Information

Do sulfate reducing bacteria ingest their sulfate as solid, or as dissolved in water?


Do sulfate reducing bacteria ingest their sulfate as solid, or liquid or gas?

I understand that almost all forms of sulfate are solid… and sulfate can be dissolved in water(thus no longer solid, but liquid).

So, do sulfate reducing bacteria ingest sulfate in its solid form, or do they require it to be dissolved in water?

A similar question in fact, for nitrifying bacteria, though if that's complex I might make it a separate question.

Also, even though it's respiration(which tends to be associated either with the breathing process - in the case of physiological respiration, or with having breathed prior - in the case of cellular respiration), here it's not using a gas. So i'm curious if biologists ever use the word "breathing" for such a process. wikipedia for example, says https://en.wikipedia.org/wiki/Sulfate-reducing_bacteria " In a sense, these organisms "breathe" sulfate… " I wonder if biologists use the term 'breath' in such a general sense(to include consuming a substance dissolved in water), or only in a specific sense of gas or air, or if they don't use the term 'breath' at all. So, how strictly is the term breath defined in biology.


Sulfates in water would not be liquid. Their melting points are far to high. When a sulfate dissolves into sulfate ions and some cation such as potassium, we say it is solvated, not liquid. Sulfates would also not be present as gasses due to their ionic nature as well as high molecular weights. So that leaves solvated sulfate ions and solid sulfates that have not completely dissolved.

To be ingested in bulk as a solid would require endocytosis which is not performed by prokaryotes: http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/proeu/proeu.html

Further research indicates sulfate, as ions, enters the cell through permeases: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.532.943&rep=rep1&type=pdf

Edit: Here's some files to help with the idea of how ions and other molecules enter cells. Again, bacteria do not engulf substances whole, in the process of endocytosis, such as eukaryotes are capable of. Here is an image of a nitrate ion within a transport protein. Only a few amino acids of the protein are shown here, the actual protein is much larger and spans across the entire membrane creating a channel for the ion to enter. http://www.rcsb.org/pdb/ngl/ngl.do?pdbid=4U4W&preset=ligandInteraction&sele=[NO3]

Here's another channel, aquaporin, which allows transport of water across the membrane. http://pdb101.rcsb.org/motm/173 The top left image shows the channel in the protein leading to the interior. The third image down shows the interaction between the water and the amino acids (backbones only are shown) which facilitate the water's passage.


Hydrogen Sulfide and Sulfate

Drinking water with high levels of hydrogen sulfide can cause nausea and stomach pain. However, it is highly unlikely that a person could consume a harmful dose of hydrogen sulfide from drinking water because water becomes unpalatable due to its unpleasant taste and odor long before hydrogen sulfide reaches a harmful level. In well water, it is usually just a nuisance.

High levels of sulfate (above 250 ppm) may have a laxative effect, cause dehydration and be especially detrimental to the health of infants and young animals. Sulfate levels in excess of 250 parts per million require treatment of drinking water before use.


Abstract

Background: Arsenic (As) toxicity is primarily based on its chemical speciation. Although inorganic and methylated As species are well characterized in terms of metabolism and formation in the human body, the origin of thiolated methylarsenicals is still unclear.

Objectives: We sought to determine whether sulfate-reducing bacteria (SRB) from the human gut are actively involved in the thiolation of monomethylarsonic acid (MMA V ).

Methods: We incubated human fecal and colon microbiota in a batch incubator and in a dynamic gut simulator with a dose of 0.5 mg MMA V in the absence or presence of sodium molybdate, an SRB inhibitor. We monitored the conversion of MMA V into monomethyl monothioarsonate (MMMTA V ) and other As species by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry analysis. We monitored the sulfate-reducing activity of the SRB by measuring hydrogen sulfide (H2S) production. We used molecular analysis to determine the dominant species of SRB responsible for As thiolation.

Results: In the absence of sodium molybdate, the SRB activity—primarily derived from Desulfovibrio desulfuricans (piger)—was specifically and proportionally correlated (p < 0.01) to MMA V conversion into MMMTA V . Inactivating the SRB with molybdate did not result in MMA V thiolation however, we observed that the microbiota from a dynamic gut simulator were capable of demethylating 4% of the incubated MMA V into arsenous acid (iAs III ), the trivalent and more toxic form of arsenic acid (iAs V ).

Conclusion: We found that SRB of human gastrointestinal origin, through their ability to produce H2S, were necessary and sufficient to induce As thiolation. The toxicological consequences of this microbial As speciation change are not yet clear. However, given the efficient epithelial absorption of thiolated methylarsenicals, we conclude that the gut microbiome—and SRB activity in particular—should be incorporated into toxicokinetic analysis carried out after As exposure.

Citation: DC.Rubin SS, Alava P, Zekker I, Du Laing G, Van de Wiele T. 2014. Arsenic thiolation and the role of sulfate-reducing bacteria from the human intestinal tract. Environ Health Perspect 122:817–822 http://dx.doi.org/10.1289/ehp.1307759

Introduction

Arsenic (As), particularly inorganic arsenic acid (iAs V ), is a ubiquitous contaminant, a nonthreshold class 1 carcinogen (Cantor and Lubin 2007 Mandal and Suzuki 2002). Global impacts of geogenic As increase the risk for elevated As exposure through the consumption of contaminated drinking water and food (Francesconi 2010 Sun et al. 2012). Although orally ingested and bioavailable, As was previously thought to be mainly biotransformed in the liver (Watanabe and Hirano 2012) however, the literature also suggests that As can be converted presystemically during gastrointestinal transit (Kubachka et al. 2009a Rowland and Davies 1981 Van de Wiele et al. 2010). Presystemic As metabolism is defined as the occurrence of As speciation changes due to physicochemical, enzymatic, or microbial metabolic processes in the gut before intestinal absorption and eventual bioavailability. Given the fact that As toxicity is primarily determined by its speciation, incorporating presystemic speciation changes into the risk evaluation process is warranted.

Analyses of human urine after iAs V exposure revealed sulfur-containing As metabolites such as monomethyl monothioarsonic acid (MMMTA V ) and dimethyl monothioarsinic acid (DMMTA V ) (Hansen et al. 2004 Raml et al. 2007). Sulfur-containing arsenicals have also been detected in the urine and feces of experimental animals (Conklin et al. 2006 Kubachka et al. 2009b), in water (Fisher et al. 2007), and in vegetables (Yathavakilla et al. 2008). In addition, thioarsenicals have been produced within the headspace of a reaction tube containing a human fecal slurry and arsenate (Diaz-Bone et al. 2009). Furthermore, significant As thiolation has been observed with in vitro digestion of iAs V under gastric conditions and with human colon microbiota (Van de Wiele et al. 2010). More recently, Pinyayev et al. (2011) showed that arsenate can be converted into methyl- and thioarsenicals by the anaerobic microbiota of the mouse cecum. However, the microbial mechanism of thioarsenical formation is not well understood. Moreover, the toxicity profiles remain under discussion (Dopp et al. 2010).

Given the importance of sulfate reduction by sulfate-reducing bacteria (SRB) in the human colon (Ley et al. 2006 Marchesi 2011), previous studies hypothesized that the SRB community in the gut may play an important role in the thiolation of arsenicals (Conklin et al. 2006 Van de Wiele et al. 2010). In the present study, we investigated to what extent thiolation of methylarsonic acid relies on the presence and metabolic activity of SRB from the human gut. Our findings suggest an active involvement of sulfate-reducing activity toward the gastrointestinal formation of thiolated methylarsenicals.

Materials and Methods

Chemicals, media, and microbial cultures. Degassed and ultrapure 18 mΩ water [double-distilled ionized water (DDI) Millipore, Bedford, MA, USA)] was used to prepare the chromatographic mobile phase and the standard stock solutions. American Chemical Society–grade ammonium nitrate and ammonium dihydrogen phosphate (Fisher Scientific, Pittsburgh, PA, USA) and technical-grade EDTA, tetrasodium salt dihydrate (Fisher Scientific, Fair Lawn, NJ, USA), were used in the chromatographic mobile phase. Certified stock solutions of monomethylarsonic acid (MMA V ) and sodium arsenate (Na2HAsO4·7H2O) were purchased from Chem Service (West Chester, PA, USA) and sodium molybdate (Na2MoO4·2H2O) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Molybdate is not considered to be a bactericidal agent. Rather, it is a bacteriostatic agent: This compound merely inhibits the metabolic activity, limiting SRB in their growth and production of hydrogen sulfide (H2S). MMA V and iAs V stock solutions were prepared in DDI water at 0.1 g As/L and stored at –4°C.

MMMTA V was synthesized using a mixture of MMA V and H2S solutions. In a 1-mL glass vial, 900 μL of a 40-μg As/mL MMA V solution and 100 μL of a saturated H2S solution were combined. The mixture was left overnight on a mechanical shaker for thorough mixing. Progress of the reaction was verified by liquid chromatography coupled with inductively coupled plasma mass spectrometry (LC-ICP-MS). Molecular identity of the product was checked by LC-LTQ-XL-MS [LC coupled with linear ion trap MS] and MS/MS (Alava et al. 2012). The MMA V and H2S solutions were made as described by Alava et al. (2012). Briefly, we prepared a 40-μg MMA V /mL solution by combining 60 μL of a 1,850-μg MMA V /mL solution and 2.94 mL of a 10% vol/vol formic acid solution. Preparation of the saturated H2S solution was conducted in a 100-mL round-bottom flask. One gram of iron(II)sulfide (Harshaw Scientific, Cleveland, OH, USA) was supplemented with 2 mL of hydrochloric acid along with 4 mL of DDI. The mixture started to bubble instantly, releasing H2S gas. The H2S was bubbled into 15 mL of DDI water until the effervescence in the round-bottom flask subsided, creating the saturated H2S solution.

