha chain (mmoX) indicated the phylotypes differed between the soil and groundwater metagenomes. For the pMMO/AMO alpha subunit (pmoA/amoA), Nitrosospira was important for the soil metagenomes, while Methylosinus and Methylocystis were dominant for the groundwater metagenomes. The majority of pmoA alignments from both metagenomes were from uncultured bacteria. High quality MAGs were obtained from the groundwater data. Four MAGs (Methylocella and Cypionkella) contained sMMO subunits. Another three MAGs, within the order Pseudomonadales, contained all three pMMO subunits. All PH subunits were detected in seven MAGs (Azonexus, Rhodoferax, Aquabacterium). In those seven, all contained catechol 2,3-dioxagenase, and Aquabacterium also contained catechol 1,2-dioxygenase. T4MO subunits were detected in eight MAGs (Azonexus, Rhodoferax, Siculibacillus) and all, except one, contained all six subunits. Four MAGs (Rhodoferax and Azonexus) contained all subunits for PH and T4MO, as well as catechol 2,3-dixoygenase. The detection of T4MO and PH in groundwater metagenomes and MAGs has important implications for the potential oxidation of groundwater contaminants.
... Cattle, for instance, produce methane through enteric fermentation (accounting for 85 to 90% of the total) and fecal excretion. In the case of cattle, a significant portion (95%) of the methane generated in the rumen is expelled through eructation (i.e., burping) and the intestines, while only a small fraction (5%) is excreted through the anus [80,81]. The trend and legal obligation of mitigating greenhouse gas emissions will likely directly impact the improved efficiency of livestock systems, including animal nutrition, productivity, handling, and management. ...
... Reducing methane emissions from coal mines is quite possible, as methane must be primarily extracted from coal mines for use in different applications [80,104]. These applications comprise a basic conversion of methane into CO2, which can decrease the greenhouse effect by 20 times [105]. ...
... In the late 1960s, Whittenbury provided a taxonomic framework for methanotrophs. It is based on cell morphology, intracellular membrane structure, carbon assimilation pathways, and cell wall components, Reducing methane emissions from coal mines is quite possible, as methane must be primarily extracted from coal mines for use in different applications [80,104]. These applications comprise a basic conversion of methane into CO 2 , which can decrease the greenhouse effect by 20 times [105]. ...
Methanotrophy is a biological process that effectively reduces global methane emissions by utilizing microorganisms that can utilize methane as a source of energy under both oxic and anoxic conditions, using a variety of different electron acceptors. Methanotrophic microbes, which utilize methane as their primary source of carbon and energy, are microorganisms found in various environments, such as soil, sediments, freshwater, and marine ecosystems. These microbes play a significant role in the global carbon cycle by consuming methane, a potent greenhouse gas, and converting it into carbon dioxide, which is less harmful. However, methane is known to be the primary contributor to ozone formation and is considered a major greenhouse gas. Methane alone contributes to 30% of global warming; its emissions increased by over 32% over the last three decades and thus affect humans, animals, and vegetation adversely. There are different sources of methane emissions, like agricultural activities, wastewater management, landfills, coal mining, wetlands, and certain industrial processes. In view of the adverse effects of methane, urgent measures are required to reduce emissions. Methanotrophs have attracted attention as multifunctional bacteria with potential applications in biological methane mitigation and environmental bioremediation. Methanotrophs utilize methane as a carbon and energy source and play significant roles in biogeochemical cycles by oxidizing methane, which is coupled to the reduction of various electron acceptors. Methanotrophy, a natural process that converts methane into carbon dioxide, presents a promising solution to mitigate global methane emissions and reduce their impact on climate change. Nonetheless, additional research is necessary to enhance and expand these approaches for extensive use. In this review, we summarize the key sources of methane, mitigation strategies, microbial aspects, and the application of methanotrophs in global methane sinks with increasing anthropogenic methane emissions.
... Considering the lower hydrate formation pressure of CH 4 compared to other components at the same temperature value, CH 4 converts to the hydrate form earlier and is finally recovered purely after the decomposition of hydrates, resulting in its separation from the initial gas mixture. Nonetheless, some challenges are considerable in this approach, such as performance in streams with high flow rates of VAM and the existence of contaminants and coal dust in the inlet gas stream [7,11,12]. While it is true that the presence of other compounds could complicate the process of methane uptake using clathrate hydrates, several promoters have been studied to increase the selectivity towards methane separation [13][14][15]. ...
... Solid adsorbents such as zeolites, Metal-Organic Frameworks (MOFs), and activated carbons can selectively adsorb methane from a gas mixture, allowing for the separation and purification of the gas. This technology is energy-efficient, environmentally friendly, and has the potential to significantly reduce Processes 2023, 11,1496 4 of 32 the cost of natural gas purification. However, the performance of the adsorbents is highly dependent on their intrinsic properties, such as pore size, surface area, and selectivity. ...
... Processes 2023,11, 1496 ...
Ventilation Air Methane (VAM) refers to the release of fugitive methane (CH4) emissions into the atmosphere during underground coal mining operations. Growing concerns regarding the greenhouse effects of CH4 have led to a worldwide effort in developing efficient and cost-effective methods of capturing CH4. Among these, absorption-based processes, particularly those using Ionic Liquids (ILs) are appealing due to their advantages over conventional methods. In this study, the solubility of CH4 in various ILs, expressed by Henry’s law constant, is first reviewed by examining a wide range of experimental techniques. This is followed by a review of thermodynamic modelling tools such as the extended Henry’s law model, extended Pitzer’s model, Peng–Robinson (PR) equation of state, and Krichevsky−Kasarnovsky (KK) equation of state as well as computational (Artificial Neural Network) modelling approaches. The comprehensive analysis presented in this paper aims to provide a deeper understanding of the factors that significantly influence the process of interest. Furthermore, the study provides a critical examination of recent advancements and innovations in CH4 capture by ILs. ILs, in general, have a higher selectivity for methane compared to conventional solvents. This means that ILs can remove methane more effectively from VAM, resulting in a higher purity of the recovered methane. Overall, ILs offer several advantages over conventional solvents for the after treatment of VAM. They are more selective, less volatile, have a wider temperature range, are chemically stable, and can be made from renewable materials. As a result of their many advantages, ILs are becoming increasingly popular for the after treatment of VAM. They offer a more sustainable, efficient, and safe alternative to conventional solvents, and they are likely to continue gaining market share in the coming years.
... Also, methane is released from waste-water treatment plants, manure storage pits, ruminants (e.g., cattle) or natural soil such as wetlands. It is in these habitats where also methane-consuming microorganisms, so-called methanotrophic bacteria [30][31][32][33] can be found. They can be isolated [34] and be deployed for various target products. ...
... Said methanotrophs are gram-negative bacteria that use methane as their only source of carbon and energy [31], making them unique and interesting for biotechnological applications. Apart from being hosts for biotechnological production with native and engineered microorganisms in industry, they could be used for geoengineering purposes to fight global warming by methane, e.g., from thawing permafrost soil, fugitive anthropogenic emissions or direct air capturing of methane. ...
... Apart from being hosts for biotechnological production with native and engineered microorganisms in industry, they could be used for geoengineering purposes to fight global warming by methane, e.g., from thawing permafrost soil, fugitive anthropogenic emissions or direct air capturing of methane. Methanotrophs have also been described to co-metabolize toxic compounds [31]. Table 2 lists several methane-utilizing bacteria. ...
Methanotrophic bacteria can use methane as their only energy and carbon source, and they can be deployed to manufacture a broad range of value-added materials, from single cell protein (SCP) for feed and food applications over biopolymers such as polyhydroxybutyrate (PHB) to value-added building blocks and chemicals. SCP can replace fish meal and soy for fish (aquacultures), chicken and other feed applications, and also become a replacement of meat after suitable treatment, as a sustainable alternative protein. Polyhydroxyalkanoates (PHA) like PHB are a possible alternative to fossil-based thermoplastics. With ongoing and increasing pressure towards decarbonization in many industries, one can assume that natural gas consumption for combustion will decline. Methanotrophic upgrading of natural gas to valuable products is poised to become a very attractive option for owners of natural gas resources, regardless of whether they are connected to the gas grids. If all required protein, (bio)plastics and chemicals were made from natural gas, only 7, 12, 16–32%, and in total only 35–51%, respectively, of the annual production volume would be required. Also, that volume of methane could be sourced from renewable resources. Scalability will be the decisive factor in the circular and biobased economy transition, and it is methanotrophic fermentation that can close that gap.
... Methanotrophs contain methane monooxygenase (MMO), which catalyzes the oxidation of methane to methanol. MMO exists in two forms, a soluble cytoplasmic form (sMMO) and a particulate membraneassociated form (pMMO) (Jiang et al., 2011). While pMMO has been found in all methanotrophs, except for the genera Methylocella (Theisen et al., 2005) and Methyloferula (Vorobev et al., 2011), sMMO is present in fewer strains (Murrell et al., 2000). ...
... sMMO contains a hydroxylase with an (αβγ) 2 structure, a regulatory protein and a reductase (Jiang et al., 2011). The genes encoding sMMO in Methylococcus capsulatus Bath (Csaki et al., 2003;Stainthorpe et al., 1990;Stainthorpe et al., 1989) and Methylosinus trichosporium OB3b (Cardy et al., 1991a;Cardy et al., 1991b) have been determined. ...
... The genes encoding sMMO in Methylococcus capsulatus Bath (Csaki et al., 2003;Stainthorpe et al., 1990;Stainthorpe et al., 1989) and Methylosinus trichosporium OB3b (Cardy et al., 1991a;Cardy et al., 1991b) have been determined. They are found in a six-gene operon (mmoXYBZDC), which encodes the α, β, and γ subunits of the hydroxylase (mmoXYZ), the reductase (mmoC) and a regulatory or coupling protein (mmoB) (Jiang et al., 2011). MMOD is a regulatory element to repress expression of sMMO (Kim et al., 2019;Koo and Rosenzweig, 2021;Merkx and Lippard, 2002;Sazinsky et al., 2004;Semrau et al., 2013). ...
Cometabolic oxidation involves the oxidation of chemicals often by monooxygenases or dioxygenases and can be a removal process for environmental contaminants such as trichloroethene (TCE) or 1,4-dioxane. Information on the occurrence of these genes and their associated microorganisms in environmental samples has the potential to enhance our understanding of contaminant removal. The overall aims were to 1) ascertain which genes encoding for monooxygenases (from methanotrophs, ammonia oxidizing bacteria and toluene/phenol oxidizers) and other key enzymes are present in soil microcosms and 2) determine which phylotypes are associated with those genes.
The approach involved a predictive tool called PICRUSt2 and 16S rRNA gene amplicon datasets from two previous soil microcosm studies. The following targets from the KEGG database were examined: pmo/amo, mmo, dmp/pox/tomA, tmo/tbu/tou, bssABC (and downstream genes), tod, xylM, xylA, gst, dhaA, catE, dbfA1, dbfA2 and phenol 2-monooxygenase.
