sulphate reducers are anaerobic microorganisms that are able to use sulphate as a terminal electron acceptor . they are widespread in anoxic habitats   and can use numerous substrates as electron donor for growth . these include sugars [ 3 , 4 ] , amino acids [ 5 , 6 ] , hydrogen , and one - carbon compounds , such as methanol [ 811 ] , carbon monoxide [ 1214 ] , and methanethiol . even alkanes [ 1619 ] , alkenes , and short - chain alkanes , as well as aniline , benzoate , phenol , aromatic hydrocarbons [ 2325 ] , and phosphite   are used as electron donor . industries that use sulphuric acid , sulphate - rich feedstocks , or reduced sulphur compounds generate wastewaters rich in sulphate . sulphate is removed from wastewater by the combined activity of srb that generate sulphide and the subsequent partial oxidation of sulphide to insoluble elemental sulphur by sulphide oxidizing bacteria . biotechnological applications of sulphate reduction further include sox abatement from the flue gas of coal fueled power plants   and treatment of sulphate - rich , heavy metal contaminated wastewaters . heavy metals such as cu , zn , cd , pb , ni , and fe can be removed from waste streams by precipitation with biogenic sulfide . because of differences in solubility of products , the metals can be selectively precipitated , which enables their recovery and reuse as demonstrated at full - scale for a zinc smelting plant . if sulphate - rich wastewaters contain no or insufficient amounts of suitable electron and carbon donors for sulphate reduction , external addition becomes a prerequisite . examples of such wastewaters are waste streams generated in galvanic processes , in the detoxification of metal - contaminated soils , in the mining of heavy metals and coal , and in flue gas desulphurization . the selection of the most appropriate electron donor depends on its sulphate reduction efficiency , costs , and residual pollution . the use of simple organic compounds ( e.g. , ethanol or methanol ) or h2/co2 is highly preferred over complex wastes , for example , molasses , tannery effluents , solid waste materials , and manure . especially h2 is often used for biotechnological sulphate reduction . van houten et al .   demonstrated that with a mixture of h2/co2 ( 80%  :  20% ) , sulphate reduction rates as high as 30  g so4  l  d are achieved within 10 days of operation in gas lift bioreactors . alternatively , synthesis gas or methane can be used ; however , the sulphate reduction rates with methane are still extremely low . synthesis gas , a mixture of h2 , co and co2 and some other trace gases , has been considered a cheap alternative for high - purity h2 . synthesis gas can be used either directly or after enriching its h2 content by means of a water - gas shift reaction , that is , the catalytic reaction in which co is forced to react with water yielding carbon dioxide and hydrogen . in addition , residual electron donors or side products should be low or easily removed . here some pure cultures of hydrogen - utilizing sulphate - reducing bacteria are able to use carbon monoxide as a carbon and energy source [ 3436 ] . a few species of mesophilic sulphate - reducers can grow at low concentrations ( 4.520% ) of co. the most tested co - utilizing srb are species of genera desulfovibrio and desulfotomaculum ( table 1 ) . the following reactions   ( gibbs energy , g ) , calculated according to   play a role during growth of srb on co :  
 ( 1)4co+so42+4h2o4hco3+hs+3h+g=37.1  kj / mol  co , 
 ( 2)4co+4h2o  acetate+2hco3+3h+g=28.2  kj / mol  co , 
 ( 3)co+2h2ohco3+h2+h+g=  15  kj / mol  co , 
 ( 4)4h2+so42+h+hs+4h2og=45.2  kj / mol  h2 , 
 ( 5)4h2 + 2hco3+h+acetate  + 4h2og=36.4  kj / mol  h2 . 