The Simulator of the Human Intestinal Microbial Ecosystem (SHIME) is a dynamic multicompartment simulator of the human gastrointestinal tract, mimicking the digestive processes of the stomach, small intestine, and ascending, transverse, and descending colon. The model has been validated against human in vivo conditions both in terms of gut microbial composition and metabolic activity (i.e., short-chain fatty acid profile) (Molly et al. 1994 Possemiers et al. 2006). The nutritional medium for the SHIME was prepared as described by Boever et al. (2000) and enabled the microbial communities of the different colon compartments to adapt to the nutritional and physicochemical conditions that prevail in the ascending, transverse, and descending colon. Briefly, 1 L of SHIME medium contained 1 g arabinogalactan, 2 g pectin, 1 g xylan, 3 g starch, 0.4 g glucose, 3 g yeast extract, 1 g pepsin, 4 g mucin, and 0.5 g cystein, at pH 7.

Postgate medium C (Grossman and Postgate 1953) was used to enrich SRB. It consisted of 4.5 g sodium sulfate, 0.5 g potassium dihydrogen phosphate, 0.06 g magnesium sulfate, 1.0 g ammonium chloride, 0.06 g calcium chloride, 1 g yeast extract, 0.1 g ascorbic acid, 0.004g ferrous sulfate, 6 g sodium lactate, and 0.3 g sodium citrate at pH 7.5. Modified Postgate medium C with different sulfate concentrations was obtained using a 4-fold dilution series in the concentration range: 0.007, 0.032, 0.125, and 0.5 M of sodium sulfate.

A pure culture of Desulfovibrio desulfuricans LMG 7529 was purchased from the Belgian Co-ordinated Collections Of Micro-organisms (BCCM-LGM http://bccm.belspo.be/index.php) and was grown in the recommended medium 104 (BCCM-LMG). This strain is equivalent to ATCC 29577 ( http://www.atcc.org). D. desulfuricans is still considered D. piger (Castro et al. 2000).

Batch incubations of enriched, nonenriched, and pure cultures. A first set of experiments was used to check to what extent the in vitro–cultured gut microbiota from the human inoculum was capable of performing iAs V biotransformation in a manner similar to some of our previous findings (Alava et al. 2012 Van de Wiele et al. 2010). Briefly, 2 mL of descending colon suspension from the SHIME (see below) was anaerobically incubated with 30 μg iAs V /L for 48 hr and then analyzed for its As speciation profile with HPLC-ICP-MS as detailed by Van de Wiele et al. (2010).

The second set of experiments was more specifically targeted at evaluating the potential of gut microbiota and SRB to thiolate MMA V . We anaerobically incubated 2 mL of nonenriched SRB descending colon suspension from the SHIME for 48 hr with 0.5 mg/L MMA V . To favor SRB enrichment, 2 mL was sampled from the SHIME descending colon suspension and anaerobically incubated for 48 hr with 0.5 mg/L MMA V in 18 mL Postgate medium C. The contribution of a reference sulfate-reducing strain toward MMA V (0.5 mg/L) thiolation was assessed by incubating 2 mL of a pure culture of Desulfovibrio desulfuricans (piger) in 18 mL of culture medium 104 (BCCM-LMG).

A third set of experiments was performed to test the interindividual variability in MMA V thiolation by human fecal microbiota. Fecal microbiota from seven different human individuals with no history of antibiotic treatment in the 6 months before the study (De Weirdt et al. 2010) and descending colon samples from three different SHIMEs were separately incubated with 0.5 mg/L MMA V in Postgate medium C.

All incubation experiments with enriched and nonenriched SRB SHIME descending colon samples, human fecal microbiota and with D. desulfuricans (piger) were performed in the absence or presence of sodium molybdate (20 mM), a specific SRB inhibitor. In addition, heat-sterilized (120°C) incubations were used as an abiotic control. Incubations were performed under anaerobic conditions by capping the serum bottles with butyl rubber stoppers that are impervious to oxygen and subsequently flushing the bottles with nitrogen gas for 25 min. Cultures were then incubated at 37°C on a rotary shaker (180 rpm) for 48 hr. Aliquots of 2 mL/analysis were collected at four time points—0, 6, 24, and 48 hr—to monitor SRB activity, As speciation changes, and the molecular analysis of the microbiota. This study was approved by Ghent University’s ethical committee and registered by Belgian authorities (no. B670201214538).

Continuous incubations in a SHIME. Although the former batch experiments were conducted under SRB-favoring conditions, we performed a SHIME run to verify whether MMA V speciation changes, particularly thiolation, also occurred under more representative conditions for the human gut that do not favor SRB. Moreover, the SHIME reactor also allows addressing colon-region–specific differences in the MMA V thiolation potential.

The treatment consisted of a daily supplementation of 0.5 mg MMA V /L during 4 days. 20 mM of sodium molybdate was added on the third and fourth days of the SHIME run to inhibit SRB activity. Aliquots of 2 mL/analysis were collected from the ascending, transverse, and descending colon in order to monitor the conversion of MMA V as well as SRB activity.

SRB activity analysis and sample preparation for speciation analysis. SRB activity was monitored by measuring H2S production using an analytical kit for detection of sulfide (Hach, Loveland, CO, USA) in an automated spectrophotometer (Nanocolor 500D Macherey-Nagel, Düren, Germany) in 1:1 and 1:2 dilutions with anoxic water. To preserve the samples for further As speciation analysis, all samples were flash frozen with liquid nitrogen upon incubation and subsequently stored at –80°C. Before analysis with HPLC-ICP-MS, the samples were thawed and dissolved with ammonium carbonate (20 mM, pH 9.0) to minimize any sulfur–oxygen exchange while awaiting analysis (Conklin et al. 2006). Upon complete thawing, the sample was vortexed and centrifuged for 10 min at 10,400 ×g with an Eppendorf 5810R centrifuge (Brinkman Instruments, Westburg, NY, USA) to separate soluble As species from insoluble As (e.g., As sorbed to microbial biomass). The supernatant was filtered through a Millex-LCR 0.45 μm filter (Millipore) with a Luer-Lok 10-mL syringe (Becton, Dickinson and Co., Franklin Lakes, NJ, USA).

As speciation analysis by HPLC-ICP-MS. As speciation changes, and especially the conversion of MMA V into MMMTA V and arsenous acid (As III ), were monitored with HPLC-ICP-MS matching the retention time and by comparing fragmentation pattern of prepared MMMTA V on electrospray ionization–MS/MS with previously published conditions (Van de Wiele et al. 2010), using the limits of detection and quantification for the different As species indicated in Supplemental Material, Table S1. Briefly, 2 mL of the supernatant of each incubated sample was filtered using a 0.45-μm syringe-type PVDF (polyvinylidene difluoride) membrane filter, and the filtrate was diluted into 25 mL using DDI water. This filtrate was analyzed for total As content using ICP-MS. The same filtrate was used for speciation analysis using HPLC and optimized instrumental parameters for ICP-MS (PerkinElmer, Sunnyvale, CA, USA). Filtrates were diluted with the mobile phase and injected into the HPLC. We considered the sum of the As species in the filtrate observed chromatographically to be the bioaccessible fraction. We measured total As concentration in the digest filtrates using ICP-OES (ICP–optical emission spectroscopy) according to Alava et al. (2012, 2013). The applicable detection limit was 0.5 μg/L.

Molecular analysis. We performed polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) to obtain a general profile of the microbial community, used qPCR (quantitative PCR) to quantify the SRB (see Supplemental Material, Table S2), and created a clone library to identify the most dominant SRB species in the enriched and nonenriched SRB incubation experiments, as well as Illumina (San Diego, CA, USA) sequencing of nonenriched fecal samples. DNA extraction was carried out using the UltraClean® DNA Isolation Kit following the manufacturer’s instructions (Mo Bio Laboratories Inc., Carlsbad, CA, USA). PCR-DGGE of the 16S rRNA genes for all bacteria were amplified by PCR using the Taq-Polymerase Kit (Fermentas Inc., Hanover, MD, USA) with the general bacterial primers P338F and P518R and a GC-clamp of 40 bp on the forward primer (Muyzer et al. 1993). DGGE was performed using the Bio-Rad D gene system (Bio-Rad, Hercules, CA, USA). Clustering was based on the densitometric curves according to the Pearson correlation using BioNumerics software (version 5.1 http://www.applied-maths.com). (For the clone library, see Supplemental Material, p. 2.) Briefly, the PCR amplification of 16S rRNA gene fragments was carried out with the universal primers 63F and 1378R and cloned into the pCR®-TOPO® Vector of the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). The qPCR specific for SRB’s target gene, dsrB (dissimilatory sulfite reductase subunit beta the gene for the key enzyme in dissimilatory sulfate reduction and phylogenetic marker for identification of SRBs) was carried out as described by Vermeiren (2011), adapted from Spence et al. (2008) (See Supplemental Material, Figure S1).

