First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus

The antimicrobial activity of copaiba oils was tested against Gram-positive and Gram-negative bacteria, yeast, and dermatophytes. Oils obtained from Copaifera martii, Copaifera officinalis, and Copaifera reticulata (collected in the state of Acre) were active against Gram-positive species (Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus faecalis) with minimum inhibitory concentrations ranging from 31.3-62.5 µg/ml. The oils showed bactericidal activity, decreasing the viability of these Gram-positive bacteria within 3 h. Moderate activity was observed against dermatophyte fungi (Trichophyton rubrum and Microsporum canis). The oils showed no activity against Gram-negative bacteria and yeast. Scannning electron microscopy of S. aureus treated with resin oil from C. martii revealed lysis of the bacteria, causing cellular agglomerates. Transmission electron microscopy revealed disruption and damage to the cell wall, resulting in the release of cytoplasmic compounds, alterations in morphology, and a decrease in cell volume, indicating that copaiba oil may affect the cell wall.

Antibacterial and antifungal activity of extracts and exudates of the Amazonian medicinal tree Himatanthus articulatus (Vahl) Woodson (common name: sucuba)

Himatanthus articulatus (Vahl) Woodson is a tree found in the northern Amazon savannahs (common name: sucuba) that is used in local Amerindian medicine. Leaf, bark and branch wood methanol extracts, sequentially obtained hexane, ethyl acetate and methanol extracts and latex were evaluated for antifungal and antibacterial activities against American Type Culture Collection (ATCC) and local clinical strains using the disc diffusion method. Methanol extracts and latex inhibited Candida albicans, leaf methanol extracts inhibited Staphylococcus aureus and Bacillus subtilis and bark methanol extracts inhibited B. subtilis. Active extracts inhibited the ATCC and clinical strains. Polar antifungal and antibacterial principles in latex and extracts are thought to be responsible for the inhibition.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

Determinació de l'eficàcia de soques de Bacillus sp. en el control de Stemphylium vesicarium en assajos in vitro i ex vivo

L’estemsiliosi de la perera és una malaltia fúngica d’una gran importància econòmica a la zona del centre i sud d’Europa. És comparable al motejat de la pomera i pot arribar a assolir el 90 % de pèrdues en pressions elevades de la malaltia. S’ha comprovat que els tractaments amb fungicides presenten una eficàcia de control limitada, i que les mesures sanitàries i de control biològic generen resultats que plantegem com a una eina més a utilitzar en en la integració de mètodes de control. L’objectiu del treball és determinar l’eficàcia de control de Stemphylium vesicarium a partir de diferents soques de Bacilus subtilis en assatjos en situacions controlades

Development of bacteria-based bioassays for arsenic detection in natural waters.

Arsenic contamination of natural waters is a worldwide concern, as the drinking water supplies for large populations can have high concentrations of arsenic. Traditional techniques to detect arsenic in natural water samples can be costly and time-consuming; therefore, robust and inexpensive methods to detect arsenic in water are highly desirable. Additionally, methods for detecting arsenic in the field have been greatly sought after. This article focuses on the use of bacteria-based assays as an emerging method that is both robust and inexpensive for the detection of arsenic in groundwater both in the field and in the laboratory. The arsenic detection elements in bacteria-based bioassays are biosensor-reporter strains; genetically modified strains of, e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Rhodopseudomonas palustris. In response to the presence of arsenic, such bacteria produce a reporter protein, the amount or activity of which is measured in the bioassay. Some of these bacterial biosensor-reporters have been successfully utilized for comparative in-field analyses through the use of simple solution-based assays, but future methods may concentrate on miniaturization using fiberoptics or microfluidics platforms. Additionally, there are other potential emerging bioassays for the detection of arsenic in natural waters including nematodes and clams.