A large number of phylotypes were associated with pmo/amo, while mmo was linked to only five. Several phylotypes were associated with both pmo/amo and mmo. The most dominant microorganism predicted for mmoX was Mycobacterium (also predicted for pmo/amo). A large number of phylotypes were associated with all six genes from the dmp/pox/tomA KEGG group. The taxonomic associations predicted for the tmo/tbu/tou KEGG group were more limited. In both datasets, Geobacter was a key phylotype for benzylsuccinate synthase. The dioxygenase-mediated toluene degradation pathway encoded by todC1C2BA was largely absent, as were the genes (xylM, xylA) encoding for xylene monooxygenase. All other genes investigated were predicted to be present and were associated with a number of microorganisms. Overall, the analysis predicted the genes encoding for sMMO (mmo), T3MO/T3MO/ToMO (tmo/tbu/tou) and benzylsuccinate synthase (bssABC) are present for a limited number of phylotypes compared to those encoding for pMMO/AMO (pmo/amo) and phenol monooxygenase/T2MO (dmp/poxA/tomA). These findings suggest in soils contaminant removal via pMMO/AMO or phenol monooxygenase/T2MO may be common because of the occurrence of these enzymes with a large number of phylotypes.
... In methanotrophs possessing both sMMO and pMMO, expression of these enzymes is regulated by the availability of copper ions: during bacterial growth, pMMO is expressed when the proportion of copper to the biomass is high, while sMMO is induced at a low copper to biomass ratio (Murrell et al., 2000). The ability of sMMO to oxidize a broad range of substrates, including carbon monoxide, some alkanes, alkenes, halogenated methane derivatives, and cyclic compounds (altogether, more than 100 sMMO substrates have been recorded; see Murrell and Smith, 2010, for review) fuels research into the possibilities of using methanotrophic bacteria for the purposes of bioremediation and degradation of various pollutants, e.g., trichloroethylene (TCE) and chlorinated hydrocarbons (Smith and Nichol, 2018), as well as for the synthesis of homochiral epoxides (Jiang et al., 1996(Jiang et al., , 2010Khider et al., 2021). ...
... Cas9 endonuclease makes a double-strand break in the DNA chain, which is targeted by means of a so-called single-guide RNA (gRNA) containing a 20-bp protospacer complementary to the target sequence next to the protospacer-adjacent motif (PAM) 5'-NGG-3'. This double-strand break can be repaired with a specially designed template, which makes it possible to introduce the desired genetic modifications (Garneau et al., 2010;Gasiunas et al., 2012;Jinek et al., 2012). However, only one study on CRISPR/Cas9-mediated editing of a methanotroph genome has been performed to date (Tapscott et al., 2019). ...
... By contrast, type II methanotrophs dominate in environments with relatively high levels of CH 4 , low dissolved oxygen, and limited N and Cu [16,21]. Methanotrophs, convert CH 4 to methanol (CH 3 OH) via the methane monooxygenase (MMO) enzyme; they then initiate a sequential transformation to formaldehyde, formate, and CO 2 through methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase, respectively [5,[22][23][24] (Fig. 1). MMO is an oxidative enzyme that inserts a single O atom into an organic compound: it is considered an attractive biocatalyst in industrial and environmental biotechnology due to its high chemo, regio and enantio-selective characteristics [25]. ...
... of pollutants [22]. Regardless, OB3b has been used in bioremediation studies of chlorinated hydrocarbons for over 20 years. ...
Rising greenhouse gas concentrations in the atmosphere are having deleterious effects on biotic and abiotic systems, causing serious global concerns. Bioprocess technology is able to utilize these gases to manufacture high-value products and provide an excellent alternative to currently expensive conventional carbon sources in use. Methanotrophs are ubiquitous and utilize reduced carbon substrates without C−C bonds, such as methane, methanol, methylamine, and formaldehyde. Notably, methanotrophs produce single-cell proteins, polyhydroxyalkanoates, methanol, and exopolysaccharides, using a unique metabolic pathway during their growth process. Many industries have developed copyright-protected bioprocesses to utilize methane through methanotrophic pathways and manufacture high-value products. Among methanotrophs, Methylosinus trichosporium OB3b is one of the most important methane oxidizing bacteria; it has been studied extensively to identify potential applications including methanol and biopolymer production, and the bioremediation of environmental contaminants. This paper summarizes the characteristics and versatile role of methanotrophs and Methylosinus trichosporium OB3b. In addition, commercially established biological conversions of methane were constituted.
... Both UF and MF membrane systems work under low pressure and therefore require low TMP (transmembrane pressure) (Farahah . Scenedesmus quadricauda was harvested through cross-flow ultrafiltration technique which resulted in a harvesting efficiency of 46.01 g/m 2 /h with an average flux of 45.50 L/m 2/ h (Zhang et al., 2010). Through this method, the concentration of Arthrospira platensis suspension was also found to increase from 50 mg/L to 1 g/L. ...
... Methane monooxygenase (MMO) as a platform for epoxidation, bioremediation, and cell-free methane conversion. The use of methane monooxygenase as an enzyme for epoxidation or bioremediation is one of the most sought-after areas of methanotrophic protein implementation (Smith et al., 2002;Jiang et al., 2010;Lawton & Rosenzweig, 2016;Chan & Yu, 2019;Khider et al., 2021). The heterologous expression of methane monooxygenase in non-methanotrophic hosts has been challenging; however, the methanotrophs themselves represent an attractive production system. ...
... 2024, 25, 12469 2 of 17 oxygen availability, while high cellular growth promptly depletes nitrogen sources. As a result, this negative impact from these conditions decreases the methanotroph's growth by restricting methane oxidation and essential protein synthesis, resulting in reduced biomass production [2,11]. Operational management of these nutrients is crucial for sustaining high growth rates in bioreactors. ...
Biotechnology continues to drive innovation in the production of pharmaceuticals, biofuels, and other valuable compounds, leveraging the power of microbial systems for enhanced yield and sustainability. Genome-scale metabolic (GSM) modeling has become an essential approach in this field, which enables a guide for targeting genetic modifications and the optimization of metabolic pathways for various industrial applications. While single-species GSM models have traditionally been employed to optimize strains like Escherichia coli and Lactococcus lactis, the integration of these models into community-based approaches is gaining momentum. Herein, we present a pipeline for community metabolic modeling with a user-friendly GUI, applying it to analyze interactions between Methylococcus capsulatus, a biotechnologically important methanotroph, and Escherichia coli W3110 under oxygen- and nitrogen-limited conditions. We constructed models with unmodified and homoserine-producing E. coli strains using the pipeline implemented in the original BioUML platform. The E. coli strain primarily utilized acetate from M. capsulatus under oxygen limitation. However, homoserine produced by E. coli significantly reduced acetate secretion and the community growth rate. This homoserine was taken up by M. capsulatus, converted to threonine, and further exchanged as amino acids. In nitrogen-limited modeling conditions, nitrate and ammonium exchanges supported the nitrogen needs, while carbon metabolism shifted to fumarate and malate, enhancing E. coli TCA cycle activity in both cases, with and without modifications. The presence of homoserine altered cross-feeding dynamics, boosting amino acid exchanges and increasing pyruvate availability for M. capsulatus. These findings suggest that homoserine production by E. coli optimizes resource use and has potential for enhancing microbial consortia productivity.
... The urgent need to reduce methane (CH 4 ) emissions and to remove CH 4 from the atmosphere has sparked interest in a variety of abiotic catalytic processes for removing atmospheric CH 4 and in the biotechnology potential to increase rates of methanotrophy (microbial CH 4 oxidation) in a variety of engineered and natural systems [1][2][3][4] . Some progress has been made in the application of engineered methanotrophy systems to environments with elevated CH 4 concentrations, such as animal barns, landfill covers, wastewater treatment, and coal mine vents [5][6][7][8] . ...
... The methanotrophs (CH 4 -oxidizing bacteria) use CH 4 as their sole carbon and energy source [25], which regulates CH 4 emissions from rice paddy ecosystems [26]. In rice paddy fields, CH 4 is oxidized mainly aerobically by methanotrophs in the topsoil and rhizosphere, where O 2 and CH 4 correspond [1]. ...
Biogas digestive effluent (BDE) is a nutrient-enriched source that can be utilized as an organic fertilizer for rice cultivation without synthetic fertilizer (SF) application. However, a primary concern is the stimulation of methane (CH4) emissions due to the enrichment of the labile organic carbon, a favorite substrate of methanogenic archaea. Methanotrophs potentially reduce greenhouse gas (GHG) emissions from rice fields owing to metabolizing CH4 as a carbon source and energy. We therefore examined the effect of the application of methanotroph-inoculated BDE to the rice cultivated paddy soil on GHG emissions and rice productivity under a pot experiment. Methanotrophs (Methylosinus sp. and Methylocystis sp.), isolated from the Vietnamese Mekong Delta’s rice fields, were separately inoculated to the heated BDE, followed by a 5-day preincubation. Methanotroph-inoculated BDE was supplied to rice cultivation to substitute SF at 50% or 100% in terms of nitrogen amount. The results showed that the total CH4 emissions increased ~34% with the application of BDE. CH4 emissions were significantly reduced by ~17–21% and ~28–44% under the application of methanotroph-inoculated BDE at 100% and 50%, respectively. The reduction in CH4 was commensurate with the augmentation of pmoA transcript copy number under methanotroph-inoculated BDE. In addition, methanotroph-inoculated BDE application did not increase nitrous oxide (N2O) emissions and adversely affect rice growth and grain productivity. This study highlighted the BDE-recirculated feasibility for a lower CH4 emission rice production based on methanotrophs where high CH4-emitting fields were confirmed.
... Solid medium When preparing the solid medium, add 2% agar to the culture solution, dissolve it by heating, and inoculate the bacteria source after sterilization and cooling (Jiang et al. 2010). ...
In a semi-closed visualization pipeline, this experiment studied the inhibitory effect of ultra-fine pure water mist, ultra-fine water mist containing inorganic salt and ultra-fine water mist containing bacteria-inorganic salt on 9.8% methane explosion under five different quality of spray volume. Combined with the methane explosion suppression experiment, the ability of methane-oxidizing bacteria to degrade 9.8% of methane was studied in a simulated pipeline. Experiments showed that the addition of inorganic salt and the degradation of methane-oxidizing bacteria could improve the suppression explosion effect of ultra-fine water mist, and the suppression explosion effect was related to the volume of water mist. Under the same ultra-fine water mist condition, with the increase of the volume of water mist, the explosion suppression effect was improved. Compared with pure methane, pure water ultra-fine water mist, and inorganic salt ultra-fine water mist, the maximum explosion overpressure and flame propagation speed under the condition of bacteria-inorganic salt ultra-fine water mist were significantly reduced. Compared with the explosion of pure methane, due to the degradation of methane by methane-oxidizing bacteria, when the degradation time was 10 h, and the volume of ultra-fine water mist containing bacteria-inorganic salt was 12.5 mL, the maximum explosion overpressure dropped significantly from 0.663 to 0.343 MPa, a decrease of 48.27%. The appearance time of the maximum explosion overpressure was delayed from 208.8 to 222.6 ms. The peak flame velocity was 4 m s⁻¹, which was 83.3% lower than that of 9.8% pure methane explosion. This study will contribute to the development of efficient ultrafine water mist synergistic inhibitors for the prevention of methane explosion disasters.
... In practice, lag phases or low efficiencies in the CH 4 oxidation of traditional LCS have been commonly found due to the slow growth of MOB in the medium (Yargicoglu and Reddy, 2018). To solve the problem, maximizing the cell density of MOB through an enrichment process and applying their cultivation is promising (Jiang, et al., 2010). However, in addition to MOB abundance, the MOB community structure is another major factor regulating methanotrophic activity (Malghani et al., 2016). ...