  first information on co oxidation by thermophilic desulfotomaculum species was published in 1985 by klemps et al . . these moderately thermophilic desulfotomaculum species could grow slowly with co ( 420%  v / v ) as the only energy source . four thermophilic sulphate - reducing bacteria : desulfotomaculum thermoacetoxidans strain camz , thermodesulfovibrio yellowstonii , desulfotomaculum kuznetsovii , and desulfotomaculum thermobenzoicum subsp . thermosyntrophicum were studied in pure culture and in coculture with the thermophilic carboxydotrophic hydrogenogenic bacterium carboxydothermus hydrogenoformans . in pure culture , thermosyntrophicum were able to grow with co. in the presence of hydrogen and carbon dioxide , co concentrations as high as 5070% were utilized . the latter two srb coupled co oxidation to sulphate reduction without intermediate h2 production ( reaction ( 1 ) ) , but a large part of the co was converted to acetate ( reaction ( 2 ) ) . when grown in coculture with the carboxydotrophic bacterium c. hydrogenoformans , dtm . thermosyntrophicum could grow with 100% co ( pco = 120  kpa ) . in that experiment , hydrogen was formed from co by c. hydrogenoformans ( reaction ( 3 ) ) and subsequently the sulphate - reducers used the generated hydrogen as electron donor ( reaction ( 4 ) ) . . the bacterium could grow with 100% co in the gas phase . in the presence of sulphate , co was converted to h2 and co2 and the generated h2 was used for sulphate reduction . in the absence of sulphate , co was completely converted to h2 and co2 . nigrificans could grow up to 20% co maximally , as was found before by klemps et al . . archaeoglobus fulgidus is a thermophilic sulphate reducing archaeon able to grow on several organic substrates [ 42 , 43 ] . recently , the ability of a. fulgidus to grow with co and to couple growth on co with sulphate reduction was demonstrated . sulphate is reduced with co as electron donor without intermediate h2 production , but with transient formate formation . additionally a. fulgidus grows acetogenically with co in the presence and absence of sulphate and sulphate reduction to sulphide is not inhibited by co . it was found that desulfovibrio desulfuricans , growing on lactate and sulphate , tolerated a co concentration up to 20% , although such a co concentration caused a reversible type of growth inhibition [ 40 , 48 ] ( table 1 ) . desulfovibrio   africanus , desulfovibrio baculatus , and some desulfovibrio   desulfuricans strains grow with lactate in the presence of 20% or less of co. higher co concentrations inhibited growth completely . co concentrations exceeding 20%  ( v / v ) inhibited growth of desulfosporosinus orientis completely . thermoacetoxidans and t. yellowstonii with pyruvate was completely inhibited at co concentrations as low as 2% in the gas phase , whereas dtm . thermosyntrophicum were capable of growth in the presence of co ( table 1 ) . synthesis gas was used as an electron donor of sulphate reduction in bioreactors [ 4952 ] . the major restriction of synthesis gas utilization is the presence of co , which can range from 5 to over 50%   and its toxicity towards srb . du preez and maree   reported a sulphate reduction rate of 2.4  g so4  l  d with pure co. van houten et al . [ 51 , 54 ] achieved a sulphate reduction rate in the range of 68  g so4  l  d , with a feed gas containing maximally 20% co. van houten et al . [ 51 , 55 ] showed that co exerted a toxic effect on the srb present in the biomass at gas phase co concentrations between 5 and 20% as the sulphate reduction rates were limited to 8  g so4  l  d , whereas with h2/co2 the maximum sulphate reduction rate achieved was 30  g so4  l  d . in the presence of co , a layered biomass structure developed with acetogenic bacteria located at the outside of the biofilm , and srb located deeper inside the biofilm probably as a protection mechanism towards co toxicity [ 51 , 55 ] . carboxydivorans were not inhibited by the highest tested pco of 200 and 124  kpa , respectively [ 14 , 56 ] . presence of a co - dehydrogenase in a sulphate reducer does not necessarily mean that it is less sensitive towards co. cell extracts of dtm . thermoacetoxidans revealed a co - dehydrogenase activity   and the whole - genome sequence of t. yellowstonii contains a co - dehydrogenase gene as part of an acs / codh cluster ( table 2 ) . however , both bacteria are completely inhibited by less than 2% of co in the gas phase . it might be that the co - dehydrogenase of t. yellowstonii is inactive , since no co - oxidizing activity was found in cell extracts of this bacterium and it needs acetate as carbon source for growth with h2 or formate as electron donors . in recent reviews devoted to co - utilization , genomics and co - sensing mechanisms of anaerobic , aerobic bacteria and archaea , it was concluded that there is no single mechanism of co - utilization and co - inhibition [ 5961 ] . the mechanism of co - inhibition in particular of sulphate reducers is not completely understood as well . hydrogenases play a key role in the metabolism of hydrogenotrophic sulphate reducers , and also in the hydrogen - cycling mechanism that was proposed for desulfovibrio spp . it has been shown that co is a competitive inhibitor of [ fefe]-hydrogenases where it binds to iron in the active site [ 62 , 63 ] . interestingly , co also serves , together with cn , as a coordinating ligand in the active site of [ fefe]-hydrogenases [ 6467 ] . inhibition of hydrogenase therefore could potentially explain at least part of the sensitivity of some sulphate reducers towards co. other metallo - enzymes could similarly be inhibited by