Statistical analysis and sequences. Batch incubation experiments of more than four groups were conducted in triplicate and SHIME run in duplicates. All data were analyzed using SigmaPlot, version 12.0 (SYSTAT Software Inc., San Jose, CA, USA). A one-way analysis of variance (ANOVA) test was performed to investigate intergroup differences. Two-case groups were covered by a t-test. Statistical differences for ANOVA and t-tests were significant if p ≤ 0.05, and highly significant if p ≤ 0.01. The nucleotide sequences data of the clone library are available in the European Molecular Biology Laboratory–European Bioinformatics Institute public database EMBL-EBI ( http://www.ebi.ac.uk/ accession numbers HG531812 to HG531931).

Results

In our batch incubation experiments with SHIME descending colon microbiota, the human gut microbiota actively metabolized iAs V (30 μg = 100%) (Table 1). Importantly, 7% MMMTA V formation was observed upon 48 hr of incubation. In addition, iAs V was reduced to As III (18%), and further transformation toward monomethyl arsonous acid (MMA III ) (6.6%), MMA V (3.2%), and dimethylarsinic acid (DMA V ) (54%) was noted. These data show that the in vitro cultured microbial community from the human inoculum in these experiments had the potency to actively metabolize iAs V (Table 1).

Table 1 Metabolic potency of colon microbiota (percent toward iAs V speciation from a single experiment

Time (hr)As V As III MMA V MMA III MMMTA V DMA V DMA III
0100.0 a
55.132.13.950.1
84.931.23.73.71.259.8
246.718.15.64.73.464.5
486.118.23.26.67.054.3
Abbreviations: DMA III , dimethylarsinous acid DMA V , dimethylarsinic acid MMA III , monomethyl arsonous acid. a As V standard at high concentration of 30 μg (100%).

Subsequently, we investigated to what extent SRB are involved in the thiolation of MMA V toward MMMTA V . The batch incubations showed that SRB can be enriched with Postgate medium C and can be inhibited by the addition of sodium molybdate, and this was reflected in the sulfide production potential (Figure 1A). Molybdate is a well-known inhibitor of ATP-sulfurylase, thereby inhibiting SRB in their ability to produce sulfide but also limiting their ability to generate energy: Growth will therefore be reduced (Figure 1 see also Supplemental Material, Figure S1). Furthermore, we found that descending colon microbiota under enriched SRB conditions produced significantly more MMMTA V (28 μg/L) than under nonenriched conditions (15 μg/L) (p < 0.01). In contrast, no MMA V to MMMTA V conversion was observed when the SRB-inhibitor sodium molybdate was supplemented (Figure 1B). In addition, both the incubations, with enriched and nonenriched SRB cultures, displayed a positive correlation (coefficient of determination) between the formation of MMMTA V and production of H2S (R 2 = 0.978 and R 2 = 0.992, respectively). Finally, no speciation changes were observed in the abiotic control, in which heat-sterilized colon microbiota were incubated. However, the fraction of MMA V remaining in the supernatant declined because of sorption to the dead organic biomass (see Supplemental Material, Table S3). This sorption shows the necessity of the contribution of sulfate-reducing activity to the thiolation process, whereas inactivation through a specific inhibitor or heat-sterilization removes the thiolation ability. It must be noted that the presence of H2S as such suffices to chemically produce MMMTA V from MMA V (see “Materials and Methods”). Hence, the As thiolation in the gut can be considered a chemical process that requires a biological trigger, that is, sulfide production by metabolically active SRB.

Figure 1 Sulfate-­reducing activity (H2S production) (A) and MMMTA V formation (B) in enriched SRB (with and without sodium molybdate, the SRB inhibitor), non­enriched SRB, and abiotic (sterilized) cultures during 48 hr of incubation with MMA V (0.5 mg/L). *p < 0.05, and **p < 0.01, by one-way ANOVA. # p < 0.05, and ## p < 0.01, by two-case t-test based on non­enriched SRB (intermediary group).

To identify the dominant microbial species in enriched SRB cultures responsible for the thiolation of MMA V , we performed molecular analysis using the enriched and nonenriched SRB cultures. Analysis from the clone library revealed a dominant sequence over time (from 18% to 63% at 6 hr to 48 hr, respectively) with 99% of similarity to D. desulfuricans (piger) (Figure 2A). PCR-DGGE also showed an SRB-predominant band over the time observed in the enriched cultures (see Supplemental Material, Figures S2 and S3). Quantitative analysis with qPCR further confirmed the increasing abundance of SRB in the enriched cultures (Figure 2B). In addition, Illumina sequencing of nonenriched fecal incubations showed that D. desulfuricans (piger) is the most dominant SRB present in human gut (data not shown). Incubations of pure cultures of D. desulfuricans (piger) displayed sulfate-reducing activity and As-thiolation ability similar to that observed with the enriched SRB colon microbiota (see Supplemental Material, Figure S4).

Figure 2 Molecular analysis of SRB cultures. (A) Clone library of 16S rRNA at the genus level (the operational taxonomic unit at 0.03%) during the enrichments of SRB culture in Postgate medium C. (B) Relative number of copies normalized to SRB dsrB gene of enriched, non­enriched, and pure fecal samples during 48-hr of incubation. Values are mean ± SD n = 3.

Using SHIME as a dynamic simulator of the human gut (see Supplemental Material, Figure S5), we then investigated whether thiolation of 0.5-mg/L MMA V is colon-region–specific under more representative conditions for the gastrointestinal tract (Figure 3). MMA V thiolation was observed in the SHIME, with MMMTA V formation primarily taking place in the ascending and transverse colon compartments at a rate of > 30 μg/L per day (Figure 3A,B). This resulted in high amounts of MMMTA V (> 35 μg/L) in the ascending and transverse colon vessels, whereas only a minor amount was observed in the descending colon (Figure 3C). MMMTA V formation took place within the first 10 hr upon supplementation of MMA V (Figure 3). Adding sodium molybdate on the third and fourth days of the SHIME run to eliminate SRB activity did not result in a decrease in MMA V conversion. Instead of MMMTA V formation, demethylation of MMA V occurred toward iAs III (arsenous acid)—a process that primarily occurred in the distal colon regions (Figure 3C). (For information on the SHIME reactor distal colon regions and a scheme of As speciation, see Supplemental Material, Figures S5 and S6.)

Figure 3 As speciation of MMA V into MMMTA V and As III in the ascending (A), transverse (B), and descending (C) colon of the dynamic gut model SHIME. As speciation (i.e., thiolation of MMA V into MMMTA V or demethylation of MMA V into As III ), was measured daily during 4 days (the 24, 25, 26, 27th days) at two time points: 10 and 16 hr. The horizontal arrows indicate the sequence of colon compartments of the SHIME. The vertical arrow in (A) indicates the addition of the SRB inhibitor sodium molybdate.

Finally, we observed interindividual variability in the sulfate-reducing activity and MMA V thiolation between different human fecal inocula (Figure 4). The fecal microbiota from individuals A and C displayed much higher levels of H2S (> 15 mg/L H2S) in comparison with the fecal microbiota from the other individuals (Figure 4A). This H2S production from fecal microbiota A and C corresponded with a pronounced production of MMMTA V (> 20 μg/L) (Figure 4B). In contrast, for those fecal microbiota that displayed low SRB activity (around 5 mg/L H2S), only a limited amount of MMMTA V was formed over time (around 4.5 μg/L at 24 and 48 hr). Moreover, the fecal microbial inoculum G displayed the lowest H2S production (< 2.5 mg/L) and no formation of MMMTA V . Overall, MMMTA V formation and H2S production by fecal microbiota from each of the individuals were strongly correlated to one another (R 2 = 0.994) after 48 hr.

Figure 4 Interindividual variability of sulfate-­reducing activity (H2S production A) and thiolation (MMMTAV formation B) in different human fecal samples (from individuals A, B, C, G, H, I, and J) during 48 hr of incubation with MMA V . Abiotic controls are represented by the heat sterilized incubation of fecal microbiota from individual A. **p < 0.01, by one-way ANOVA and two-case t-test compared with the other groups.

Discussion

The findings of the present study show that human colon microorganisms have the potency of presystemic As metabolism, similar to results previously obtained with rodent (Conklin et al. 2006 Kubachka et al. 2009a) and human gut microbiota (Van de Wiele et al. 2010). Moreover, this study shows that the active involvement of SRB from human origin contributes to the thiolation of MMA V into MMMTA V . We observed this process both in enriched and nonenriched SRB in cultures of the descending colon, of human fecal microbiota, and of pure SRB isolates, as well as under more representative conditions for the human gut in the SHIME, a dynamic gut simulator. The active contribution of SRB was demonstrated by the high correlation between H2S production and MMMTA V formation and by the lack of MMMTA V formation and H2S production in the presence of the SRB inhibitor sodium molybdate. Moreover, our findings indicate that D. desulfuricans (piger) may be the principal microbe contributing to the As thiolation process. Although the metabolic activity of SRB has been well studied and even implicated in the methylation process of mercury (Gilmour et al. 2011), the role of metabolically active SRB, and particularly D. desulfuricans (piger), toward As thiolation is a new finding.