... The methylotrophic bacteria Methylosinus sporium, Methylosinus trichosporium, Methylocystis hirsuta, Methylocystis parvus, and Methylocella tundrae are methanotrophic microorganisms, also called methane-oxidizing bacteria (MOB), capable of generating energy through the oxidation of methane gas [61]. MOB have different biotechnological applications, mainly the biological mitigation of the methane greenhouse gas, the production of highvalue products from methane, and the bioremediation of pollutants [62,63]. Because of the ecological importance of these microorganisms, it is important to understand how different conditions can affect methane consumption. ...
Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.
... The methylotrophic bacteria Methylosinus sporium, Methylosinus trichosporium, Methylocystis hirsuta, Methylocystis parvus and Methylocella tundrae are methanotrophic microorganisms also called methane-oxidizing bacteria (MOB) capable of generating energy through the oxidation of methane gas [71]. MOB have different biotechnological applications mainly, biological mitigation of methane greenhouse gas, production of high-value products from methane, and bioremediation of pollutants [72,73]. Because of ecological importance of these microorganisms, it is important understand how different conditions can affect methane consumption. ...
Many bacteria have the ability to survive in challenging environments; however, they cannot grow on standard culture media, a phenomenon known as the Viable but Non-Culturable (VBNC) state. Bacteria commonly go into the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The revision also covered the significance and applications of beneficial bacteria, methods for evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited, however, beneficial bacteria in this state could face adverse environmental conditions, and return to culturable state when conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics, functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yield and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.
... WS11 and showed that this isoprene degrader could co-oxidise a broad range of alkenes, with preference for those with alkyl side chains. sMMO from methanotrophs transforms an extensive range of over 100 non-growth substrates including C 2 -C 9 alkanes and C 2 -C 5 alkenes, halogenated hydrocarbons, and aromatics such as styrene and toluene (Colby et al., 1977;Jiang et al., 2010;Smith & Dalton, 2004). Alkene monooxygenase from X. autotrophicus Py2 can catalyse the epoxidation of a range of alkenes, including trichloroethylene, and aromatics, but not alkanes (van Ginkel et al., 1987;Zhou et al., 1999). ...
Co‐oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene‐degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1‐alkynes (C2–C8) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1‐octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1‐octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co‐oxidation of isoprene by other oxygenase‐containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase‐expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co‐oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co‐oxidise isoprene at relatively high rates, suggesting that co‐oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.
... However, the limited solubility of hydrogen and methane in the culture media makes them less accessible to methanotrophs, resulting in slow growth, reduced biomass production, and suboptimal activity of the MMO enzyme, ultimately leading to decreased methanol production [104]. Thus, a significant challenge in this process is the effective transfer of gaseous substrates into the liquid culture medium [105]. Overcoming this limitation necessitates a coordinated effort. ...
... Methylotrophs can utilise reduced carbon substrates without carbon-carbon bonds (i.e., C1 substrates) as their source of carbon and energy [64]. Methanotrophs, the methylotrophs that can utilise the potent greenhouse gas methanol, are of special interest with regard to climate change [91]. While we detected diverse methylotrophs such as OM43 (family Methylophilaceae), Methylophaga (family Methylophagaceae), and Filomicrobium (family Hyphomicrobiaceae) in the microbiome, the genus Methyloceanibacter of family Methyloligellaceae and Leisingera of family Rhodobacteraceae were the most dominant and consistently associated. ...
Ostreobium, the major algal symbiont of the coral skeleton, remains understudied despite extensive research on the coral holobiont. The enclosed nature of the coral skeleton might reduce the dispersal and exposure of residing bacteria to the outside environment, allowing stronger associations with the algae. Here, we describe the bacterial communities associated with cultured strains of 5 Ostreobium clades using 16S rRNA sequencing. We shed light on their likely physical associations by comparative analysis of three datasets generated to capture (1) all algae associated bacteria, (2) enriched tightly attached and potential intracellular bacteria, and (3) bacteria in spent media. Our data showed that while some bacteria may be loosely attached, some tend to be tightly attached or potentially intracellular. Although colonised with diverse bacteria, Ostreobium preferentially associated with 34 bacterial taxa revealing a core microbiome. These bacteria include known nitrogen cyclers, polysaccharide degraders, sulphate reducers, antimicrobial compound producers, methylotrophs, and vitamin B12 producers. By analysing co-occurrence networks of 16S rRNA datasets from Porites lutea and Paragoniastrea australensis skeleton samples, we show that the Ostreobium-bacterial associations present in the cultures are likely to also occur in their natural environment. Finally, our data show significant congruence between the Ostreobium phylogeny and the community composition of its tightly associated microbiome, largely due to the phylosymbiotic signal originating from the core bacterial taxa. This study offers insight into the Ostreobium microbiome and reveals preferential associations that warrant further testing from functional and evolutionary perspectives.
... One promising way to mitigate GHG emissions from the waste sector is to convert methane produced by methanogens into value-added carbon. By using the metabolic capacities of methanotrophic microorganisms, 22 it is possible to convert methane into higher-value products such as methanol, polyhydroxyalkanoates (PHA), biopolymers and single-cell proteins (SCPs). 23 The first step in these conversions is methane oxidation to methanol catalysed by the soluble form of methane monooxygenase (sMMO) or its particulate form (pMMO) (Eqn 1). ...
The emission of greenhouse gases (GHGs) from the treatment of municipal, agricultural and industrial waste occurs in virtually every city on our planet. This is due to various microbial activities at different stages of waste treatment. Traditional treatment methods have a significant environmental impact, producing methane, carbon dioxide and nitrous oxide emissions, in addition to demanding high energy input and having low treatment efficiencies. To address these issues, the Australian water and waste sectors are shifting towards the adoption of next-generation, carbon-neutral treatment options. Here I discuss our current knowledge gaps in mitigating GHG emissions from waste streams, with a focus on wastewater treatment plants. I highlight the application of real-time genomics to identify sources of GHG emissions, monitor mitigation efforts, assist process operation and guide plant operations. I also emphasise recent innovations of microbial processes that capture GHG from waste and upgrade them into higher value products. Ultimately, combined effort across disciplines is required to proactively mitigate the global threat of climate change.
... Due to its low specificity, MMO can oxidize different recalcitrant compounds such as alkanes and aromatic hydrocarbons [3][4][5]. Therefore, methanotrophs are broadly used as biocatalysts in many biotechnological processes, such as in biodegradation systems for methane as a greenhouse gas and recalcitrant contaminants [6,7]. Most methanotrophs have been considered to be obligate to methane, but some are facultative methanotrophs that can utilize multi-carbon compounds including ethanol and organic acids, such as acetate, pyruvate, and malate [8,9]. ...
Microbacterium elymi KUDC0405T was isolated from the rhizosphere of Elymus tsukushiensis from the Dokdo Islands. The KUDC0405T strain was Gram-stain-positive, non-spore forming, non-motile, and facultatively anaerobic bacteria. Strain KUDC0405T was a rod-shaped bacterium with size dimensions of 0.3-0.4 × 0.7-0.8 μm. Based on 16S rRNA gene sequences, KUDC0405T was most closely related to Microbacterium bovistercoris NEAU-LLET (97.8%) and Microbacterium pseudoresistens CC-5209T (97.6%). The dDDH (digital DNA-DNA hybridization) values between KUDC0405T and M. bovistercoris NEAU-LLET and M. pseudoresistens CC-5209T were below 17.3% and 17.5%, respectively. The ANI (average nucleotide identity) values among strains KUDC0405T , M. bovistercoris NEAU-LLET , and M. pseudoresistens CC-5209T were 86.6% and 80.7%, respectively. The AAI (average amino acid identity) values were 64.66% and 64.97%, respectively, between KUDC0405T and its closest related type strains. The genome contained 3,596 CDCs, three rRNAs, 46 tRNAs, and three non-coding RNAs (ncRNAs). The genomic DNA GC content was 70.4%. The polar lipids included diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and unknown phospholipid, and the major fatty acids were anteiso-C17:0 and iso-C16:0. Strain KUDC0405T contained MK-12 as the major menaquinone. Based on genotypic, phylogenetic, and phenotypic properties, strain KUDC0405T should be considered a novel species within the genus Microbacterium, for which we propose the name M. elymi sp. nov., and the type strain as KUDC0405T (=KCTC 49411T , =CGMCC1.18472T ).
... Crenothrix polyspora and Candidatus Clonothrix fusca in the Crenotrichaceae family are also Type I methanotrophs (Stoecker et al., 2006;Hirayama et al., 2013;Knief, 2015). Type II methanotrophs are affiliated to the families Methylocystaceae and Beijerinckiaceae of Alphaproteobacteria (Dumont et al., 2014), including Methylocystis, Methylosinus, Methylocapsa, Methylocella, and Methyloferula (Hanson and Hanson, 1996;Jiang et al., 2010;Park and Lee, 2013). Some uncultivated methanotroph clusters containing the pmoA gene also belong to Type I methanotrophs, such as rice paddy clusters (RPC), upland soil clusters (USCα and USCγ), jasper ridge clusters (JR1, JR2, and JR3), freshwater cluster (FWs), deep-sea clusters (deep-sea-2) (Knief, 2015). ...
The extent to which propagule limitation can govern the responses of microbially-mediated processes (such as methane oxidation) to sudden environmental changes, is poorly understood. Here, we compared the ability of the methanotroph community in lakeshore soils of two lakes to respond to an experimental increase in salinity. One set of samples was taken from lakeshore soils of a freshwater lake (Yang Lake), the other from a slightly brackish lake (Qinghai Lake), both on the Tibetan Plateau. Samples were incubated in microcosms by adding ∼ 5 % ¹³CH4 or ¹²CH4 and different concentrations of NaCl solution. DNA stable-isotope probing (DNA-SIP) followed by high-throughput sequencing was used to determine how the active methanotrophic populations differed in lakeshore soils with different salinity levels. Samples from saline and freshwater lake initially showed much-reduced methane oxidation ability and methanotrophic activity at increased salinity. For the freshwater samples with the salinity of 25 to 50 g/L after NaCl addition, there was no adaptation and increase in methanotrophy after 7 days. By contrast, samples from the brackish lake showed an initial depression of methane oxidation, followed by greatly increased rates after several days. Sequencing revealed that this recovery of methanotrophy in the brackish lake samples was associated with a major switchover in composition of active methanotroph community. In particular, the relative abundance of Type Ⅰa methanotrophs became more abundant at increased salinity. It appears that in this freshwater lake environment, isolation from any nearby high-salinity-tolerant bacterial sources has prevented the possibility of full adaptation to a high salinity change in the environment, and only a moderate salinity adaptation is possible by species-sorting from within the existing community. By contrast, in the higher-salinity environment, the highly salinity-tolerant Methylomicrobium was able to break the establishment limitation in the high salinity environment and become the dominant methanotroph. Our study provides an instance of propagule limitation preventing adaptation to changed conditions.
... Methanotrophic bacteria can utilize single-carbon compounds such as methane as their sole source of carbon and energy. They play an important role in the global methane cycle and show promise as industrial platforms for the conversion of methane from impure sources, such as industrial waste streams, into a wide array of valuable products ranging from isoprenoids to polyhydroxyalkanoates and ectoines, among others (Cantera et al., 2018;Henard et al., 2017;Jiang et al., 2010;Khmelenina et al., 1999;Lee et al., 2016;Strong et al., 2015). Despite first being characterized in the early 1900s (Kaserer, 1905;Söhngen, 1906), much about methanotroph biology has yet to be elucidated, including the prevalence and impact of bacteriophages (phages) in these microorganisms. ...