Avaliação do crescimento microbiano em sondas de uso único para vitrectomia reprocessadas na prática assistencial

O objetivo deste estudo foi avaliar o crescimento microbiano em sondas para vitrectomia de uso único, reprocessadas na prática assistencial. Foram investigadas nove sondas reusadas e reprocessadas por diferentes métodos. As sondas foram segmentadas, individualmente, em porções de 3,5 cm, totalizando em 979 unidades amostrais (extensões, conectores e ponteiras) inoculadas em meio de cultura e incubadas a 37ºC, por 14 dias. Os resultados mostraram crescimento microbiano em 57 (5,8%) unidades amostrais, das quais, 25 foram esterilizadas por Óxido de Etileno, 16 por Plasma de Peróxido de Hidrogênio e 16 por Vapor à Baixa Temperatura e Formaldeído. Foram identificadas 17 espécies microbianas, sendo as mais prevalentes o Micrococcus spp., Staphylococcus coagulase negativa, Pseudomonas spp. e Bacillus subtilis. O reuso de sondas de uso único para vitrectomia não se mostrou seguro, portanto tal prática não é recomendada.

Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae, Apinae) - bionomia e biogeografia

This work, dedicated to the study of nesting habits of the species of the Neotropical genus Partamona Schwarz, is a sequence to the taxonomic revision recently published elsewhere. A total of 214 nests and nest aggregations of 18 species [Partamona epiphytophila Pedro & Camargo, 2003; P. testacea (Klug, 1807); P. mourei Camargo, 1980; P. vicina Camargo, 1980; P. auripennis Pedro & Camargo, 2003; P. combinata Pedro & Camargo, 2003; P. chapadicola Pedro & Camargo, 2003; P. nhambiquara Pedro & Camargo, 2003; P. ferreirai Pedro & Camargo, 2003; P. pearsoni (Schwarz, 1938); P. gregaria Pedro & Camargo, 2003; P. batesi Pedro & Camargo, 2003; P. ailyae Camargo, 1980; P. cupira (Smith, 1863); P. mulata Moure in Camargo, 1980; P. seridoensis Pedro & Camargo, 2003; P. criptica Pedro & Camargo, 2003; P. helleri (Friese, 1900)] were studied , including data about habitat, substrate, structural characteristics, construction materials and behavior. The descriptions of the nests are illustrated with 48 drawings. Partial data of the nests of P. bilineata (Say, 1837), P. xanthogastra Pedro & Camargo, 1997, P. orizabaensis (Strand, 1919), P. peckolti (Friese, 1901), P. aequatoriana Camargo, 1980, P. musarum (Cockerell, 1917) and P. rustica Pedro & Camargo, 2003 are also presented. Nests of P. grandipennis (Schwarz, 1951), P. yungarum Pedro & Camargo, 2003, P. subtilis Pedro & Camargo, 2003, P. vitae Pedro & Camargo, 2003, P. nigrior (Cockerell, 1925), P. sooretamae Pedro & Camargo, 2003 and P. littoralis Pedro & Camargo, 2003 are unknown. The species of Partamona build notable nest entrance structures, with special surfaces for incoming / exiting bees; some of them are extremely well-elaborated and ornamented, serving as flight orientation targets. All species endemic to western Ecuador to Mexico with known nesting habits (P. orizabaensis, P. peckolti, P. xanthogastra, P. bilineata, P. aequatoriana and P. musarum) build their nests in several substrates, non-associated with termitaria, such as cavities and crevices in walls, among roots of epiphytes and in bases of palm leaves, in abandoned bird nests, under bridges, and in other protected places, except P. peckolti that occasionally occupies termite nests. In South America, on the eastern side of the Andes, only P. epiphytophila and P. helleri nest among roots of epiphytes and other substrates, non-associated with termitaria. All other species studied (P. batesi, P. gregaria, P. pearsoni, P. ferreirai, P. chapadicola, P. nhambiquara, P. vicina, P. mourei, P. auripennis, P. combinata, P. cupira, P. mulata, P. ailyae, P. seridoensis, P. criptica and P. rustica) nest inside active termite nests, whether epigeous or arboreous. The only species that builds obligate subterranean nests, associated or not with termite or ant nests (Atta spp.) is P. testacea. Nests of Partamona have one vestibular chamber (autapomorphic for the genus) closely adjacent to the entrance, filled with a labyrinth of anastomosing pillars and connectives, made of earth and resins. One principal chamber exists for food and brood, but in some species one or more additional chambers are filled with food storage pots. In nests of P. vicina, there is one atrium or "false nest", between the vestibule and the brood chamber, which contains involucral sheaths, cells and empty pots. All structures of the nest are supported by permanent pillars made of earth and resins (another autapomorphy of the genus). The characters concerning nesting habits were coded and combined with morphological and biogeographic data, in order to hypothesize the evolutive scenario of the genus using cladistic methodology. The phylogenetic hypothesis presented is the following: (((((P. bilineata (P. grandipennis, P. xanthogastra)) (P. orizabaensis, P. peckolti)) (P. aequatoriana, P. musarum)) P. epiphytophila, P. yungarum, P. subtilis, P. vitae) (((((P. testacea (P. mourei, P. vicina)) (P. nigrior (P. auripennis, P. combinata))) (P. ferreirai (P. pearsoni (P. gregaria (P. batesi (P. chapadicola, P. nhambiquara)))))) ((((P. ailyae, P. sooretamae) P. cupira, P. mulata) P. seridoensis) P. criptica, P. rustica, P. littoralis)) P. helleri))). One area cladogram is presented. Dates of some vicariance / cladogenesis events are suggested. For bilineata / epiphytophila group, which inhabits the Southwestern Amazonia and the Chocó-Mexican biogeographical components, the origin of ancestral species is attributed to the Middle Miocene, when the transgressions of the Maracaibo and Paranense seas isolated the tropical northwestern South America from the eastern continental land mass. The next cladogenic event in the history of the bilineata / epiphytophila group is attributed to the Plio-Pleistocene, when the Ecuadorian Andes reached more than 3000 m, and the ancestral species was fragmented in two populations, one occupying the western Andes (ancestral species of the bilineata subgroup) and other the southwestern Amazon (ancestral species of the epiphytophila subgroup). Other aspects of the history of Partamona are also discussed.

Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae)

Neotropical Meliponini: the genus Partamona Schwarz, 1939 (Hymenoptera, Apidae). The systematics and biogeography of Partamona Schwarz, a Neotropical genus of stingless bees (Meliponini, Apinae, Apidae), are revised. Seventeen new species are described: P. epiphytophila sp. nov., P. subtilis sp. nov., P. nhambiquara sp. nov., P. batesi sp. nov., P. yungarum sp. nov., P. vitae sp. nov., P. ferreirai sp. nov., P. gregaria sp. nov., P. auripennis sp. nov., P. nigrilabris sp. nov., P. combinata sp. nov., P. chapadicola sp. nov., P. seridoensis sp. nov., P. littoralis sp. nov., P. criptica sp. nov., P. rustica sp. nov. and P. sooretamae sp. nov. Partamona pseudomusarum Camargo, 1980, is considered as junior synonym of P. vicina Camargo, 1980. Types of P. grandipennis (Schwarz, 1951), P. xanthogastra Pedro & Camargo, 1996-1997, P. pearsoni (Schwarz, 1938), P. ailyae Camargo, 1980, P. pseudomusarum, P. vicina, P. mulata Moure in Camargo, 1980, P. aequatoriana Camargo, 1980, P. mourei Camargo, 1980, P. peckolti, (Friese, 1901), P. testacea (Klug, 1807), P. helleri (Friese, 1900) and P. musarum (Cockerell, 1917) were examined. Lectotypes of P. orizabaensis (Strand, 1919), and P. cupira (Smith, 1863) are designated. An identification key for the species and drawings of morphological characters are presented. A phylogenetic hypothesis, based mainly on morphological characters is proposed. Four groups are defined, considering the shape of mandible of workers and sternum VII of males: bilineata / epiphytophila group (western Amazon to México), including P. bilineata (Say), P. grandipennis, P. xanthogastra P. orizabaensis P. peckolti P. epiphytophila sp. nov., P. subtilis sp. nov., P. nhambiquara sp. nov., P. batesi sp. nov., P. yungarum sp. nov. and P. vitae sp. nov.; musarum group (Central Brazil, north of South America to Central America), including P. musarum, P. aequatoriana, P. vicina, P. mourei, P. pearsoni, P. ferreirai sp. nov., P. gregaria sp. nov. and P. testacea; nigrior group (Central Brazil to northeast of South America) including P. nigrior (Cockerell, 1925), P. auripennis sp. nov., P. nigrilabris sp. nov., P. combinata sp. nov., P. chapadicola sp. nov., P. seridoensis sp. nov. and P. littoralis sp. nov., and cupira group (southeastern and Central Brazil), including P. cupira, P. mulata, P. ailyae, P. sooretamae sp. nov., P. criptica sp. nov., P. rustica sp. nov. and P. helleri. Some geographic distribution patterns, congruent with that of other Meliponini bees, are commented.

