Interesting anaerobes in the environment

Prokaryotes (Bacteria and Archaea) have a wide range of capacities to survive by generating energy in environments and situations lacking oxygen, which abound on Earth. Anaerobic metabolic strategies include anaerobic respiration (numerous types – e.g. nitrate reduction – Paracoccus denitrificans; sulfur respiration – Desulfuromonadales; methanogenesis – Methanosarsina spp.; iron reduction – Geobacter spp.; dehalorespiration – Dehalococcoides ethenogenes) and fermentation (sugars converted to simpler organic compounds like acids, gases and alcohols – e.g. Lactobacillus spp.). Relatively novel environmental anaerobic strategies include anaerobic ammonium oxidation (Anammox – e.g. Brocadia spp.) and anaerobic methane oxidation (AMO)¹, which is a syntrophic association between anaerobic methanotrophic archaea (ANME) and sulfate-, iron-, manganese- or nitrate-reducing bacteria². The classic anaerobic synthrophic example is interspecies hydrogen/formate transfer between a hydrogen/formate producing fatty acid oxidising bacterium (the syntroph) and a hydrogen/formate consumer (methanogen or sulfate-reducer)³. Microbes vary in their oxygen tolerance and are described as obligate anaerobes if they are killed by atmospheric levels of oxygen due to the lack of catalase and superoxide dismutase that provide oxygen radical protection.

A recent Microbiology Australia issue (The Microbial Ecology of the Environment; November 2014) covered some environmental anaerobic microbiology topics including sulfidic sediments⁴, electromicrobiology (typically comprising an anaerobic anode and an oxic cathode)⁵ and chloroethene degradation (dehalorespiration)⁶.

This short review provides a brief overview of three environmental anaerobic metabolic topics and demonstrates metabolic and environmental diversity employed by the microbes.

Corrosion of pure metals or their alloys involves ionisation of the metal (the oxidative anodic reaction) coupled with the cathodic reaction (a chemical species reduction), is relatively well understood and accounted for in design and maintenance of metallic structures. Metals are used in many terrestrial (e.g. buried pipes for transmission of many different liquids) and aquatic, particularly marine (ships hulls, steel sheet piles; Figure 1), structures. MIC is also known as biocorrosion and involves microbial processes on metal surfaces that lead to localised material degradation at rates that are orders of magnitude higher than would normally be expected from standard, abiotic corrosion^7–9. No new electrochemical processes are involved in MIC, simply corrosion is accelerated and occurs under conditions that would not normally favour it. MIC can lead to premature and unpredicted failure of a wide range of important structures that can be costly to repair and, in some cases, can have fatal consequences. The overall cost of corrosion has been estimated to be between approximately 2 and 5% of the gross national product in a range of countries, with a global value of US$50–80 billion per annum¹⁰ and approximately A$18 billion suggested for Australia in 2006¹¹.

Figure 1. An example of late stage microbiologically influenced corrosion of a sheet metal structure in a shallow marine environment. (Photo credit: Dr Scott Wade, Swinburne University of Technology.)

Microbes mass at the metalic surface to produce a ‘biofilm’ and the collective action of the different microbial species in the biofilm along with abiotic features result in the phenomenon of biocorrosion. Corrosion kinetics are modulated by the surface physico-chemical properties including oxygen, ions, pH, redox, conductivity and more. Various microbial species have been singled out as major culprits in enhancing corrosion, notably sulfate reducing bacteria (SRB)¹². However, in a natural corrosion site, there will be a complex community of microbes present with some involved in setting up and facilitating the appropriate conditions for the anaerobic corrosion organisms. Thus, although many microbes might not directly cause the corrosion, their properties might be vital for the maintenance of the corrosion species. Major biocorrosion mechanisms include acid attack, creation of anodic sites and cathodic depolarisation¹².

In MIC of steel surfaces, SRB energy production relies on elemental iron oxidation to release soluble ferrous iron (Fe –> Fe²⁺ + 2e^–) and typically sulfate is used by the SRB as their electron acceptor (SO₄^2– + 9H⁺ + 8e^– –> HS^– + 4H₂O). Acid producing bacteria and methanogens are also corrosive anaerobes, the former by way of their corrosive acid metabolites. A recent microbiological study of ‘accelerated low water corrosion’ using high throughput DNA sequencing determined that Methanococcus maripaludis, which comprised 31% of retrieved sequences, could extract electrons directly from steel and use CO₂ as a carbon source¹³.

How to mitigate MIC involves numerous strategies including use of biocides, promoting non-corrosive biofilms on surfaces to mask it from the MIC organisms and manipulation of electron acceptors to preclude MIC organisms¹⁴. Recently reported steel MIC control methods involve:

• microbiological production of an Fe²⁺-phosphate layer that has a reduced corrosion potential¹⁵, and

• the use of graphene coatings¹⁶.