Methanotrophs are a unique class of bacteria with the ability to metabolize single-carbon compounds such as methane. They play an important role in the global methane cycle and have great potential as industrial platforms for the bioconversion of methane from industrial waste streams into valuable products, such as biofuels and bioplastics. However, many aspects of methanotroph biology have yet to be elucidated, including the prevalence and impact of lysogenized bacteriophages (phages), which can greatly affect both the ecology and the industrial performance of these bacteria. The present study investigates the presence of putative prophages in three gammaproteobacterial (Methylobacter marinus A45, Methylomicrobium album BG8, Methylomonas denitrificans FJG1) and two alphaproteobacterial (Methylosinus trichosporium OB3b, Methylocystis sp. Rockwell) methanotrophs using four programs predicting putative phage sequences (PhageBoost, PHASTER, Phigaro, and Island Viewer). Mitomycin C was used to trigger induction of prophages, which was monitored through infection dynamics. Successfully induced phages from M. marinus A45 (MirA1, MirA2), M. album BG8 (MirB1), and M. trichosporium OB3b (MirO1) were isolated and characterized using transmission electron microscopy. Subsequently, bioinformatic analyses (BLAST and phylogenetics) were performed on three induced phages to obtain a profile of their respective genetic makeup. Their broad diversity and differences from previously known phages, based on whole genome and structural gene sequences, suggest they each represent a new phage family, genus and species: "Britesideviridae Inducovirus miraone", "Patronusviridae Enigmavirus miratwo", and "Kainiviridae Tripudiumvirus miroone" represented by isolates MirA1, MirA2, and MirO1, respectively.
... The unique ability of methanotrophs to metabolize CH 4 is catalyzed by an enzyme called methane monooxygenase (MMO) (Bowman, 2006). Methanotrophs are part of bigger group of bacteria called methylotrophs which can utilize wide range of C1 compounds other than CH 4 such as methanol, methylated amines, halomethanes, and methylated compounds containing sulfur (Hanson and Hanson, 1996;Bowman, 2006;Jiang et al., 2010). Landfill cover soils have shown wide diversity of methanotrophs and methylotrophs. ...
Biochar-amended soils have been explored to enhance microbial methane (CH4) oxidation in landfill cover systems. Recently, research priorities have expanded to include the mitigation of other components of landfill gas such as carbon dioxide (CO2) and hydrogen sulfide (H2S) along with CH4. In this study, column tests were performed to simulate the newly proposed biogeochemical cover systems, which incorporate biochar-amended soil for CH4 oxidation and basic oxygen furnace (BOF) slag for CO2 and H2S mitigation, to evaluate the effect of cover configuration on microbial CH4 oxidation and community composition. Biogeochemical covers included a soil cover, a biochar-amended soil cover (10% w/w), and methanotroph-enriched activated biochar amended soil covers (5% or 10% w/w). The primary outcome measures of interest were CH4 oxidation rates and the structure and abundance of methane-oxidation bacteria in the covers. All column reactors were active in CH4 oxidation, but columns containing activated biochar-amended soils had higher CH4 oxidation rates (133 to 143 μg CH4 g⁻¹ day⁻¹) than those containing non-activated biochar-amended soil (50 μg CH4 g⁻¹ day⁻¹) and no-biochar soil or control soil (43 μg CH4 g⁻¹ day⁻¹). All treatments showed significant increases in the relative abundance of methanotrophs from an average relative abundance of 5.6% before incubation to a maximum of 45% following incubation. In activated biochar, the abundance of Type II methanotrophs, primarily Methylocystis and Methylosinus, was greater than that of Type I methanotrophs (Methylobacter) due to which activated biochar-amended soils also showed higher abundance of Type II methanotrophs. Overall, biogeochemical cover profiles showed promising potential for CH4 oxidation without any adverse effect on microbial community composition and methane oxidation. Biochar activation led to an alteration of the dominant methanotrophic communities and increased CH4 oxidation.
... The isobutanol production process involves both biomass generation and isobutanol biosynthesis by either cyanobacterium cultured in closed tubular photobioreactors (PBR) with 50 m 3 working volume [26] or methanotrophs grown in 1000 m 3 bubble column bioreactors (BCB) with a working volume of 80% [25,27]. The stoichiometry equations for biomass generation by cyanobacteria (Eq.1) and methanotrophs (Eq.2) applied in Aspen simulations have been proposed based upon published literature [28,29], in which the empirical formula of "CH 1.934 O 0.473 N 0.23 " and "C 4 H 8 O 2 N" stands for the biomass from cyanobacteria and methanotrophs, respectively. The stoichiometry equation for isobutanol biosynthesis in cyanobacteria and methanotrophs has been assumed as Eq. 3 and Eq. 4, respectively. ...
Background
The dramatic increase in emissions of greenhouse gases (GHGs) has led to an irreversible effect on the ecosystem, which in turn caused significant harm to human beings and other species. Exploring innovative and effective approaches to neutralizing GHGs is urgently needed. Considering the advancement of synthetic biology and the bioconversion process, C1-utilizing cell factories (CUCFs) have been modified to be able to effectively convert C1-gases includes biogas, natural gas, and carbon dioxide (CO 2 ) into chemicals or fuels via biological routes, which greatly facilitates the inedible carbon sources used in biomanufacturing, increases the potential value of GHGs and meanwhile reduces the GHG emissions. Process design and resultsEven though the current experimental results are satisfactory in lab-scale research, the evaluation of economic feasibility as well as applications of CUCFs in industrial-scale still need to be analyzed. This study designed three scenarios of CUCFs-based conversion of biogas, natural gas, and CO 2 into isobutanol, the detailed techno-economic analyses of these scenarios were conducted with the comparisons of capital cost, operating cost, and minimum isobutanol selling price (MISP). Results revealed that direct bio-conversion of CO 2 by CUCFs into isobutanol exhibited the best economic performance with a MISP of /kg with using ideal targets. Conclusions
Our findings provide a comprehensive assessment of bio-conversion of C1-gases via CUCFs to isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and environmental impact. It is expected that this study may lead to the paradigm shift in isobutanol synthesis with C1-gases as substrates.
... Furthermore, co-metabolism results in the transformation of OMPs by nonspecific enzymes produced by microorganisms using a different substrate for their primary metabolism (Alexander, 1981;Benner et al., 2015). Nitrifying and methanotrophic bacteria are able to co-metabolically degrade OMPs though the activity of their ammonia monooxygenase enzyme (AMO) (Roh et al., 2009;Rattier et al., 2014) and methane monooxygenase enzyme (MMO) (Jiang et al., 2010;Semrau et al., 2010;Papadopoulou et al., 2019), respectively. Rod et al. (2009) reported that AMO can not only promote ammonia oxidation, but also degrades a number of hydrocarbon compounds as well. ...
The presence of organic micropollutant (OMP) in groundwater threatens drinking water quality and public health. Rapid sand filter (RSF) rely on biofilms with nitrifying and methanotrophic bacteria to remove ammonia and methane during drinking water production. Previous research observed the partial removal of OMPs with active nitrification and methane oxidation due to co-metabolic conversion of OMPs. However, the contribution of indigenous nitrifying and methanotrophic communities from RSF has yet to be fully explored. Accordingly, experiments were carried out with biofilm-covered sand collected from field-scale RSF, to assess the removal of nine OMPs by nitrifying and methanotrophic bacteria. Results indicated that stimulating nitrification resulted in significantly more removal of caffeine, 2,4-dichlorophenoxyacetic acid and bentazone. Stimulating methanotrophic conditions enhanced the removal of caffeine, benzotriazole, 2,4-dichlorophenoxyacetic acid and bentazone. Microbial community analysis based on 16 S rRNA gene sequencing revealed Nitrosomonas and Nitrospira are the dominant genus in the community under nitrifying conditions. The three genera Methylobacter, Methylomonas and Methylotenera were enriched under methanotrophic conditions. This study highlights that nitrifying and methanotrophic bacteria play important roles during OMP removal in field RSF. Furthermore, results suggest that bioaugmentation with an enriched nitrifying and methanotrophic culture is a promising approach to improve OMP removal in RSF.
Climate change and global warming are serious threats to our planet, affecting millions of people and economic development. They are the result of excess emissions of greenhouse gases (GHGs), which escalate the greenhouse effect (GE). GHGs are emitted to the atmosphere through natural biogeochemical cycles induced by different groups of organisms present in both terrestrial and marine ecosystems, which are essential for maintaining balance with the environment. However, the abrupt rise in human population has disrupted natural biogeochemical cycles and led to additional emissions from anthropogenic activities, resulting in more emissions than absorption. The anthropogenic sources of GHGs to the atmosphere, induced by the rapid increase in human population, are industrialization, transportation, heat and electricity production, manufacturing and construction, and industrial processes. Emissions from the soil ecosystem are solely due to the activities of organisms that inhabit the soil, and their emission rates rely on some important abiotic factors, especially pH, oxygen level, nutrient load, and carbon. The major GHGs associated with GE, emitted from both natural and artificial sources, include carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and fluorinated gases. Organisms not only emit GHGs, but they also absorb these gases for their metabolic pathways. Different soil microbes depend on specific substrates for their growth and development. Soil is teeming with different organisms and is by far the most biologically diverse part of Earth. The majority of important organisms in soil have yet to be explored. Exploring organisms that can utilize potent GHGs is a possible option to mitigate climate change. There are soil microorganisms that can utilize highly potent GHGs and reduce them to something that is no longer a GHG.
The COVID-19 pandemic led to an immediate lockdown declared by the government of India (March to April 2020) which saw a decrease in public activities. In view of this, a comparative study was carried out on the influence of environmental parameters on methane activities between the pre-lockdown and lockdown periods along a tropical estuarine mangrove ecosystem. Significant changes in the studied parameters specially in the organic matter and methane production rates (P < 0.05) were observed. During the lockdown as compared to the pre-lockdown period, the downstream site was impacted more than those at the upstream site with an average methane production decrease by 60X and 1.5X respectively. Both, oxidation and production of methane decreased during the lockdown and was related to organic matter availability (P < 0.05). The findings elucidate the positive impact of lockdown on the model ecosystem and the reduction in greenhouse methane activities.
Graphical abstract
Trong các năm gần đây, nồng độ khí methane tăng lên đột biến do các hoạt động sản xuất nông nghiệp, trong đó chăn nuôi có nguồn phát thải khí methane đáng kể, một trong những tác nhân chính gây ra sự phá hủy tầng ozone. Ở nghiên cứu này đã phân lập và sàng lọc được một số chủng vi khuẩn có khả năng oxy hóa methane (Methane Oxidizing Bacteria - MOB). Từ 56 mẫu bao gồm: đất bùn, mẫu đất trồng lúa, mẫu nước thải biogas, mẫu dạ cỏ và mẫu nước sông, chúng tôi đã phân lập được 370 chủng vi khuẩn có khả năng oxy hóa khí CH4 trên môi trường dAMS với CH4 là nguồn carbon duy nhất. Qua kết quả định lượng của 18/370 chủng vi khuẩn có khả năng làm giảm CH4, trong đó có hai chủng TB18 và DC11 được phân lập từ đất trồng lúa và dạ cỏ có khả năng oxy hóa methane cao nhất (32.6 ± 0.25%, 30.2 ± 0.14%). Kết quả của nghiên cứu này là tiền đề cho việc ứng dụng vi khuẩn MOB vào sản xuất chế phẩm vi sinh giảm phát thải khí CH4 trong quá trình canh tác lúa hay trong chăn nuôi gia súc ở Việt Nam.