Our observations parallel those of studies showing MMMTA V formation upon incubation of iAs V with human colon microbiota (Van de Wiele et al. 2010) or the formation of headspace thio-arsenicals when a human fecal slurry was incubated with arsenate (Diaz-Bone et al. 2009). Previous studies have reported that mouse cecal microbiota can trigger the formation of thioarsenosugars upon the incubation with arsenosugars (Conklin et al. 2006) as well as the production of methylated thioarsenicals from DMA V by rat intestinal microbiota (Yoshida et al. 2001) and DMA V conversion into trimethylarsine sulfide by mouse ceca (Kubachka et al. 2009a). Therefore, the presence of SRB in both the human and different animal gastrointestinal environments may be considered to be an important factor in the As thiolation process and, when considering the environmental presence of SRB, to also impact the biogeochemical cycles of sulfur and As (Muyzer and Stams 2008). Yet, As thiolation involves a chemical reaction that is biologically induced by metabolically active SRB. This view is supported by the possibility of chemically producing MMMTA V by reacting MMA V with a saturated H2S solution and corresponds with previous hypotheses that the presence of sulfide is sufficient to obtain interconversion between oxide and sulfide forms of MMA V , DMA V , and trimethylarsine oxide (Conklin et al. 2006).

Although the importance of As thiolation by endogenous SRB can be derived from the present data set, the results also demonstrate that the thiolation does not take place at the same rate throughout the entire gastrointestinal tract. In the present study, As thiolation appeared to be colon-region specific: Thiolation primarily occurred in the ascending and transverse colon. This observation is strongly supported by the fact that in the in vivo human colon, SRB are more abundant in the ascending and transverse colon, whereas homo-acetogens (which compete for reducing equivalents) are more abundant than SRB in the descending colon (Nava et al. 2012).

In addition, the inactivation of SRB activity by sodium molybdate in the SHIME colon compartments resulted in the demethylation of MMA V toward iAs III by descending colon microbiota. Although demethylation of MMA V was previously reported for soil microbial communities (Yoshinaga et al. 2011), this is to our knowledge the first study to report As demethylation by human colon microorganisms. These findings are of toxicological concern. On the one hand, MMA V demethylation is rather unexpected because iAs III is more toxic than MMA V (Naranmandura et al. 2011 Van de Wiele et al. 2010). On the other hand, the strongly reducing conditions that prevail in the SHIME colon compartments (–200 to –250 mV) may lead to the reduction of MMA V toward its trivalent analogue, MMA III . The ability of this As species to generate highly reactive oxygen species and to induce DNA damage makes it an order of magnitude more toxic than iAs III (Naranmandura et al. 2011). Although MMA III was not detected under the dynamic incubation conditions of the SHIME, its production as intermediate is likely, as was also indicated by the finding of considerable amounts of MMA III upon static incubation of iAs V (Table 1). Supported by reports that MMMTA V is several orders of magnitude less toxic than iAs III , and even less toxic than iAs V , we therefore consider intestinal MMA V thiolation to be a detoxification reaction however, further investigation is needed. Thiolated arsenicals display a highly variable toxicity profile. The monothiolated form of DMA V , DMMTA V —often found in the urine of iAs V -exposed individuals (Heitland and Köster 2008 Raml et al. 2007)—is one of the most toxic As species known, comparable to DMA III (dimethylarsinous acid), whereas its dithiolated analogue, DMDTA V (dimethyldithioarsinic), is almost harmless (Naranmandura et al. 2011). Whether SRB also contribute to the formation of DMMTA V , just as they do for MMMTA V , remains to be resolved.

Finally, As thiolation was characterized by a large interindividual variability. Again, the ability for a fecal microbiome to produce MMMTA V correlated with the levels of H2S produced, further supporting the role for SRB as the basis of the thiolation process. Despite the enterotypes (Arumugam et al. 2011), the human gut microbiome is known for its high interindividual variability, which might also be reflected in the variable abundance of SRB in the colon lumen or on the colon mucosal surfaces (Nava et al. 2012). Although host genetic factors have been reported to contribute to the interindividual variability in As toxicity (Hernández and Marcos 2008), we propose that the gut microbiome must be incorporated as a factor that contributes to this variability.

Conclusion

From the findings presented here, we conclude that gut microbiota of human origin can extensively metabolize As, with SRB being necessary and sufficient for the biologically induced thiolation of MMA V into MMMTA V . The variability in the thiolation potency between different fecal inocula was reflected by a large interindividual variability in SRB abundance. In addition, eliminating SRB activity (as evidenced by H2S production) may also result in MMA V demethylation to iAs III . Although the toxicological consequences of these microbial processes are not yet clear and the interindividual variability adds an extra layer of complexity over As toxicokinetics, our findings demonstrate the necessity to consider SRB and, by extension, the human gut microbiome when assessing risks from oral As exposure.

Supplemental Material

(1.6 MB) PDF Click here for additional data file.

We thank T. Lacoere and J. Debodt for their helpful technical assistance.

S.S.C.DC.R. received fellowships from the De Vlaamse Interuniversitaire Raad (VLIR), Belgium, and the Erasmus Mundus Program from the European Commission and is currently a postdoc fellow of the Science Without Borders Program, from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. I.Z. received support from the Estonian Ministry of Education and Science Project IUT20-16. This work was supported by grant BIOTRAS RF 6247 from the Belgian federal government.

The authors declare they have no actual or potential competing financial interests.


Abstract

We investigated microbial methylmercury (CH3Hg) production in sediments from the South River (SR), VA, an ecosystem contaminated with industrial mercury (Hg). Potential Hg methylation rates in samples collected at nine sites were low in late spring and significantly higher in late summer. Demethylation of 14 CH3Hg was dominated by 14 CH4 production in spring, but switched to producing mostly 14 CO2 in the summer. Fine-grained sediments originating from the erosion of river banks had the highest CH3Hg concentrations and were potential hot spots for both methylation and demethylation activities. Sequencing of 16S rRNA genes of cDNA recovered from sediment RNA extracts indicated that at least three groups of sulfate-reducing bacteria (SRB) and one group of iron-reducing bacteria (IRB), potential Hg methylators, were active in SR sediments. SRB were confirmed as a methylating guild by amendment experiments showing significant sulfate stimulation and molybdate inhibition of methylation in SR sediments. The addition of low levels of amorphous iron(III) oxyhydroxide significantly stimulated methylation rates, suggesting a role for IRB in CH3Hg synthesis. Overall, our studies suggest that coexisting SRB and IRB populations in river sediments contribute to Hg methylation, possibly by temporally and spatially separated processes.


Do sulfate reducing bacteria ingest their sulfate as solid, or as dissolved in water? - Biology

a School of Environmental Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
E-mail: [email protected]
Fax: +86-531-89631680
Tel: +86-531-89631680

b Jiangsu Key Laboratory of Anaerobic Biotechnology, Jiangnan University, Wuxi 214122, P. R. China

Abstract

Removal of sulfide from a micro-aerobic bio-reactor was studied at 10 000 mg L −1 chemical oxygen demand (COD) of inlet water, with the sulfate volumetric loading 0.75, 1.0, 1.5 and 2.0 kg (m −3 d −1 ), respectively. Tentatively, activated carbon (AC) as an adsorbent was modified in positively charged iron to adsorb bio-sulfur through electrostatic interaction. At an O2/S molar ratio of 8–10, the reactor was sufficient to decrease the sulfide in the effluent and biogas to low levels at the sulfate volumetric loading of 2 kg (m −3 d −1 ). After iron-modified, the specific surface area of AC was form 32.4 m 2 g −1 to 65.0 m 2 g −1 , and the zeta potential was 25.3 mV at pH 7.0. The XRD pattern of the iron-modified activated carbon (FeAC) explained that the metal species of iron was Fe3O4. It could be clearly seen that there was Fe3O4 on the surface of the FeAC, and sulfur particles with a large particle size were adsorbed by the FeAC on the SEM figures. And the XRD pattern of the bio-sulfur explained that the bio-sulfur was made up of S8 (91.444%), C3H4N2OS (1.491%) and CH5N3S (7.075%). The zeta potential of bio-sulfur was −25 mV and the particle size was mainly distributed at the average diameter of 1935 nm at pH 7.0.