However, more research into the microbiological mechanisms of MIC (anode/cathode metabolism, acid production) is required for additional novel, targeted, reliable control strategies to be developed. These studies will likely involve controlled experimental conditions that simulate the natural settings¹⁰ and determination of specific corrosive strains and their mechanisms.

Ammonia oxidation has been very well studied and the biochemistry understood in aerobic bacteria (e.g. Nitrosomonas spp.) and more recently in the abundant, ammonium-scavenging, aerobic, marine archaeon Nitrosopumilus spp. However, the finding of Anammox (NH₄⁺ + NO₂^– –> N₂ + 2H₂O) from nitrogen and redox balances in a denitrifying bioreactor treating effluent from a methanogenic bioreactor¹⁷ reported a novel biochemical pathway. Although the discovery was in a bioreactor with Anammox-enriched biomass, theoretical calculations had previously been used to hypothesise that Anammox could occur¹⁸ and it has been found to be environmentally widespread. Despite that for the majority of the 20th century it was thought that the nitrogen cycle was fully understood, Anammox has been found to be responsible for up to:

• 70% of marine nitrogen cycling, and

• 50% of marine N₂ production¹⁹.

The possibility of AMO (CH₄ + SO₄^2– + H⁺ –> CO₂ + HS^– + 2H₂O) only developed through the 1970s from coupled methane and sulfate profiles in marine sediments²⁰. AMO was not available in enriched biomasses and experimental evidence for AMO and its syntrophic SRB came by implementing a combination of methodological procedures including lipid biomarking, clone libraries and fluorescence in situ hybridisation (FISH)²¹. Subsequently, secondary ion mass spectrometry and FISH were used in re-confirming AMO in marine methane-seep sediments, for revealing two distinct AMO archaeal groups called ANME-1 (a distant relative of Methanosarcinales and Methanomicrobiales) and ANME-2 (in the Methanosarcinales), and for demonstrating that the AMO in the syntrophic association likely employs a ‘reversed methanogenesis’ pathway²². A third ANME-3 group (in the Methanosarcinales), typically found near mud volcanoes has been discovered²³. The ANME clades form syntrophic associations with specific partners and most that have been reported are with SRB, which are in the Deltaproteobacteria. These syntrophic associations are highly relevant to global carbon cycling since although oceans contribute only 2% of the global methane budget, it has been found that this low net total is due to the fact that AMO utilise 70–300 teragrams of methane per year²⁴. The AMO-SRB synthrophic associations typically form ‘microbial reefs’ in marine sediments at the sulfate-methane transition zone – critical for the metabolism of both syntrophic partners²⁵.

Recently, from bioreactors, a single organism (ANME-2d, ‘Candidatus Methanoperedens nitroreducens’) has been reported to be capable of independent AMO through reverse methanogenesis using nitrate as the terminal electron accepter²⁶:

• CH₄ + 4NO₃^– -> 4NO₂^– + 2H₂O (ΔG^o = –503 kJ.mol^–1 CH₄)

Another string to the AMO bow is the star-like shaped ‘Candidatus Methylomirabilis oxyfera’ (in the bacterial candidate phylum NC10), which is appears solely responsible for nitrite-dependent AMO (N-DAMO) in fresh and marine waters²⁷. The fine balance of the global carbon budget and its relationship with the nitrogen cycle is substantially held by the AMO and their syntrophic associations. Since many aspects of the biochemistry are not fully elucidated, including definitive proof of the reverse methanogensis²⁵, it is imperative that urgent research addresses this lack of knowledge².

Anaerobic processes (those lacking the requirement for or intolerance of oxygen) have many industrial applications (e.g. wastewater treatment) and implications (e.g. MIC) and also are critical to the global cycling of many elements (two relevant to nitrogen and carbon are briefly described). Despite that microbes were implicated in corrosion at least by 1910, due to the multidisciplinary nature of the field and the lack of connection between experts in the disparate fields like electromicrobiology, corrosion and process engineering, mechanisms involved in MIC are not well understood¹². Detailed studies into the role of relevant microbes in MIC and of smart biomimetic surface control strategies are required to abate this problem.

The relatively novel processes of Anammox and AMO are common in the environment but their existence was largely predicted from thermodynamic calculations. Their studies have been prolific since they are relevant to marine nutrient cycling where >75% of methane and 30–50% of ammonium are oxidised by them²⁴. Industrial application of Anammox in the wastewater treatment sphere facilitates cost and CO₂ emissions reductions but the environmental relevance of Anammox in marine and freshwater ecosystems is profound. Future research elucidating connections between sulfate, nitrite/nitrate, and metal ion (manganese and iron) electron acceptors in AMO will be valuable in improved understanding of global nitrogen and carbon cycles.