Currently the majority of carbon-based feedstocks come from fossil fuels of which there are a finite supply. Methane is an abundantly available carbon-based feedstock, with large amounts now available through fracking and renewable sources available from biogas plants. However, methane is not very chemically reactive. One of the remaining “grand challenges” in chemistry is the development of clean, efficient, affordable processes that allow methane to be converted to other high value molecules.
Highlighting the recent advances in methane activation and direct conversion processes this book discusses the progress and current state of the art for a wide variety of alternative methane activation and subsequent conversion processes, including homogeneous- and heterogeneous catalytic, electro catalytic and pyrolytic systems. It is a useful resource for anyone working in green chemistry, catalysis and chemical engineering.
Geothermal areas represent substantial point sources for greenhouse gas emissions such as methane. While it is known that methanotrophic microorganisms act as a biofilter, decreasing the efflux of methane in most soils to the atmosphere, the diversity and the extent to which methane is consumed by thermophilic microorganisms in geothermal ecosystems has not been widely explored. To determine the extent of biologically mediated methane oxidation at elevated temperatures, we set up 57 microcosms using soils from 14 Aotearoa-New Zealand geothermal fields and show that moderately thermophilic (>40°C) and thermophilic (>60°C) methane oxidation is common across the region. Methane oxidation was detected in 54% (n = 31) of the geothermal soil microcosms tested at temperatures up to 75°C (pH 1.5–8.1), with oxidation rates ranging from 0.5 to 17.4 μmol g⁻¹ d⁻¹ wet weight. The abundance of known aerobic methanotrophs (up to 60.7% Methylacidiphilum and 11.2% Methylothermus) and putative anaerobic methanotrophs (up to 76.7% Bathyarchaeota) provides some explanation for the rapid rates of methane oxidation observed in microcosms. However, not all methane oxidation was attributable to known taxa; in some methane-consuming microcosms we detected methanotroph taxa in conditions outside of their known temperature range for growth, and in other examples, we observed methane oxidation in the absence of known methanotrophs through 16S rRNA gene sequencing. Both of these observations suggest unidentified methane oxidizing microorganisms or undescribed methanotrophic syntrophic associations may also be present. Subsequent enrichment cultures from microcosms yielded communities not predicted by the original diversity studies and showed rates inconsistent with microcosms (≤24.5 μmol d⁻¹), highlighting difficulties in culturing representative thermophilic methanotrophs. Finally, to determine the active methane oxidation processes, we attempted to elucidate metabolic pathways from two enrichment cultures actively oxidizing methane using metatranscriptomics. The most highly expressed genes in both enrichments (methane monooxygenases, methanol dehydrogenases and PqqA precursor peptides) were related to methanotrophs from Methylococcaceae, Methylocystaceae and Methylothermaceae. This is the first example of using metatranscriptomics to investigate methanotrophs from geothermal environments and gives insight into the metabolic pathways involved in thermophilic methanotrophy.
Methane is abundant in nature, and excessive emissions will cause the greenhouse effect. Methane is also an ideal carbon and energy feedstock for biosynthesis. In the review, the microorganisms, metabolism, and enzymes for methane utilization, and the advances of conversion to value-added bioproducts were summarized. First, the physiological characteristics, classification, and methane oxidation process of methanotrophs were introduced. The metabolic pathways for methane utilization and key intermediate metabolites of native and synthetic methanotrophs were summarized. Second, the enzymatic properties, crystal structures, and catalytic mechanisms of methane-oxidizing and metabolizing enzymes in methanotrophs were described. Third, challenges and prospects in metabolic pathways and enzymatic catalysis for methane utilization and conversion to value-added bioproducts were discussed. Finally, metabolic engineering of microorganisms for methane biooxidation and bioproducts synthesis based on different pathways were summarized. Understanding the metabolism and challenges of microbial methane utilization will provide insights into possible strategies for efficient methane-based synthesis.
In this work, the novel N-damo (Nitrite dependent anaerobic methane oxidation) process was investigated at high biomass activities for its potential to remove simultaneously nitrite and methane, as well as selected antibiotics commonly found in sewage in trace amounts. For this purpose, two MBRs were operated at three high nitrite loading rates (NLRs), namely 76 ± 9.9, 161.5 ± 11.4 and 215.2 ± 24.2 mg N-NO⁻2 L-1 d-1, at long-term operation. The MBRs performance achieved a significantly high nitrite removal activity for an N-damo process (specific denitrifying activity of up to 540 mg N-NO⁻2 g-1 VSS d-1), even comparable to heterotrophic denitrification values. In this study, we have implemented a novel operational strategy that sets our work apart from previous studies with similar bioreactors. Specifically, we have introduced Cerium as a trace element in the feeding medium, which serves as a key differentiating factor. It allowed maintaining a stable reactor operation at high NLRs. Microbial community composition evidenced that both MBRs were dominated with N-damo bacteria (67-87% relative abundance in period III and I, respectively). However, a decrease in functional N-damo bacteria (Candidatus Methylomirabilis) abundance was observed during the increase in biomass activity and concentration, concomitantly with an increase of the other minor families (Hypomicrobiaceae and Xanthobacteraceae). Most of the selected antibiotics showed high biotransformation such as sulfamethoxazole, trimethoprim, cefalexin and azithromycin, whereas others such as roxithromycin and clarithromycin were only partially degraded (20-35%). On the contrary, ciprofloxacin showed almost no removal. Despite the metabolic enhancement, no apparent increase on the antibiotic removal was observed throughout the operation, suggesting that microbiological composition was of greater influence than its primary metabolic activity on the removal of antibiotics.
Flaring of associated gas from oil wells and the excess gas from gas-processing units and oil refineries is one of the most prominent producers of greenhouse gas emissions. Flaring, by definition, is a method used to burn unwanted flammable gas, which produces significant amounts of methane, carbon dioxide, nitrogen oxide, and sulfur oxide. The petroleum industry adds millions of tons of CO2 annually into the atmosphere by flaring gas, which presents a serious risk due to the environmental and economic impacts associated with it. In light of the increasing awareness of this threat, the industry is investigating economical means to reduce the anthropogenic release of greenhouse gases into the atmosphere. Oil production from the Bakken and Three Forks formations has significantly increased over the last ten years without commensurate augmentation of gas capture infrastructure, which, consequently, resulted in increased flaring of the associated gas. The North Dakota Industrial Commission (NDIC) has set rigorous regulations to reduce flaring. However, operating companies are experiencing challenges to meet NDIC gas capturing limit of 95%, which leads to oil production being curtailed. This paper presents an overview of the latest technologies implemented worldwide to reduce gas flaring and discusses their applicability as well as the advantages and disadvantages of each method. Then, to evaluate North Dakota’s flaring situation, the amount of gas flaring within the recent years is provided. It is discussed that implementation of underground gas storage and methanol portable units may be cost-effective measures to meet the North Dakota gas-capturing objective. A successful implementation of gas recovery technologies can significantly reduce gas emissions and gain potential economic profit.
Methanotrophs play key roles in global methane cycling and are promising platforms for methane bioconversion. However, major gaps existing in fundamental knowledge undermines understanding of these methane-consuming microorganisms. To associate genes with a phenotype at the genome-wide level, we developed a Cre/lox-mediated method for constructing a large-scale CRISPRi library in a model methanotroph Methylotuvimicrobium buryatense 5GB1C. The efficiency of this Cre mediated integration method was up to a level of 10⁵ CFU/μg DNA. Targeting 4,100 predicted protein-coding genes, our CRISPRi pooled screening uncovered 788 core genes for the growth of strain 5GB1C using methane. The core genes are highly consistent with the gene knockout results, indicating the reliability of the CRISPRi screen. Insights from the core genes include that annotated isozymes generally exist in metabolic pathways and many core genes are hypothetical genes. This work not only provides functional genomic data for both fundamental research and metabolic engineering of methanotrophs, but also offers a method for CRISPRi library construction.
IMPORTANCE Due to their key role in methane cycling and their industrial potential, methanotrophs have drawn increasing attention. Genome-wide experimental approaches for gene-phenotype mapping accelerate our understanding and engineering of a bacterium. However, these approaches are still unavailable in methanotrophs. This work has two significant implications. First, the core genes identified here provide functional genetic basics for complete reconstruction of the metabolic network and afford more clues for knowledge gaps. Second, the Cre-mediated knock-in method developed in this work enables large-scale DNA library construction in methanotrophs; the CRISPRi library can be used to screen the genes associated with special culture conditions.
Ostreobium, the major algal symbiont of the coral skeleton, remains understudied despite extensive research on the coral holobiont. The enclosed nature of the coral skeleton might reduce the dispersal and exposure of residing bacteria to the outside environment, allowing stronger associations with the algae. Here, we describe the bacterial communities associated with cultured strains of 5 Ostreobium clades using 16S rRNA sequencing. We shed light on their likely physical associations by comparative analysis of three datasets generated to capture (1) all algae associated bacteria (2) enriched tightly attached and potential intracellular bacteria and (3) bacteria in spent media. Our data showed that while some bacteria may be loosely attached, some tend to be tightly attached or potentially intracellular. Although colonised with diverse bacteria, Ostreobium preferentially associated with 34 bacterial taxa revealing a core microbiome. These bacteria include taxa known as nitrogen cyclers, polysaccharide degraders, sulphate reducers, antimicrobial compound producers, methylotrophs and vitamin B12 producers. By analysing co-occurrence networks of 16S rRNA datasets from Porites lutea and Paragoniastrea australensis skeleton samples, we show that the Ostreobium-bacterial associations present in the cultures are likely to also occur in their natural environment. Finally, our data show significant congruence between the Ostreobium phylogeny and the community composition of its tightly associated microbiome, largely due to the phylosymbiotic signal originating from the core bacterial taxa. This study offers insight into the Ostreobium microbiome and reveals preferential associations that warrant further testing from functional and evolutionary perspectives.
Background
The dramatic increase in greenhouse gas (GHG) emissions, which causes serious global environmental issues and severe climate changes, has become a global problem of concern in recent decades. Currently, native and/or non-native C1-utilizing microbes have been modified to be able to effectively convert C1-gases (biogas, natural gas, and CO2) into isobutanol via biological routes. Even though the current experimental results are satisfactory in lab-scale research, the techno-economic feasibility of C1 gas-derived isobutanol production at the industrial scale still needs to be analyzed and evaluated, which will be essential for the future industrialization of C1-gas bioconversion. Therefore, techno-economic analyses were conducted in this study with comparisons of capital cost (CAPEX), operating cost (OPEX), and minimum isobutanol selling price (MISP) derived from biogas (scenario #1), natural gas (scenario #2), and CO2 (scenario #3) with systematic economic assessment.
Results
By calculating capital investments and necessary expenses, the highest CAPEX (67 MM) were projected in scenario #1 and scenario #2, respectively. Because of the lower CAPEX and OPEX from scenario #3, the results revealed that bioconversion of CO2 into isobutanol temporally exhibited the best economic performance with an MISP of 0.99/kg in a long-term case study.