Results

Sulfate reduction, H2 uptake, cell growth, and iron sulfide precipitation

In all assays of both enrichments, an increase in pH was always concomitant to a decrease in sulfate. Assays at pHini 4.2, 4.8, and 5.8 of enrichments I behaved similarly to assays pHini 4.8 and 5.9 of enrichments II with respect to the time courses of sulfate and pH. While for both enrichments, a delay in sulfate reduction at pHini < 4 was noticed, those of enrichments II showed a steeper decline in sulfate concentrations and a prolonged sulfate reduction phase. In the following, we focus on enrichment II as the influence of the original sediment was marginal and a distinct sulfate-reducing bacterial community had developed in the more acidic assays. Replicate assays behaved similarly and averaged values of replicate assays are depicted in Figs 0001 and 0002 with the exception of replicate B at pHini 3.8 where reactions occurred earlier on in comparison to the other two replicates. Overall, pronounced metabolic activity and cell growth started off later under more acidic conditions with the lag phase increasing from about 3 to 34 days (Fig. 0001 ). However, free sulfide was already detectable about 2 weeks earlier than the onset of the main sulfate reduction phase (Fig. 0002 a and b). An increase of cell numbers was also observed in this early phase of incubation (Fig. 0001 a and b). This indicated that sulfate reduction and cell growth occurred at low pH conditions, though at a very low rate. In all assays, sulfate removal and H2 uptake showed almost identical time courses and the ratios between the amount of H2 consumed and the amount of sulfate removed approximated 4 : 1 (Table 0002 ). A ratio close to 4 : 1 suggests that microbial sulfate reduction with H2 as an electron donor accounted for most of the sulfate removal. A ratio slightly lower than 4 : 1 in the more acidic assays indicated that additional abiotic processes, such as the precipitation of sulfate minerals, might have taken place. The formation of alunite and gypsum was predicted, but their presence in the final samples could not be confirmed by electron microscopy and microanalysis. A linear increase of the amount of sulfate removed (and H2 consumed) indicated H2 limitation because of a low gas transfer rate. Average sulfate reduction rates in replicate assays were 450, 400, 240, and 320 μmol sulfate L −1 day −1 at pHini 3.3, 3.8 (excluding replicate B), 4.8, and 5.9, respectively. Cell numbers increased by a factor of approximately 120 in the more acidic assays but only by a factor of 20 at pHini 4.8 and of 30 at pHini 5.9. Estimates for growth yields were generally very low ranging from 0.23 to 0.52 g dry mass per mol (Table 0002 ). Using the cell numbers obtained at the end of the main sulfate reduction phase (Table 0002 ), the cellular sulfate reduction rates were 8.6, 6.6, 12.7, and 17.7 fmol sulfate cell −1 day −1 at pHini 3.3, 3.8, 4.8, and 5.9, respectively. Taking, however, the cell numbers determined at the onset of the main sulfate reduction phase, cellular sulfate reduction rates corresponded to 88, 72, 285, and 417 fmol sulfate cell −1 day −1 , respectively.

Sulfate removal, H2 uptake, concentrations of dissolved Fe(II), cell density, and pH as a function of time for enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Scale of H2 uptake is four times the scale of sulfate removal. Values from replicate assays were averaged bars indicate minimum and maximum values. Replicate B at pHini 3.8 is not included in (b).

Sulfate removal, H2 uptake, concentrations of dissolved Fe(II), cell density, and pH as a function of time for enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Scale of H2 uptake is four times the scale of sulfate removal. Values from replicate assays were averaged bars indicate minimum and maximum values. Replicate B at pHini 3.8 is not included in (b).

Concentrations of free sulfide (H2S/HS − ) over the first 40 days of incubation compared to changes in concentrations of dissolved Fe(II) and pH (from Fig. 0001 ) for enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Values from replicate assays were averaged (excluding replicate B at pHini 3.8) bars indicate minimum and maximum values (only shown for free sulfide).

Concentrations of free sulfide (H2S/HS − ) over the first 40 days of incubation compared to changes in concentrations of dissolved Fe(II) and pH (from Fig. 0001 ) for enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Values from replicate assays were averaged (excluding replicate B at pHini 3.8) bars indicate minimum and maximum values (only shown for free sulfide).

Final pH, total amounts of sulfate removed and H2 consumed by the end of the experiment, maximum cell densities, and estimated biomasses obtained for enrichment II

pHini 3.3 pHini 3.8 pHini 4.8 pHini 5.9
pHfinal6.61 (6.59/6.64) 6.75 (6.61/6.96) 7.01 (6.97/7.09) 7.36 (7.29/7.40)
(mM) 17.20 (17.14/17.26) 16.19 (15.36/17.67) 12.67 (12.47/13.02) 13.96 (13.46/14.36)
H2 total (mM) 68.09 (67.29/69.15) 65.73 (60.50/73.82) 57.49 (55.48/58.68) 57.92 (56.78/58.68)
Ratio H2 total/ 3.96 (3.93/4.02) 4.06 (3.94/4.18) 4.54 (4.26/4.71) 4.15 (4.09/4.22)
Maximum cell number (mL −1 ) 5.25 × 10 7 (4.26/5.99 × 10 7 ) 6.11 × 10 7 (4.48/6.95 × 10 7 ) 1.89 × 10 7 (1.44/2.41 × 10 7 ) 1.81 × 10 7 (1.61/1.97 × 10 7 )
Average cell volume (μm 3 ) 0.158 0.134 0.747 1.025
Biomass (mg dry mass L −1 ) 3.90 4.39 6.64 7.25
Yield (g dry mass mol −1 ) 0.23 0.27 0.52 0.52
pHini 3.3 pHini 3.8 pHini 4.8 pHini 5.9
pHfinal6.61 (6.59/6.64) 6.75 (6.61/6.96) 7.01 (6.97/7.09) 7.36 (7.29/7.40)
(mM) 17.20 (17.14/17.26) 16.19 (15.36/17.67) 12.67 (12.47/13.02) 13.96 (13.46/14.36)
H2 total (mM) 68.09 (67.29/69.15) 65.73 (60.50/73.82) 57.49 (55.48/58.68) 57.92 (56.78/58.68)
Ratio H2 total/ 3.96 (3.93/4.02) 4.06 (3.94/4.18) 4.54 (4.26/4.71) 4.15 (4.09/4.22)
Maximum cell number (mL −1 ) 5.25 × 10 7 (4.26/5.99 × 10 7 ) 6.11 × 10 7 (4.48/6.95 × 10 7 ) 1.89 × 10 7 (1.44/2.41 × 10 7 ) 1.81 × 10 7 (1.61/1.97 × 10 7 )
Average cell volume (μm 3 ) 0.158 0.134 0.747 1.025
Biomass (mg dry mass L −1 ) 3.90 4.39 6.64 7.25
Yield (g dry mass mol −1 ) 0.23 0.27 0.52 0.52

Values of replicate assays are averaged with minimum and maximum values in parentheses.

Removal of sulfate and uptake of H2 in μmol per ml culture liquid accumulated over time.

Average cell volume calculated from measurements performed on 30 cells at late growth phase to early stationary phase.

Biomass was converted from total biovolume (see explanations in the main text).

Final pH, total amounts of sulfate removed and H2 consumed by the end of the experiment, maximum cell densities, and estimated biomasses obtained for enrichment II

pHini 3.3 pHini 3.8 pHini 4.8 pHini 5.9
pHfinal6.61 (6.59/6.64) 6.75 (6.61/6.96) 7.01 (6.97/7.09) 7.36 (7.29/7.40)
(mM) 17.20 (17.14/17.26) 16.19 (15.36/17.67) 12.67 (12.47/13.02) 13.96 (13.46/14.36)
H2 total (mM) 68.09 (67.29/69.15) 65.73 (60.50/73.82) 57.49 (55.48/58.68) 57.92 (56.78/58.68)
Ratio H2 total/ 3.96 (3.93/4.02) 4.06 (3.94/4.18) 4.54 (4.26/4.71) 4.15 (4.09/4.22)
Maximum cell number (mL −1 ) 5.25 × 10 7 (4.26/5.99 × 10 7 ) 6.11 × 10 7 (4.48/6.95 × 10 7 ) 1.89 × 10 7 (1.44/2.41 × 10 7 ) 1.81 × 10 7 (1.61/1.97 × 10 7 )
Average cell volume (μm 3 ) 0.158 0.134 0.747 1.025
Biomass (mg dry mass L −1 ) 3.90 4.39 6.64 7.25
Yield (g dry mass mol −1 ) 0.23 0.27 0.52 0.52
pHini 3.3 pHini 3.8 pHini 4.8 pHini 5.9
pHfinal6.61 (6.59/6.64) 6.75 (6.61/6.96) 7.01 (6.97/7.09) 7.36 (7.29/7.40)
(mM) 17.20 (17.14/17.26) 16.19 (15.36/17.67) 12.67 (12.47/13.02) 13.96 (13.46/14.36)
H2 total (mM) 68.09 (67.29/69.15) 65.73 (60.50/73.82) 57.49 (55.48/58.68) 57.92 (56.78/58.68)
Ratio H2 total/ 3.96 (3.93/4.02) 4.06 (3.94/4.18) 4.54 (4.26/4.71) 4.15 (4.09/4.22)
Maximum cell number (mL −1 ) 5.25 × 10 7 (4.26/5.99 × 10 7 ) 6.11 × 10 7 (4.48/6.95 × 10 7 ) 1.89 × 10 7 (1.44/2.41 × 10 7 ) 1.81 × 10 7 (1.61/1.97 × 10 7 )
Average cell volume (μm 3 ) 0.158 0.134 0.747 1.025
Biomass (mg dry mass L −1 ) 3.90 4.39 6.64 7.25
Yield (g dry mass mol −1 ) 0.23 0.27 0.52 0.52

Values of replicate assays are averaged with minimum and maximum values in parentheses.

Removal of sulfate and uptake of H2 in μmol per ml culture liquid accumulated over time.

Average cell volume calculated from measurements performed on 30 cells at late growth phase to early stationary phase.

Biomass was converted from total biovolume (see explanations in the main text).