Fitoplancton del mar peruano en el verano 2008

La biomasa planctónica en promedio fue 0,36 mL.m-3; el 95% de los volúmenes fue <0,5 mL.m-3, el fitoplancton fue 35% y se caracterizó por el dominio de diatomeas de afloramiento (Skeletonema costatum, Lithodesmium undulatum, Chaetoceros spp., Thalassiosira subtilis y Thalassionema nit schioides) entre Salaverry- Chancay y Pisco-Ilo (30 mn) y abundancia de diatomeas oceánicas y dinoflagelados termófilos (Guinardia striata, G. flaccida, Cerataulina pelagica, Coscinodiscus wailesii, Proboscia spp., Ceratium massiliense, C. tripos v. atlanticum y Goniodoma polyedricum) entre Puerto Pizarro-Chicama y Punta Mendieta–Ilo hasta 120 mn en la zona norte. La distribución de los indicadores biológicos estuvieron acorde a las condiciones ambientales, Protoperidinium obtusum, indicador de Aguas Costeras Frías se registró desde Punta Falsa hasta Ilo (30 mn). Ceratium praelongum y C. incisum, indicadores de Aguas Subtropicales Superficiales se registraron frente a Punta Falsa y San Juan (60 mn) ampliando su distribución en Atico (120 mn). Ceratium breve, indicador de AES estuvo ampliamente distribuido al norte de 10°S (120 mn), con acercamientos a la costa en Puerto Pizarro y Punta Falsa. Messodinium rubrum se registró desde Punta Mendieta hasta Matarani, concentración de 8756x103 cel.L-1.

El fitoplancton en la primavera 2008. Crucero Oceanográfico BIC Olaya 0810-11

Los volúmenes de plancton fluctuaron entre 0,015 mL.m-3 (Pisco) y 4,648 mL.m-3 (Chimbote), promedio 0,838 mL.m-3, el 79% de estaciones tuvo volúmenes <1,0 mL.m-3. La predominancia del fitoplancton alcanzó 34%. El análisis de comunidades discriminó la formación de dos grupos: 1) estaciones con especies en fases iniciales de sucesión dentro de las 50 mn (Chaetoceros spp., Detonula pumila y Thalassiosira subtilis) y 2) con especies de fases intermedias y típicas de aguas cálidas (Planktoniella sol, Thalassiosira cf. parteneia y Thalassiothrix longissima) asociadas a dinoflagelados cosmopolitas, que se distribuyeron por fuera de las 50 mn. Indicadores biológicos: Protoperidinium obtusum (ACF) se registró hasta las 60 mn; Ceratium breve, indicador de AES se registró frente a Chimbote (75 mn) y Paita (190 mn); Ceratium praelongum, indicador de ASS, se registró a 60 mn, llegando hasta 30 mn frente a Paita.