Conclusions
This study provides a comprehensive assessment of the bioconversion of C1-gases into isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and minimum selling price. Compared with isobutanol derived from biogas and natural gas, the CO2-based isobutanol showed better economic feasibility. A market competitive isobutanol derived from CO2 is predicable with lower CO2 cost, better isobutanol titer, and higher annual capacity. This study will help researchers and decision-makers explore innovative and effective approaches to neutralizing GHGs and focus on key economic-driving forces to improve techno-economic performance.
This study examined soil, sediment and groundwater microbial communities for a set of key functional genes important for contaminant biodegradation. This involved PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) predictions based on 16S rRNA gene amplicon datasets from three separate studies with different inocula and incubation conditions, as follows: aerobic soils, oxygen-limited microcosms containing sediments and groundwater, as well as methanogenic microcosms with different inocula. PICRUSt2 predicts functional profiles of microbial communities based on marker gene (16S rRNA gene) data. The relative abundances of genera previously associated with the biodegradation of chlorinated solvents/metabolites and/or 1,4-dioxane were also determined.
Predicted values for each functional gene varied between the three datasets. In all, values were high for propane monooxygenase and low for soluble methane monooxygenase. Common phylotypes associated with propane monooxygenase in two of the three datasets included Mycobacterium, Rhodococcus and Pseudonocardia. Toluene monooxygenase predicted values were greater in the oxygen-limited microcosms compared to the other two datasets. The methanogenic microcosms exhibited the highest predicted values for particulate methane/ammonia monooxygenase. The most common genera detected, previously reported as chlorinated solvents/metabolites and/or 1,4-dioxane degraders, included Pseudomonas, Sphingomonas, Rhodococcus and Rhodanobacter. Eighteen of the queried genera were not detected. As expected, more potential anaerobic degrading genera were found in the oxygen-limited and methanogenic microcosms compared to the aerobic soils. The results provide key insights as to which genes and genera may be important for biodegradation over a range of inocula and redox conditions.
This review provides an overview of different C1 building blocks as substrates of enzymes, or part of their cofactors, and the resulting functionalized products. There is an emphasis on the broad range of possibilities of biocatalytic one-carbon extensions with C1 sources of different oxidation states. The identification of uncommon biosynthetic strategies, many of which might serve as templates for synthetic or biotechnological applications, towards one-carbon extensions is supported by recent genomic and metabolomic progress and hence we refer principally to literature spanning from 2014 to 2020.
1 Introduction
2 Methane, Methanol, and Methylamine
3 Glycine
4 Nitromethane
5 SAM and SAM Ylide
6 Other C1 Building Blocks
7 Formaldehyde and Glyoxylate as Formaldehyde Equivalents
8 Cyanide
9 Formic Acid
10 Formyl-CoA and Oxalyl-CoA
11 Carbon Monoxide
12 Carbon Dioxide
13 Conclusions
Increasing environmental concentrations of methane have a huge impact on the Earth's climate, owing to its high global warming potential. Thus, its biological conversion by methane oxidizing bacteria, i.e., methanotrophs offer an environmentally friendly and cost-efficient solution to combat climate change. Advances in the omics-based study of methanotrophs inhabiting different environments have greatly improved our understanding of their diversity and metabolic potential. This chapter provides a detailed insight into the emerging technologies for biological conversion of methane by methanotrophs, their immense potential of methane utilization to mitigate climate change along with the production of high-value bioproducts and biomaterials such as biofuels, biodiesel, biopolymers, single-cell protein, ectoine, surface layers, organic acids, etc. Further, challenges and opportunities associated with technology upgradation, genetic engineering, and the application of synthetic biology for methane bioconversion processes and biorefineries are discussed.
Diatoms are among the opaquest photosynthetic microorganism found in oceans, rivers, and freshwaters. They play a major role in reducing global warming as they fix more than 25% of atmospheric carbon di oxide (CO2). They are a reservoir of untapped potential with the multifaceted application including CO2 mitigation, play a vital role in the aquatic food web as primary producers, and wastewater remediation by quenching pollutants originating from diverse sources such as industries, agricultural, and human sources. Despite their abundance and diversity in nature, only a few species are currently used for biotechnological applications. Diatom biorefinery has gained importance in recent years as more and more algae are identified and explored as a source for lipids, pigments, and other biomolecules. In this chapter, the role of diatom biorefinery has been elaborated extensively displaying the potential of diatoms in carbon dioxide (CO2) mitigation, lipid production for biofuel, nutraceutical potential, and development of new-age drug molecules for therapeutic applications.
Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.
The intracellular location of methane mono-oxygenase (MMO) (soluble or particulate) in Methylosinus trichosporium OB3b is dependent on the availability of copper in the growth medium. Raising the Cu2+ concentration from 1 μM to 5 μM effected a transition from soluble to particulate MMO activity, and changes in major cell polypeptides were observed on SDS-polyacrylamide gels. Organisms containing soluble MMO oxidized a wide range of substrates including n-alkanes, n-alkenes, aromatic and alicyclic compounds. By contrast, organisms containing particulate MMO did not oxidize aromatic or alicyclic compounds. These observations provide further evidence that the two types of MMO are fundamentally different.
~~ ~~ ~ ~ In cicv 13C NMR has been used to observe metabolism of exogenously supplied methanol by suspensions of Methylosinus trichosporium OB3b grown under a variety of conditions. Formaldehyde, formate and bicarbonate ions were the only metabolites of methanol to be detected. Accumulation of formaldehyde was observed only with suspensions grown under conditions which yield particulate, membrane-bound, methane mono-oxygenase (MMO). Ethyne abolished MMO activity, partially inhibited methanol oxidation in whole organisms, and prevented growth of the organism on methanol (1 %, v/v) in batch culture. Oxidation of ethanol, a substrate of methanol dehydrogenase, was not affected by ethyne. Ethyne caused accumulation of formaldehyde in all suspensions of the organism incubated with methanol, although oxidation of exogenously added formaldehyde was not affected. These observations are consistent with the proposal that in M. frichosporium OB3b both MMO and methanol dehydrogenase oxidize exogenously supplied methanol and suggest that the further oxidation of formaldehyde is stimulated by the consumption of reducing equivalents by MMO.
An in situ microbial filter technology is being tested and developed
for remediating migrating subsurface plumes contaminated with
low concentrations of trichloroethylene (TCE). The current focus is the
establishment of a replenishable bioactive zone (catalytic filter) along
expanding plume boundaries by the injection of a representative methanotrophic
bacterium, Methylosinus trichosporium OB3b. This microbial
filter strategy has been successfully demonstrated using emplaced,
attached resting cells (no methane additions) in a 1.1 m flow-through test
bed loaded with water-saturated sand. Two separate 24 h pulses of TCE
(109 ppb and 85 ppb), one week apart, were pumped through the system
at a flow velocity of 15 mm h"1; no TCE ( < 0.5 ppb) was detected on the
downstream side of the microbial filter. Subsequent excavation of the wet
sand confirmed the existence of a TCE-bioactive zone 21 days after it
had been created. An enhanced longevity of the cellular, soluble-form
methane monooxygenase produced by this methanotroph is a result of the
laboratory bioreactor culturing conditions. Additional experiments with
cells in sealed vials and emplaced in the 1.1 m test bed yielded a high
resting-cell finite TCE biotransformation capacity of about 0.25 mg per
mg of bacteria; this is suitable for a planned sand-filled trench field
demonstration at a Lawrence Livermore National Laboratory site.
1] Following almost a decade with little change in global atmospheric methane mole fraction, we present measurements from the Advanced Global Atmospheric Gases Experiment (AGAGE) and the Australian Common-wealth Scientific and Industrial Research Organisation (CSIRO) networks that show renewed growth starting near the beginning of 2007. Remarkably, a similar growth rate is found at all monitoring locations from this time until the latest measurements. We use these data, along with an inverse method applied to a simple model of atmospheric chemistry and transport, to investigate the possible drivers of the rise. Specifically, the relative roles of an increase in emission rate or a decrease in concentration of the hydroxyl radical, the largest methane sink, are examined. We conclude that: 1) if the annual mean hydroxyl radical concentration did not change, a substantial increase in emissions was required simultaneously in both hemispheres between 2006 and 2007; 2) if a small drop in the hydroxyl radical concentration occurred, consistent with AGAGE methyl chloroform measurements, the emission increase is more strongly biased to the Northern Hemisphere.
Trichloroethylene was shown to degrade aerobically to carbon dioxide in an unsaturated soil column exposed to a mixture of natural gas in air (0.6%).
One-carbon compounds (C1) at all oxidation levels between methane and carbon dioxide occur abundantly throughout nature. Methane is present in fossil deposits and is formed by methanogenic bacteria. Methanol arises by the hydrolysis of methyl esters and ethers such as pectin and lignin, which are present in plants. Methylated amines occur in plants and animals and are produced by microbial degradation of choline derivatives present in plant membrane material and animal tissue. Formate is a major end-product of mixed-acid fermentation and carbon dioxide is present in the atmosphere and, as carbonates, in natural waters and soil. It is not surprising, therefore, that microorganisms are found in nature which are capable of growth on such compounds as carbon and energy sources.
The current focus is the establishment of a replenishable bioactive zone (catalytic filter) along expanding plume boundaries by the injection of a representative methanotrophic bacterium, Methylosinus trichosporium OB3b. This microbial filter strategy has been successfully demonstrated using emplaced, attached resting cells (no methane additions) in a 1.1 m flow-through test bed loaded with water-saturated sand. Two separate 24 h pulses of TCE (109 ppb and 85 ppb), one week apart, were pumped through the system at a flow velocity of 15 mm h-1; no TCE (<0.5 ppb) was detected on the downstream side of the microbial filter. Subsequent excavation of the wet sand confirmed the existence of a TCE-bioactive zone 21 days after it had been created. Additional experiments with cells in sealed vials and emplaced in the 1.1 m test bed yielded a high resting-cell finite TCE biotransformation capacity of about 0.25 mg per mg of bacteria. -from Authors
A batch culture enrichment programme was used to isolate a methanol-oxidizing microbial association from landfill final covering soil. Aerobic closed cultures were also used to determine the critical substrate concentration and the catabolic ranges and optima for pH and temperature. The association was found to be active between 20 and 45°C, with an optimum at 35°C. The thermal death times at different temperatures were determined by standard methods. The association was active at pH < 8.5, with an optimum at pH 7. The maximum specific growth rate (μmax) was determined in chemostat culture at 30°C. The results of this study will be used to assess the potential efficacy of the association to remove methanol vapour and methane gas in a biofilter and, thus, to develop an operational protocol for landfill gas remediation by biofiltration.
The methane monooxygenase of methanotrophs is capable of catalyzing propene to produce epoxypropane directly in one step, without formation of by-products except water. Compared with the chemical synthesis method for production of epoxypropane, the biosynthesis has promising potential due to the mild reaction conditions, high selectivity and environmental friendliness. In this paper, the conditions were optimized for production of epoxypropane from propene with Methylosinus trichosporim OB3b whole cells cultivated by the traditional method, including temperatures, initial propene concentrations, sodium formate and MgCl2 concentrations, which are important factors affecting the epoxypropane biosythesis. Based on the optimization results, the performance of epoxypropane production carried out with M. trichosporium OB3b of high cell density was investigated, which was obtained by a novel rapid cultivation method developed by the authors. The final concentration of epoxypropane was almost four times of the highest productivity reported so far.