Sulfate removal was followed by a decrease of dissolved Fe(II) concentrations (Fig. 0001 ), which was accompanied by the formation of black precipitates, i.e., iron sulfides, as described below. At pHini 3.3 and pHini 3.8, the formation of black precipitates was preceded by the formation of whitish/greyish precipitates that corresponded most likely to aluminum precipitates. In the less acidic assays, the formation of iron sulfides started shortly after sulfate reduction, whereas in the more acidic assays, the formation of iron sulfides was delayed by almost 2 weeks starting at an average pH of 4.5. At this point, 5–6 μmol of sulfate per mL culture had already been reduced. At pH < 5, more than 99% of the free sulfide was present as H2S. As estimated by chemical equilibrium calculations, the amount of sulfate reduced corresponded to 2.2–2.5 mM H2S in the aqueous phase with the remaining H2S in the gas phase. In assays at pHini 4.8, the measured concentrations of free sulfide quickly rose to > 300 μM despite iron sulfide precipitation (Fig. 0002 c). In contrast, in assays at pHini 5.9, free sulfide concentrations > 50 μM were only observed after 20 days (Fig. 0002 d). During iron precipitation, the rate of pH increase generally slowed down because of the release of protons during iron sulfide formation (Fe 2+ + H2S → FeS(s) + 2H + ) (Fig. 0001 ). In addition, iron precipitation had no influence on the rate of sulfate reduction or H2 uptake.

At the end of the experiment, the pH values were in the circumneutral pH-range, and final redox potentials were around −100 to −140 mV. Concentrations of dissolved Al, Fe, Zn, and P were below detection limit while the concentrations of Ca and Si decreased and the concentrations of Na, K, Mg, and Mn remained unchanged in comparison to the original medium. H2 and CO2 volume percentages of the gas phase decreased to 7–14% and 3–7%, respectively. Methane was not detected in the headspace of any assay.

Mineral formation

After initial pH adjustment, media set to pH ≥ 3.8 showed the formation of a white fluffy precipitate that was more pronounced at higher pH values. As shown for media of enrichments II, the concentrations of dissolved Al decreased with increasing pH and were below detection limit at pHini 5.9 (Table 0001 ). At the same time, concentrations of sulfate were lower by 0.9 mM and 0.5 mM at pHini 4.8 and pHini 5.9, respectively, compared to media set to pHini 3.8. This indicated the formation of Al hydoxides and Al sulfates. Chemical equilibrium calculations predicted the formation of alunite [KAl3(SO4)2(OH)6)] in all media and of gypsum [CaSO4∙2H2O] at pHini 4.8 and 5.9 (see Supporting Information, Tables S1, S2 and S3, for the modeled concentrations of Al and sulfate species). The precipitation of alunite and gypsum, however, was not supported by our experimental data as a decrease in dissolved K or Ca was not observed (Table 0001 ). Furthermore, the formation of a phosphate mineral [(Ca2PO4Fe) 3+ ] was predicted (see Tables S1, S4, and S5 for the modeled concentrations of phosphate and Fe(II) species). Accordingly, the concentrations of dissolved P determined by ICP-AES were below total P concentrations of the medium at pHini 3.3 and 3.8 and below detection limit at pHini 4.8 and 5.9 (Table 0001 ).

Precipitates formed in enrichments II were studied by electron microscopy and microanalytic tools at the end of the incubation (Fig. 0003 Figs S1 and S2 show the element composition as determined by EDS and electron diffraction patterns, respectively). Precipitates enriched in iron and sulfur were identified as iron sulfides. They appeared as very thin ‘translucent’ sheets or as more electron dense scales in whole mounts (Fig. 0003 l). Thin sections showed the iron sulfide scales in cross section revealing their irregular surfaces (Fig. 0003 a, f and m). EDS performed on whole mounts revealed a Fe : S atomic ratio of 1.13 ± 0.03 (arithmetic mean ± standard deviation n = 6). Chemical equilibrium calculations predicted the formation of mackinawite (FeS1–x 0.07 > x > 0.04) (Tables S6 and S7). Electron diffraction showed that the precipitates had a crystalline structure (Fig. S2). Iron sulfides were mainly found in the bulk phase and were rarely in direct contact with bacterial cells. In addition to iron sulfides, Al-rich precipitates were detected. These were always slightly enriched in phosphorous and silicon, but not in sulfur. Al precipitates appeared as two morphological types. At pHini 3.3 and pHini 3.8, Al precipitates had a more spherical structure and were always associated with Thermodesulfobium-like cells (Fig. 0003 a–g). They appeared to be attached to the cell surfaces forming either small spheres (Fig. 0003 c and d) or covering the cell surface completely hereby enclosing a single cell (Fig. 5e) or a cell cluster (Fig. 0003 a and b). At pHini 3.8, 4.8, and 5.9, a second type of Al precipitate was observed. It consisted of a very fine mesh of very small crystals, which was found in the bulk phase or seemed to be only loosely attached to cell surfaces (Fig. 0003 f–k, n, o). For both morphological types of Al precipitates, electron diffraction did not reveal a crystalline structure. Few cells showed electron dense particles inside the cells (Fig. 0003 i, j and o) however, these were too small to identify their elemental composition by EDS.

TEM images from thin sections (a, c, e, f, h–j, m–o) and whole mounts (b, d, d, k, l) showing mineral precipitates (Al or Fe S) and cells (c) from enrichments II at the end of the experiment. (a)–(e) pHini 3.3, (f)–(h) pHini 3.8, (i)–(k) pHini 4.8, (l)–(o) pHini 5.9.

TEM images from thin sections (a, c, e, f, h–j, m–o) and whole mounts (b, d, d, k, l) showing mineral precipitates (Al or Fe S) and cells (c) from enrichments II at the end of the experiment. (a)–(e) pHini 3.3, (f)–(h) pHini 3.8, (i)–(k) pHini 4.8, (l)–(o) pHini 5.9.

Microbial community analyses

The amplification of both, archaeal and bacterial 16S rRNA genes, was attempted, but only amplification of the bacterial genes was successful. The T-RFLP profiles revealed a marked change in bacterial community composition from enrichments I to enrichments II at low initial pH values (Fig. 0004 ). The most frequent T-RF in enrichments II at low initial pH had a length of 520 nt which was also characteristic for cloned 16S rRNA genes with a highest sequence similarity to Thermodesulfobium narugense Na82 T (Table 0003 ). Correspondingly, more than 90% of the analyzed clones from the more acidic assays of enrichments II were affiliated to the genus Thermosdesulfobium (40 of 42 clones at pHini 3.3 and 36 of 39 clones at pHini 3.8). Only one clone related to Thermodesulfobium spp. was found at pHini 4.8, and none at pHini 5.9. T-RFLP analysis of cloned 16S rRNA genes revealed multiple fragments for Desulfosporosinus-affiliated clones (Table 0003 ). Virtual digestion of the 16S rRNA gene of Desulfosporosinus auripigmenti DSM 13351 T suggested that additionally to these fragments, the fragments of 301 nt and 513 nt may have resulted from Desulfosporosinus-affiliated genes. T-RFs that were characteristic for Desulfosporosinus-affiliated clones dominated in all enrichments I and in enrichments II at pHini 4.8 and 5.9. Correspondingly, the majority of clones in the less acidic assays of enrichments II showed highest sequence similarities to Desulfosporosinus spp. (41 of 43 clones at pHini 4.8 and 35 of 40 clones at pHini 5.9). Three clones (out of 39) related to Desulfosporosinus spp. were found at pHini 3.8 and none at pHini 3.3. Besides those two genera of known sulfate-reducing bacteria, other bacterial species were also found in enrichments II. At pHini 3.3, one clone each related to Acidithiobacillus ferrooxidans and Acidocella sp. was found. In the less acidic assays of enrichments II, species of the Acidaminococcaceae (one clone) and Clostridiaceae (three clones), as well as Thiomonas sp. (one clone), were present. The majority of clones obtained from enrichments I was identical to those of enrichments II. Fourteen clones were additionally identified as Desulfosporosinus-related gene sequences two clones were affiliated to species of the Syntrophobacteraceae. However, it has to be noted that 25–33% of the clones from enrichments I were unique and not further analyzed.

T-RFLP profiles based on bacterial 16S rRNA genes digested with Msp1 obtained from all replicate systems of enrichment I (a) and enrichment II (b). Red: T-RF of 520 nt characteristic for Thermodesulfobium-affiliated 16S rRNA genes blue shades: T-RFs characteristic for Desulfosporosinus-affiliated 16S rRNA genes.

T-RFLP profiles based on bacterial 16S rRNA genes digested with Msp1 obtained from all replicate systems of enrichment I (a) and enrichment II (b). Red: T-RF of 520 nt characteristic for Thermodesulfobium-affiliated 16S rRNA genes blue shades: T-RFs characteristic for Desulfosporosinus-affiliated 16S rRNA genes.