Avaliação de populações de possíveis rizobactérias em solos sob espécies florestais

Embora estudos recentes relatem a utilização de RCPC (Rizobactérias Promotoras de Crescimento de Plantas) no Brasil, raríssimos trabalhos avaliam a presença natural dessas espécies bacterianas no solo. O objetivo deste trabalho foi avaliar a ocorrência de RPCP em duas amostras de solo sob diferentes tipos de manejo, através da construção e do seqüenciamento de bibliotecas de DNA metagenômico. Utilizaram-se oligonucleotídeos específicos para amplificação da região hipervariável do espaço intergênico dos genes ribossomais 16S-23S de DNA extraído de diferentes solos, sob Eucalyptus sp. e sob mata. Os fragmentos obtidos foram inseridos em vetor e clonados. As bibliotecas geraram 495 clones, que foram seqüenciados e identificados através de comparações realizadas pelo software Blast. O solo sob Eucalyptus sp. apresentou maior número de RPCP do que sob mata. Os filos Actinobacteria e Proteobacteria eram maiores no solo sob Eucalyptus sp., estando o filo Firmicutes ausente no solo sob mata. Somente oito espécies diferentes de RPCP foram detectadas: Bacillus subtilis, Bacillus megaterium, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium sp., Frankia sp., Pseudomonas fluorescens e Pseudomonas gladioli. O trabalho forneceu valiosos dados sobre a presença de RPCP em solos com espécies florestais e sua possível utilização em reflorestamentos, assim como para o melhor conhecimento desses microrganismos nos solos do Brasil.

Sinergismo Bacillus, Brevibacillus e, ou, Paenibacillus na simbiose Bradyrhizobium-caupi

O feijão-caupi Vigna unguiculata [L.] Walp. é a principal cultura de subsistência do semiárido brasileiro, sendo fonte de proteínas de baixo custo, notadamente, para populações carentes. A produção dessa cultura no Nordeste é baixa devido à não utilização de insumos agrícolas - entre eles, o fertilizante nitrogenado. Por outro lado, bactérias promotoras de crescimento em plantas (BPCPs) vêm sendo estudadas, de forma a maximizar a fixação de N2, disponibilizar nutrientes como P ou fito-hormônios e inibir doenças. Os objetivos deste trabalho foram verificar a viabilidade da coinoculação das sementes de feijão-caupi usando Paenibacillus, Brevibacillus e, ou, Bacillus e Bradyrhizobium-caupi; caracterizar as estirpes quanto à produção de ácido indol acético (AIA) e solubilização de fosfato; e avaliar o sinergismo entre os microrganismos como alternativa para otimizar a FBN. Os experimentos foram conduzidos em laboratório e em casa de vegetação do Instituto Agronômico de Pernambuco (IPA), utilizando o feijão-caupi cv. "IPA - 206". As estirpes utilizadas foram: Bacillus sp. - 449, 450, 451, 461 e ANBE 31; B. cereus - 440; B. subtilis - 438, 441, 454, 455 e 459; B. pumilus - 444, 445 e 448; B. megaterium - 462; Brevibacillus brevis - 447; Paenibacillus brasilensis - 24, 172 e 177; P. graminis - MC 04.21, MC 22.13 e BR 60106; P. polymyxa - S21; e P. durus - RBN4. Os microrganismos não apresentaram capacidade para produzir AIA nem solubilizar fosfato. Ocorreu sinergismo das estirpes de Bacillus sp. (449) e Bacillus pumilus (444) coinoculadas com a estirpe de Bradyrhizobium sp. (BR 3267) no feijão-caupi.

Functional genomics of intracellular bacteria.

During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

Bacterial variations on the methionine salvage pathway.

BACKGROUND: The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism. RESULTS: This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase), belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate). CONCLUSION: A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria (and in Eukarya), with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and RuBisCO, as an enolase, in some Firmicutes). Many are highly dependent on the presence of oxygen, suggesting that the ecological niche may play an important role for the existence and/or metabolic steps of the pathway, even in phylogenetically related bacteria. Further work is needed to uncover the corresponding steps when dioxygen is scarce or absent (this is important to explore the presence of the pathway in Archaea). The thermophile T. tengcongensis, that thrives in the absence of oxygen, appears to possess the pathway. It will be an interesting link to uncover the missing reactions in anaerobic environments.