Methane monooxygenase is the enzyme responsible for the initial oxygenation of methane to methanol. The methanotrophs (methane-oxidizing bacteria) are divided into two types depending on whether they assimilate carbon via the ribulose monophosphate pathway (RMP) (type I) or serine pathway (type II), or both. The range of substrates oxidized and products observed from M. capsulatus and M. trichosporium extracts imply that the monooxygenases from these two organisms essentially are similar. The monooxygenase from M. methanica does not oxidize n-alkanes with more than six carbon atoms, or alicyclic, heterocyclic, and aromatic compounds. Experiments have shown that the extracts of M. methanica would catalyze the O2 and NADPH-dependent disappearance of bromomethane. They presented evidence that the methane monooxygenase was responsible for the catalysis by showing that the enzyme was particulate and stable to freezing but unstable at 2°C.
Until recently, pure cultures of only mesophilic and thermotolerant strains of methanotrophs were isolated. Studies of psychrophilic methane-oxidizing organisms are important for the elucidation of the mechanism of the low methane emission from the northern regions of Russia. This work provides the first description of a psychrophilic methanotroph. This bacterium was classified as the new species Methylobacter psychrophilus sp. nov.
Laboratory experiments were conducted with a sequencing, packed-bed bioreactor that was seeded with the methanotroph, Methylosinus trichosporium OB3b. The reactor cycled between a growth mode, in which methane and oxygen were supplied from the gas phase,
and a degradation mode, in which water containing chloroform at 100 µg/L was treated in the absence of methane. When the influent was supplemented with formate, chloroform degradation was as great as 90% at an empty bed retention time of approximately 90 minutes. The degradation
rate remained stable for several days, but steadily declined thereafter. In the absence of formate, the initial degradation rate was smaller and declined more rapidly than in the presence of formate. Mathematical modeling demonstrated that the pseudo-first-order rate constants for chloroform
degradation in the reactor were about two orders of magnitude smaller than those measured in suspended growth, batch kinetic studies. The sequencing reactor performed better than a packedbed, continuous-flow reactor, because a larger, more evenly distributed biomass could be established in
the sequencing reactor.
Biological oxidation of CH4 is an important constraint on the emission of this gas from areas, such as landfills to the atmosphere. We studied the effect of temperature on methanotrophic bacteria in three different landfill cover soils, incubated in the laboratory. In samples of a young cover, consisting of wood chips and sewage sludge, the phospholipid fatty acids (PLFAs), regarded as biomarkers for type I methanotrophs (16:1ω5t, 16:1ω6c, 16:1ω8c), primarily increased at low temperatures (5–10 °C). On the other hand, the PLFA marker for type II methanotrophs (18:1ω8c) was highly elevated only at 20 °C. These results suggest that temperature can determine the selection of methanotroph populations.
The enhanced biotransformation was accomplished by stimulating the growth of indigenous methane-oxidizing bacteria (methanotrophs), which transform chlorinated aliphatic compounds by a cometabolic process to stable, nontoxic end products. Experiments were performed in the presence and absence of biostimulation by means of controlled chemical addition, frequent sampling, and quantitative analysis. The degree of biotransformation was assessed using mass balances and comparisons with bromide as a conservative tracer. Biostimulation of the test zone was successfully achieved by injecting methane- and oxygen-containing ground water in alternating pulses under induced gradient conditions. After a few weeks of stimulation, methane concentrations gradually decreased below the detection limit within two meters of travel. Under active biostimulation conditions, 20 to 30% of the trichloroethylene was biotransformed during the first season of testing. -from Authors
Methanotrophic and nitrifying bacteria are both able to oxidize CH4 as well as NH4 +. To date it is not possible to estimate the relative contribution of methanotrophs to nitrification and that of nitrifiers to CH4 oxidation and thus to assess their roles in N and C cycling in soils and sediments. This study presents new options for discrimination between the activities of methanotrophs and nitrifiers, based on the competitive inhibitor CH3F and on recovery after inhibition with C2H2. By using rice plant soil as a model system, it was possible to selectively inactivate methanotrophs in soil slurries at a CH4/CH3F/NH4 + molar ratio of 0.1:1:18. This ratio of CH3F to NH4 + did not affect ammonia oxidation, but methane oxidation was inhibited completely. By using the same model system, it could be shown that after 24 h of exposure to C2H2 (1,000 parts per million volume), methanotrophs recovered within 24 h while nitrifiers stayed inactive for at least 3 days. This gave an “assay window” of 48 h when only methanotrophs were active. Applying both assays to model microcosms planted with rice plants demonstrated a major contribution of methanotrophs to nitrification in the rhizosphere, while the contribution of nitrifiers to CH4 oxidation was insignificant.
Use of methanotrophic bacteria for reactor treatment of contaminated groundwater is a bioremediaton alternative to physical-thermal destructive methods. The cometabolic degradation rates of trichloroethylene (TCE) and 1,1,1-trichloroethane (TCA) were determined using mixed culture, methanotrophic biofilms grown on glass beads in a complete-mixed, laboratory reactor sparged with methane gas. The TCA and TCE degradation rates were approximated by a first-order model, with rate constants of 5.4 at concentrations below 55 μg/L and 0.40 at concentrations below 1000 μg/L. Maximum degradation rates of approximately 302 and 400 μg/L·h, were observed for TCA and TCE, respectively. For TCE concentrations below 760 μg/L, TCA was degraded faster than TCE. TCA degradation rates in mixtures were not significantly inhibited by the presence of TCE up to 870 μg/L. Small amounts of a chlorinated metabolite, presumed to be 2,2,2-trichloroethanol, were formed during degradation of TCA. An example design calculation is presented, suggesting that useful reductions of TCA and TCE could be obtained in similar reactors with retention times of 1-12 hours.
Aerobic cometabolism of chlorinated aliphatic solvents in biofilm reactors is a potential treatment technology for contaminated water and air streams. This research investigated cometabolism by pure and mixed cultures of methanotrophs and mixed cultures of phenol-degrading bacteria. Initial experiments with continuous-flow, packed-bed bioreactors proved unsuccessful; therefore, the major focus of the work was on sequencing biofilm reactors, which cycle between two modes of operation, degradation of chlorinated solvents and rejuvenation of the microbial population. Particular success was obtained with a mixed culture of phenol degraders in the treatment of chlorinated ethenes (e.g., trichloroethylene - TCE). Under the best operating conditions, 90% removal of TCE occurred at a 14-minute packed-bed hydraulic residence time. The bioreactors required only two, 1.5 h biomass rejuvenation periods per day to sustain this removal. Experiments with Methylosinus trichosporium OB3b were less successful because of the organism's slow growth rate, relatively poor ability to attach to surfaces, and its inability to successfully compete with other methanotrophs in the bioreactor environment Overall, however, the research demonstrated the potential attractiveness of sequencing biofilm reactors in treating water contaminated with chlorinated solvents.
The kinetics of methane utilization and the biodegradation of trichloroethylene (TCE) and 1,1,1-trichloroethane (TCA) by a mixed, methanotrophic bacterial culture were studied in a closed-system reactor. Methane oxidation followed Michaelis-Menten kinetics. TCE and TCA degradation followed first-order kinetics with rate constants of 3.7 à 10â»â´ and 8.8 à 10â»âµ L/mg VSS {center dot} h, respectively, for concentrations less than 3,000 μg/L. Oxidative activity of the methanotrophic culture ceased at a dissolved TCE concentration of 7,770 μg/L. TCA, but not TCE, biodegradation was inhibited by dissolved methane concentrations in excess of 0.25 mg/L. In the absence of methane, the culture continued to degrade TCE and TCA, but degradation ceased after 104 hours. Lower biodegradation rates were observed when treating a mixture of TCE and TCA.
Abstract Methylosinus trichosporium OB3b is a wild type, obligate methanotroph that grows only on one-carbon compounds and, in the absence ofcopper, produces high levels of soluble methane,monooxygenase ,(sMMO) to metabolize methane ,to methanol. , SMMO has gained a great deal of attention in the bioremediation and chemical industries because of its low substrate specificity and its ability to ,oxidize chlorinated hydrocarbons. Much literature exists on cultivating this organism on methane, however no one has achieved dry cell weight ,densities exceeding ,18 g/L. Biomass growth ,is limited ,due to mass transfer of methane,to cells. This study investigated the growth of M. trichosporium on
Aerobic cometabolism of chlorinated aliphatic solvents in biofilm reactors is a potential treatment technology for contaminated water and air streams. This research investigated cometabolism by pure and mixed cultures of methanotrophs and mixed cultures of phenol-degrading bacteria. Initial experiments with continuous-flow, packed-bed bioreactors proved unsuccessful; therefore, the major focus of the work was on sequencing biofilm reactors, which cycle between two modes of operation, degradation of chlorinated solvents and rejuvenation of the microbial population. Particular success was obtained with a mixed culture of phenol degraders in the treatment of chlorinated ethenes (e.g., trichloroethylene - TCE). Under the best operating conditions, 90% removal of TCE occurred at a 14-minute packed-bed hydraulic residence time. The bioreactors required only two, 1.5 h biomass rejuvenation periods per day to sustain this removal. Experiments with Methylosinus trichosporium OB3b were less successful because of the organism's slow growth rate, relatively poor ability to attach to surfaces, and its inability to successfully compete with other methanotrophs in the bioreactor environment. Overall, however, the research demonstrated the potential attractiveness of sequencing biofilm reactors in treating water contaminated with chlorinated solvents.
CH4 oxidation activities from various soils and freshwater sediments were measured at low (=1000 ppmv) CH4 mixing ratios. Most of the tested soils acted as sinks for atmospheric CH4. A correlation between the CH4 oxidation activity and the numbers of methanotrophs was only observed at high (1000 ppmv) CH4 mixing ratios. This indicates that the counted methanotrophs were not the bacteria which are oxidizing atmospheric CH4 (3%. Undisturbed, stratified soils, and freshwater sediments showed vertical profiles of CH4 oxidation activities with a distinct maximum. Sediments showed an exact correspondence between the number of methanotrophs and the maximum of CH4 oxidation both being localized at the surface sediment layer. The oxic soils showed maxima of CH4 oxidation activities generally located in subsurface layers. The maxima of CH4 oxidation activities were slightly shifted below the maxima of the numbers of methanotrophs indicating that the counted bacteria (incubation under 20% CH4) might not represent the active population which oxidizes atmospheric CH4. Plowed, agricultural soils showed no distinct maxima, neither of the CH4 oxidation activities nor of the numbers of methanotrophs. The grain size fractionation by centrifugation or wet sieving of slurries of two forest soils showed that the bulk (80-96%) of the CH4 oxidizing activity was attached to the smaller mineral fractions (clay, silt, fine sand) of these soils. Within the mineral fractions, greater particles had higher specific activities of CH4 oxidation than smaller particles.