Phylogenetic affiliation and corresponding T-RFs of representative clones from bacterial 16S rRNA genes retrieved from enrichments II

Clone id. Accession number Frequency Closest cultured relative (sequence similarity/SAB) T-RF (relative peak area)
Thermodesulfobium spp. (in total 13 ARDRA patterns)
II_1b_5 EU755163 40 T. narugense strain Na82 T AB077817 (96%/0.832) 520 nt
II_2b_8 EU755171 15 T. narugense strain Na82 T AB077817 (96%/0.817) 520 nt
II_1b_9 EU755165 11 T. narugense strain Na82 T AB077817 (95%/0.820) 520 nt
II_2b_15 EU755172 2 T. narugense strain Na82 T AB077817 (96%/0.827) 520 nt
Desulfosporosinus spp. (in total 33 ARDRA patterns)
II_3b_5 EU755181 24 Desulfosporosinus sp. PFB AY007667 (97%/0.883) 138 nt (90%), 170 nt (10%)
II_3b_14 EU755185 10 Desulfosporosinus sp. PFB AY007667 (98%/0.930) 138 nt (74%), 170 nt (26%)
II_3b_10 EU755184 8 Desulfosporosinus sp. PFB AY007667 (98%/0.929) 138 nt (92%), 170 nt (8%)
II_4b_1II EU755195 4 Desulfosporosinus sp. 44a-T3a AY082482 (96%/0.884) 136 nt (90%), 168 nt (10%)
II_3b_61 EU755191 3 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.746) nd
II_3b_62 EU755192 3 Desulfosporosinus sp. PFB AY007667 (98%/0.927) nd
II_3b_9 EU755183 2 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.787) nd
II_3b_1II EU755177 2 Desulfosporosinus sp. PFB AY007667 (95%/0.858) nd
II_4b_2 EU755196 2 Desulfosporosinus sp. A10 AJ582756 (97%)/Desulfosporosinus sp. 44a-T3a (0.882) nd
II_2b_17N EU755176 2 Desulfosporosinus sp. PFB AY007667 (95%/0.656) 226 nt (97%), 258 nt (3%)
Clone id. Accession number Frequency Closest cultured relative (sequence similarity/SAB) T-RF (relative peak area)
Thermodesulfobium spp. (in total 13 ARDRA patterns)
II_1b_5 EU755163 40 T. narugense strain Na82 T AB077817 (96%/0.832) 520 nt
II_2b_8 EU755171 15 T. narugense strain Na82 T AB077817 (96%/0.817) 520 nt
II_1b_9 EU755165 11 T. narugense strain Na82 T AB077817 (95%/0.820) 520 nt
II_2b_15 EU755172 2 T. narugense strain Na82 T AB077817 (96%/0.827) 520 nt
Desulfosporosinus spp. (in total 33 ARDRA patterns)
II_3b_5 EU755181 24 Desulfosporosinus sp. PFB AY007667 (97%/0.883) 138 nt (90%), 170 nt (10%)
II_3b_14 EU755185 10 Desulfosporosinus sp. PFB AY007667 (98%/0.930) 138 nt (74%), 170 nt (26%)
II_3b_10 EU755184 8 Desulfosporosinus sp. PFB AY007667 (98%/0.929) 138 nt (92%), 170 nt (8%)
II_4b_1II EU755195 4 Desulfosporosinus sp. 44a-T3a AY082482 (96%/0.884) 136 nt (90%), 168 nt (10%)
II_3b_61 EU755191 3 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.746) nd
II_3b_62 EU755192 3 Desulfosporosinus sp. PFB AY007667 (98%/0.927) nd
II_3b_9 EU755183 2 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.787) nd
II_3b_1II EU755177 2 Desulfosporosinus sp. PFB AY007667 (95%/0.858) nd
II_4b_2 EU755196 2 Desulfosporosinus sp. A10 AJ582756 (97%)/Desulfosporosinus sp. 44a-T3a (0.882) nd
II_2b_17N EU755176 2 Desulfosporosinus sp. PFB AY007667 (95%/0.656) 226 nt (97%), 258 nt (3%)

Clone labels: II = secondary enrichment 1b = pHini 3.3, replicate B 2b = pHini 3.8, replicate B 3b = pHini 4.8, replicate B 4b = pHini 5.9, replicate B.

Frequency of appearance in clone libraries from enrichments II (in total 164 clones).

Highest sequence similarity match in GenBank using blastn and SAB-values in Ribosomal Database Project-II Release 9 using Sequence Match.

Phylogenetic affiliation and corresponding T-RFs of representative clones from bacterial 16S rRNA genes retrieved from enrichments II

Clone id. Accession number Frequency Closest cultured relative (sequence similarity/SAB) T-RF (relative peak area)
Thermodesulfobium spp. (in total 13 ARDRA patterns)
II_1b_5 EU755163 40 T. narugense strain Na82 T AB077817 (96%/0.832) 520 nt
II_2b_8 EU755171 15 T. narugense strain Na82 T AB077817 (96%/0.817) 520 nt
II_1b_9 EU755165 11 T. narugense strain Na82 T AB077817 (95%/0.820) 520 nt
II_2b_15 EU755172 2 T. narugense strain Na82 T AB077817 (96%/0.827) 520 nt
Desulfosporosinus spp. (in total 33 ARDRA patterns)
II_3b_5 EU755181 24 Desulfosporosinus sp. PFB AY007667 (97%/0.883) 138 nt (90%), 170 nt (10%)
II_3b_14 EU755185 10 Desulfosporosinus sp. PFB AY007667 (98%/0.930) 138 nt (74%), 170 nt (26%)
II_3b_10 EU755184 8 Desulfosporosinus sp. PFB AY007667 (98%/0.929) 138 nt (92%), 170 nt (8%)
II_4b_1II EU755195 4 Desulfosporosinus sp. 44a-T3a AY082482 (96%/0.884) 136 nt (90%), 168 nt (10%)
II_3b_61 EU755191 3 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.746) nd
II_3b_62 EU755192 3 Desulfosporosinus sp. PFB AY007667 (98%/0.927) nd
II_3b_9 EU755183 2 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.787) nd
II_3b_1II EU755177 2 Desulfosporosinus sp. PFB AY007667 (95%/0.858) nd
II_4b_2 EU755196 2 Desulfosporosinus sp. A10 AJ582756 (97%)/Desulfosporosinus sp. 44a-T3a (0.882) nd
II_2b_17N EU755176 2 Desulfosporosinus sp. PFB AY007667 (95%/0.656) 226 nt (97%), 258 nt (3%)
Clone id. Accession number Frequency Closest cultured relative (sequence similarity/SAB) T-RF (relative peak area)
Thermodesulfobium spp. (in total 13 ARDRA patterns)
II_1b_5 EU755163 40 T. narugense strain Na82 T AB077817 (96%/0.832) 520 nt
II_2b_8 EU755171 15 T. narugense strain Na82 T AB077817 (96%/0.817) 520 nt
II_1b_9 EU755165 11 T. narugense strain Na82 T AB077817 (95%/0.820) 520 nt
II_2b_15 EU755172 2 T. narugense strain Na82 T AB077817 (96%/0.827) 520 nt
Desulfosporosinus spp. (in total 33 ARDRA patterns)
II_3b_5 EU755181 24 Desulfosporosinus sp. PFB AY007667 (97%/0.883) 138 nt (90%), 170 nt (10%)
II_3b_14 EU755185 10 Desulfosporosinus sp. PFB AY007667 (98%/0.930) 138 nt (74%), 170 nt (26%)
II_3b_10 EU755184 8 Desulfosporosinus sp. PFB AY007667 (98%/0.929) 138 nt (92%), 170 nt (8%)
II_4b_1II EU755195 4 Desulfosporosinus sp. 44a-T3a AY082482 (96%/0.884) 136 nt (90%), 168 nt (10%)
II_3b_61 EU755191 3 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.746) nd
II_3b_62 EU755192 3 Desulfosporosinus sp. PFB AY007667 (98%/0.927) nd
II_3b_9 EU755183 2 Desulfosporosinus sp. LauIII AJ302078 (93%)/D. acidophilus SJ4 T FJ951625 (0.787) nd
II_3b_1II EU755177 2 Desulfosporosinus sp. PFB AY007667 (95%/0.858) nd
II_4b_2 EU755196 2 Desulfosporosinus sp. A10 AJ582756 (97%)/Desulfosporosinus sp. 44a-T3a (0.882) nd
II_2b_17N EU755176 2 Desulfosporosinus sp. PFB AY007667 (95%/0.656) 226 nt (97%), 258 nt (3%)

Clone labels: II = secondary enrichment 1b = pHini 3.3, replicate B 2b = pHini 3.8, replicate B 3b = pHini 4.8, replicate B 4b = pHini 5.9, replicate B.

Frequency of appearance in clone libraries from enrichments II (in total 164 clones).

Highest sequence similarity match in GenBank using blastn and SAB-values in Ribosomal Database Project-II Release 9 using Sequence Match.

Microscopic analysis of enrichments II revealed different cell types that were typical for the different pHini-values (Fig. 0005 ) and that were present throughout the incubation, i.e., from the early growth phase until the end of the experiment, when samples were withdrawn for 16S rRNA gene analysis. Small slender rods (1–2 × 0.3 μm), very similar to cells of T. narugense strain Na82 T (Mori et al., 2003), were dominant in systems at pHini 3.3 and pHini 3.8. These cells tended to form cell clumps with a diameter of up to 20 μm. At pHini 3.8, the small rods were accompanied by large rods (5–10 × 0.6 μm) that were present in much lower numbers and that formed central spores. At pHini 4.8, smaller spore-forming rods (2–6 × 0.5 μm) with terminal spores were observed. At pHini 5.9, large, curved to vibrioid cells of variable length (3–12 × 0.5 μm) forming terminal spores were dominant.

Representative micrographs of cells from enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Cells were stained with DNA stain SYBRGreen I and imaged at 1000× magnification. Scale bar is the same for all micrographs.

Representative micrographs of cells from enrichment II: pHini 3.3 (a), pHini 3.8 (b), pHini 4.8 (c), and pHini 5.9 (d). Cells were stained with DNA stain SYBRGreen I and imaged at 1000× magnification. Scale bar is the same for all micrographs.