CH4 fluxes and various soil properties were measured over three successive years at a field site on a loamy sand soil in eastern Scotland, to determine which factors influence CH4 oxidation rate. This site included three adjacent areas with contrasting land use: woodland, arable land and set aside land. The CH4 oxidation rates in the arable soil were less than half the corresponding rates in the woodland soil. The CH4 oxidation rates in the set aside soil were even lower, indicating that there is no immediate recovery when cultivation and fertilisation are abandoned. In the woodland and set aside soils, a seasonal variation in CH4 oxidation rate was found, but in the arable soil there was no such trend. The CH4 oxidation rate was negatively correlated with soil moisture content (P < 0.001) in the woodland soil and positively correlated with soil temperature (P < 0.001) in the set aside soil. In the arable soil, CH4 oxidation rate was related to moisture content only in dry summer conditions, when the relationship was positive (P < 0.001). These relationships suggest that CH4 oxidation was controlled partly by diffusion and partly by biological activity. A negative correlation was found between soil ammonium concentration and CH4 oxidation rate in the woodland soil (P < 0.001), indicating that ammonium inhibited CH4 oxidation in that environment.
A fluidized-bed bioreactor (FBB) containing Methylocystis sp. strain M (strain M) isolated from Japanese soil was developed for treating synthetic groundwater containing trichloroethylene (TCE). Strain M bacteria were immobilized in 2% calcium alginate gel beads and introduced into the reactor, which was supplied with a methane/air gas mixture. TCE concentrations in the reactor were reduced from 0.9–1.6 to 0.1–0.2 mg/litre after a residence time of 2.56 h. Thus, 80–90% of the influent TCE was degraded in the reactor, and the resulting effluent gas contained only 0.02–0.04 mg/litre TCE. The ability of the gel-immobilized cells to degrade TCE declined rapidly during the TCE degradation process. To compensate for this decrease in activity, the reactor was operated for TCE degradation and reactivation of the immobilized strain M cells on alternate days. TCE degradation activity was completely recovered within 24 h after supplying a methane/air mixture (2:8 v/v) and a mineral salt solution to the bacteria. The degradation capacity of the reactor was kept in a steady state for 10 days in this way, but the rate of recovery of TCE degradation activity gradually decreased. TCE degradation activity fell from 6.0 litres/g dry weight (gdw) per hour on the sixth day to 1.4 litres/gdw per hour on the 24th day. Interestingly, there was no corresponding decrease in methane consumption by the bacteria
A model describing the growth of bacteria and the degradation of methane and trichloroethylene (TCE) based on the concept of competitive inhibition is proposed. The model has been applied to laboratory batch experiments representing different initial TCE concentrations (50–4300 μg/l) and initial methane concentrations (0.53–3.2 mg/l). The proposed model simulated successfully the data obtained for initial methane concentration (less than 1.8 mg/l), causing constant experimental growth conditions during the experiments. This indicates that the interactions between methane and TCE degradation can be explained as competitive inhibition. The model simulations of the results from the experiments with the highest initial methane concentration of 3.2 mg/l failed, supposedly because the growth conditions changed during the experiments.The proposed model is a useful engineering tool for design of treatment processes and in situ bioremediation schemes for degradation of TCE by methane-oxidizing bacteria.
The steady-state concentration of M. trichosporium OB3b increased about two-fold in the continuous culture when the feeding medium was supplemented by ferrous sulphate (50mg/L) and citric acid (100mg/L) at a steady state. In batch and continuous cultures, the cell growth was significantly inhibited by excess N-sources (NH4OH, NH4Cl, NH4NO3, HNO3, and NaNO3) and ammonium N-sources were more inhibitory. Both volumetric O2 transfer coefficient and specific O2 uptake rate increased monotonously in an extensive range of air flow rate (0.1–7 vvm) and the methane interfered with the O2 transfer even at very low flow rates (0.01–0.1 vvm).
The mutant methanotroph, Methylosinus trichosporium OB3b PP358, which constitutively expresses soluble methane monooxygenase (sMMO), was used to study the degradation kinetics of individual chlorinated solvents and binary solvent mixtures. Although sMMO's broad specificity permits a wide range of chlorinated solvents to be degraded, it creates the potential for competitive inhibition of degradation rates in mixtures because multiple chemicals are simultaneously available to the enzyme. To effectively design both ex-situ and in-situ groundwater bioremediation systems using strain PP358, kinetic parameters for chlorinated solvent degradation and accurate kinetic expressions to account for inhibition in mixtures are required. Toward this end, the degradation parameters for six prevalent chlorinated solvents and the verification of enzyme competition model for binary mixtures were the focus of this investigation. M. trichosporium OB3b PP358 degraded trichloroethylene (TCE), chloroform, cis-1,2-dichloroethylene (c-DCE), trans-1,2-dichloroethylene (t-DCE), and 1,1-dichloroethylene (1,1-DCE) rapidly, with maximum substrate transformation rates of >20.8, 3.1, 9.5 24.8, and >7.5 mg/mg-day, respectively. 1,1,1-trichloroethane (TCA) was not significantly degraded. Half-saturation coefficients ranged from 1 to greater than 10 mg/L. Competition experiments were carried out to observe the effect of a second solvent on degradation rates and to verify the applicability of the Monod model adjusted for competitive inhibition. Binary mixtures of 0.3–>0.5 mg/L TCE with up to 5 mg/L c-DCE and up to 7 mg/L 1,1,1-TCA were studied with 20 mM of formate and no growth substrate. No competition was observed at any of these concentrations. Additional competition experiments, using binary mixtures of t-DCE with TCE and t-DCE with c-DCE, were conducted at higher concentrations (i.e., 7–18 mg/L) and enzyme competition was observed. Predictions from a competitive inhibition model compared well with experimental data for these mixtures. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 100–107, 1999.
Air streams contaminated with chlorinated solvents are increasingly common as by-products of air-stripping and soil-vapor-extraction operations. This research investigated treatment of such gas streams with a bioreactor that supported the growth of methanotrophic bacteria. These bacteria cometabolize many chlorinated solvents. Trichloroethylene (TCE) and 1,2-dichloroethane (DCA) were selected as model contaminants. Removals ranged from 20% to 80% at influent concentrations of 300-1,000 [mu]g/L of air and packed-bed gas-resident times of 5-12 min. Biofilm models were able to describe bioreactor performance well. Pseudo-first-order rate constants from reactor modeling were considerably smaller than those measured in batch systems, suggesting that much of the biofilm was inactive, which also was supported by methane-removal data. Enzyme competition between methane and chlorinated solvents and toxicity from chlorinated solvent metabolites both appeared significant.
The potential of using the methanotrophic bacterium Methylomonas methanica to remove methane from coal mine atmospheres was investigated using a bench scale, gas phase bioreactor. The bioreactor was constructed from a cylindrical glass column packed with bio-rings to support bacterial growth. M. methanica grew readily on the bio-rings with a total of 133.4 g (wet weight) of the bacterium being contained in the bioreactor. Methane in air mixtures ranging from 0 to 50.9% were recirculated through the 4.5 1 (void volume) bioreactor at a rate of 200 ml min−1, and significant levels of methane removal were noted. With a 35% methane/air mixture, 90.4% of the methane was removed in 24 h. This equated to a removal rate of ~38 mg methane h−1. Using a 12% methane/air mixture the removal rate was ~23 mg methane h−1. From these results, it was concluded that methanotrophic bacteria grown in gas phase bioreactors are capable of removing significant amounts of methane from methane/air mixtures and may be of practical use in removing methane from coal mine atmospheres.
The obligate methylotroph Methylosinus trichosporium OB3 b was capable of growth on methanol as sole carbon source at concentrations as high as 4% (v/v), and viability was maintained over many successive transfers. Methane mono-oxygenase, detected by epoxidizing and hydroxylating activity, was retained. The gross morphology of the organism on this substrate was dependent on culture conditions. It varied from organisms containing extensive peripheral membrane systems to those with extensive inclusions, the latter representing a poorly developed membrane system which predominated under most growth conditions.
Prior to a down-hole-column treatability test of a Methylosinus trichosporium OB3b attached-resting-cell in situ biofilter strategy, a set of three sequential laboratory experiments were carried out to define several key operational parameters and to evaluate the likely degree of success at a NASA Kennedy Space Center site. They involved the cell attachment to site-specific sediments, the intrinsic resting-cell biotransformation capacities for the contaminants of interest plus their time-dependent extents of biodegradative removal at the concentrations of concern, and a scaled in situ mini-flow-through-column system that closely mimics the subsurface conditions during a field-treatability or pilot test of an emplaced resting-cell filter. These experiments established the conditions required for the complete metabolic removal of a vinyl chloride (VC), cis-dichlororthylene (cis-DCE) and trichloroethylene (TCE) mixture. However, the gas chromatographic (GC) procedures that we utilized and the mini-flow-through column data demonstrated that, at most, only about 50--70% of the site-water VC, cis-DCE, and TCE would be biodegraded. This occurred because of a limiting level of dissolved oxygen, which was exacerbated by the simultaneous presence of several additional previously unrecognized groundwater components, especially methane, that are also competing substrates for the whole-cell soluble methane monooxygenase (sMMO) enzyme complex. Irrespective, collectively the simplicity of the methods that we have developed and the results obtainable with them appear to provide relevant laboratory-based test-criteria before taking our microbial filter strategy to an in situ field treatability or pilot demonstration stage at other sites in the future.
Oxidation of methane by methanotrophs in the landfill cover soil provides a source reduction for methane. Full factorial 23 experimental design using heterogeneous batch reactors was conducted to investigate statistically the individual and combined effects of soil moisture content, nutrient addition, and cover thickness on the CH4 oxidation process, during the migration through a landfill cover soil. Adding fertilizer as nutrient source to the 200 mm layer thickness of the landfill cover soil that contained 30% moisture content increased the CH4 oxidation efficiency from 38% to 81%. While, adding nutrients to the soil with less moisture content (15%) affected negatively the bacterial performance of methane oxidation, possibly as a result of toxicity or microbial water stress. Kinetic constants were reported and statistical design model was developed to describe the expected methane oxidation efficiencies under different levels of moisture content and nutrient addition that occur in a typical landfill cover soil.Key words: methane oxidation, landfill cover soil, moisture content, nutrients, soil thickness.
Metabolite toxicity production during trichloroethylene (TCE) degradation by methanotrophic bacteria can lead to partial or complete inactivation of the TCE-degrading enrichment. The general objectives of this research were to determine if TCE and methane feeding strategies could be selected that would reduce metabolite toxicity effects and determine operating conditions that would sustain TCE-degrading methanotrophic enrichments. In batch treatment of TCE with and without methane, the presence of methane doubled the amount of TCE degraded per unit biomass (0.051 mg TCE/mg biomass). In a closed complete-mix methanotrophic enrichment reactor continuously fed TCE and methane, reactor failure occurred at a loading of only 0.005 mg TCE/mg volatile suspended solids (VSS) · d. In the same type of reactor operation with intermittent TCE feeding and continuous methane feeding, TCE degradation was sustained at daily loadings of 0.03 mg TCE/mg VSS · d. The intermittent TCE-fed reactor could produce an effluent TCE concentration below the drinking water standard (0.005 mg/L) at a higher TCE loading than the continuous-fed system (0.03 versus 0.0025 mg TCE/mg VSS · d). The system with intermittent TCE feeding provided more efficient use of methane relative to TCE degradation. The amount of methane consumed per unit of TCE degraded was 33 and 167 mg methane/mg TCE for the intermittent and continuous TCE-fed operations, respectively. With continuous TCE exposure, the methanotrophic population in the continuous TCE-fed reactor changed to a type that is characterized by having less soluble TCE-oxidizing enzymes and slower TCE oxidation rates.