While sulfur bacteria are not harmful, hydrogen sulfide gas in the air can be harmful at high levels. It is important to remove the gas from the water, or vent the gas to the atmosphere. Venting prevents the gas from collecting in low-lying spaces (such as well pits and basements) or enclosed spaces (such as well houses). Only well professionals should enter a well pit or other enclosed space where hydrogen sulfide gas may be present.

  • Bacterial slime may be white, grey, black, or reddish brown if associated with iron bacteria (signs of sulfur bacteria).
  • Black stains on silverware and plumbing fixtures (signs of hydrogen sulfide gas).
  • Corrosion on pipes and metal components of the water distribution system (signs of hydrogen sulfide gas).
  • Have your water tested at a laboratory.

Author information

Affiliations

U.S. Geological Survey, California Water Science Center, 4165 Spruance Road, San Diego, CA, 92101, USA

U.S. Department of Agriculture, Agricultural Research Service, Curtin Road, Building 3702, University Park, PA, 16802, USA

U.S. Environmental Protection Agency, National Risk Management Research Laboratory, 919 Kerr Research Drive, Ada, OK, 74820, USA

U.S. Geological Survey, California Water Science Center, 6000 J Street, Sacramento, CA, 95819, USA

U.S. Geological Survey, Denver Federal Center, Mailstop 963, Denver, CO, 80225, USA

U.S. Geological Survey, Suite 127, 3215 Marine Street, Boulder, CO, 80303, USA

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Contents

"Sulfate" is the spelling recommended by IUPAC, but "sulphate" was traditionally used in British English.

The sulfate anion consists of a central sulfur atom surrounded by four equivalent oxygen atoms in a tetrahedral arrangement. The symmetry is the same as that of methane. The sulfur atom is in the +6 oxidation state while the four oxygen atoms are each in the −2 state. The sulfate ion carries an overall charge of −2 and it is the conjugate base of the bisulfate (or hydrogen sulfate) ion, HSO −
4 , which is in turn the conjugate base of H
2 SO
4 , sulfuric acid. Organic sulfate esters, such as dimethyl sulfate, are covalent compounds and esters of sulfuric acid. The tetrahedral molecular geometry of the sulfate ion is as predicted by VSEPR theory.

The first description of the bonding in modern terms was by Gilbert Lewis in his groundbreaking paper of 1916 where he described the bonding in terms of electron octets around each atom, that is no double bonds and a formal charge of +2 on the sulfur atom. [1] [a]

Later, Linus Pauling used valence bond theory to propose that the most significant resonance canonicals had two pi bonds involving d orbitals. His reasoning was that the charge on sulfur was thus reduced, in accordance with his principle of electroneutrality. [2] The S−O bond length of 149 pm is shorter than the bond lengths in sulfuric acid of 157 pm for S−OH. The double bonding was taken by Pauling to account for the shortness of the S−O bond. Pauling's use of d orbitals provoked a debate on the relative importance of pi bonding and bond polarity (electrostatic attraction) in causing the shortening of the S−O bond. The outcome was a broad consensus that d orbitals play a role, but are not as significant as Pauling had believed. [3] [4]

A widely accepted description involving pπ – dπ bonding was initially proposed by Durward William John Cruickshank. In this model, fully occupied p orbitals on oxygen overlap with empty sulfur d orbitals (principally the dz 2 and dx 2 –y 2 ). [5] However, in this description, despite there being some π character to the S−O bonds, the bond has significant ionic character. For sulfuric acid, computational analysis (with natural bond orbitals) confirms a clear positive charge on sulfur (theoretically +2.45) and a low 3d occupancy. Therefore, the representation with four single bonds is the optimal Lewis structure rather than the one with two double bonds (thus the Lewis model, not the Pauling model). [6] In this model, the structure obeys the octet rule and the charge distribution is in agreement with the electronegativity of the atoms. The discrepancy between the S−O bond length in the sulfate ion and the S−OH bond length in sulfuric acid is explained by donation of p-orbital electrons from the terminal S=O bonds in sulfuric acid into the antibonding S−OH orbitals, weakening them resulting in the longer bond length of the latter.

However, the bonding representation of Pauling for sulfate and other main group compounds with oxygen is still a common way of representing the bonding in many textbooks. [5] [7] The apparent contradiction can be cleared if one realizes that the covalent double bonds in the Lewis structure in reality represent bonds that are strongly polarized by more than 90% towards the oxygen atom. On the other hand, in the structure with a dipolar bond, the charge is localized as a lone pair on the oxygen. [6]

Methods of preparing metal sulfates include: [7]

There are numerous examples of ionic sulfates, many of which are highly soluble in water. Exceptions include calcium sulfate, strontium sulfate, lead(II) sulfate, and barium sulfate, which are poorly soluble. Radium sulfate is the most insoluble sulfate known. The barium derivative is useful in the gravimetric analysis of sulfate: if one adds a solution of most barium salts, for instance barium chloride, to a solution containing sulfate ions, barium sulfate will precipitate out of solution as a whitish powder. This is a common laboratory test to determine if sulfate anions are present.

The sulfate ion can act as a ligand attaching either by one oxygen (monodentate) or by two oxygens as either a chelate or a bridge. [7] An example is the complex [Co(en)2(SO4)] + Br − [7] or the neutral metal complex PtSO4(P(C6H5)3)2 where the sulfate ion is acting as a bidentate ligand. The metal–oxygen bonds in sulfate complexes can have significant covalent character.

Commercial applications Edit

Sulfates are widely used industrially. Major compounds include:

    , the natural mineral form of hydrated calcium sulfate, is used to produce plaster. About 100 million tonnes per year are used by the construction industry. , a common algaecide, the more stable form (CuSO4) is used for galvanic cells as electrolyte , a common form of iron in mineral supplements for humans, animals, and soil for plants (commonly known as Epsom salts), used in therapeutic baths , produced on both plates during the discharge of a lead–acid battery , or SLES, a common detergent in shampoo formulations , hydrated K2Ca2Mg-sulfate, used as fertiliser.

Occurrence in nature Edit

Sulfate-reducing bacteria, some anaerobic microorganisms, such as those living in sediment or near deep sea thermal vents, use the reduction of sulfates coupled with the oxidation of organic compounds or hydrogen as an energy source for chemosynthesis.

Some sulfates were known to alchemists. The vitriol salts, from the Latin vitreolum, glassy, were so-called because they were some of the first transparent crystals known. [8] Green vitriol is iron(II) sulfate heptahydrate, FeSO4·7H2O blue vitriol is copper(II) sulfate pentahydrate, CuSO4·5H2O and white vitriol is zinc sulfate heptahydrate, ZnSO4·7H2O. Alum, a double sulfate of potassium and aluminium with the formula K2Al2(SO4)4·24H2O, figured in the development of the chemical industry.

Sulfates occur as microscopic particles (aerosols) resulting from fossil fuel and biomass combustion. They increase the acidity of the atmosphere and form acid rain. The anaerobic sulfate-reducing bacteria Desulfovibrio desulfuricans and D. vulgaris can remove the black sulfate crust that often tarnishes buildings. [9]

Main effects on climate Edit

The main direct effect of sulfates on the climate involves the scattering of light, effectively increasing the Earth's albedo. This effect is moderately well understood and leads to a cooling from the negative radiative forcing of about 0.4 W/m 2 relative to pre-industrial values, [10] partially offsetting the larger (about 2.4 W/m 2 ) warming effect of greenhouse gases. The effect is strongly spatially non-uniform, being largest downstream of large industrial areas. [11]

The first indirect effect is also known as the Twomey effect. Sulfate aerosols can act as cloud condensation nuclei and this leads to greater numbers of smaller droplets of water. Many smaller droplets can diffuse light more efficiently than a few larger droplets. The second indirect effect is the further knock-on effects of having more cloud condensation nuclei. It is proposed that these include the suppression of drizzle, increased cloud height, [12] [ full citation needed ] to facilitate cloud formation at low humidities and longer cloud lifetime. [13] [ full citation needed ] Sulfate may also result in changes in the particle size distribution, which can affect the clouds radiative properties in ways that are not fully understood. Chemical effects such as the dissolution of soluble gases and slightly soluble substances, surface tension depression by organic substances and accommodation coefficient changes are also included in the second indirect effect. [14]

The indirect effects probably have a cooling effect, perhaps up to 2 W/m 2 , although the uncertainty is very large. [15] [ full citation needed ] Sulfates are therefore implicated in global dimming. Sulfate is also the major contributor to stratospheric aerosol formed by oxidation of sulfur dioxide injected into the stratosphere by impulsive volcanoes such as the 1991 eruption of Mount Pinatubo in the Philippines. This aerosol exerts a cooling effect on climate during its 1-2 year lifetime in the stratosphere.


Figure 4.4.1 Tremetol, a metabolic poison found in white snake root plant, prevents the metabolism of lactate. When cows eat this plant, Tremetol is concentrated in the milk. Humans who consume the milk become ill. Symptoms of this disease, which include vomiting, abdominal pain, and tremors, become worse after exercise. Why do you think this is the case?

The illness is caused by lactic acid build-up. Lactic acid levels rise after exercise, making the symptoms worse. Milk sickness is rare today, but was common in the Midwestern United States in the early 1800s.


Watch the video: Reduction of Sulfate. Shipwrecks and Salvage (December 2021).