co. in dsv . desulfuricans strain b-1388 , the effect of co on metabolism was studied in more detail . in this bacterium , 5% resulting in a lower growth rate , increased content of cytochrome c , increased content of reduced pyridine nucleotides , and a low atp concentration [ 6870 ] . therefore , it has been suggested that the function of the co - dehydrogenase in this organism is to detoxify co . it was shown that hydrogenases of clostridium pasteurianum are inhibited by binding of co to iron in the active site , but whether this is the sole mechanism of inhibition is not clear . recent interesting results in this field - co - tolerant hydrogenases exist that exclude co from the active site by means of a narrow tunnel that allows hydrogen to pass , but not co . since co binds readily to metals , such as iron , other metal bearing proteins may be similarly affected . the genome sequence of dsv . desulfuricans strain b-1388 has not been solved , but the genome of seven other desulfovibrio spp . and of ten other dissimilatory sulphate reducers has been sequenced ( table 2 ) . of the seven available desulfovibrio genomes , only desulfovibrio piger does not contain a co - dehydrogenase gene homologue . the co - dehydrogenase gene context appears conserved in desulfovibrio spp . with cooa and cooc homologues , respectively , up- and down - stream of the coos , the gene fragment encoding for co - dehydrogenase . cooa is a co - binding transcriptional regulator   and cooc a co - dehydrogenase maturation protein [ 75 , 76 ] . interestingly , cooa seems only present in the genomes of desulfovibrio spp . , but in none of the other available genomes of sulphate reducers . further , co - dehydrogenase gene context reveals little information about its possible physiological role in the metabolism . a hyd   [ fefe]-hydrogenase deletion mutant of desulfovibrio vulgaris hildenborough formed transient amounts of co up to 6000  ppm as part of a fermentation burst . likely its co - dehydrogenase is first involved in the production and then in the consumption of co by this mutant , with electron transfer possibly linked to a general electron pool .   for dsv . desulfuricans b-1388 , it was also shown that co - dehydrogenase activity in cell extracts increases over time in batch cultures of cells grown with lactate and sulphate as substrates [ 35 , 78 ] . in late growth stages , the degradation of cellular material , for example , porphyrins , may release co and this results in an increased co - dehydrogenase expression through the action of cooa . in the remainder of fully sequenced genomes of sulphate reducers ( table 2 ) that contain one or more co - dehydrogenase genes , the co - dehydrogenase is either part of an acs / codh cluster or is located alone or together with unexpected genes . in this group of sulphate reducers , a. fulgidus contains three co - dehydrogenase genes , two of the type typically found in archaeal acs / codh complexes and one homologous to coos found in bacteria . the archaeal type of co - dehydrogenases is paired together only with the epsilon subunit of acs / codh in a. fulgidus . the beta , gamma , and delta subunits of acs / codh appear only once in the genome , separate from the alpha epsilon pairs . it has been speculated that both archaeal type co - dehydrogenases are involved in the acetyl - coa pathway and that oxidation of co by coos provides electrons for reduction of co2 to formyl methanofuran and possibly in the reduction of sulphate . the presence of multiple codh genes in a single genome suggests that multiple physiological roles exist for co - dehydrogenase . besides a role in the acs / codh complex , co could be oxidized by codh and serve as an electron donor for sulfate reduction or co - dehydrogenase could serve to detoxify co , but clear experimental evidence is lacking . clearly , the different sulphate reducers respond differently to co. while based on available whole genome sequences it appears that most sulphate reducers contain a co - dehydrogenase gene , their sensitivity towards co allows speculation that levels of co that are encountered by these microorganisms in their natural habitat are limited . it seems that only few sulphate reducers are insensitive towards co , although more may be found . according to a cost estimation concerning the use of ethanol and h2 as electron donors for sulphate reduction , ethanol turned out to be cheaper for small - scale installations ( < 5  kmol h ) , whereas h2 is cheaper for larger installations , assuming the use of a high - purity h2 . synthesis gas , produced by , for example , steam reforming of natural gas or thermal gasification of coal , oil , biomass , or other organic matter and widely available as by - product of coal burners is a cheap alternative for high - purity h2 . furthermore , operational costs could be greatly reduced in case of on - site production of synthesis gas from coal , thus minimizing transportation costs . several mesophilic full - scale anaerobic sludges , when incubated at 55c , revealed the capability of hydrogenogenic co conversion , whereas direct acetate production was not observed . as mentioned earlier , especially hydrogenogenic co conversion holds a promise for the use of synthesis gas in biotechnological sulphate reduction , for example , in thermophilic flue gas desulfurization . therefore , the utilization of co as a sole external electron donor in thermophilic ( 55c ) sulphate reduction was investigated in lab - scale gas lift bioreactors [ 8082 ] . these reactors were inoculated with mesophilic granular sludge , with a high hydrogenogenic activity on co at 55c , from which the hydrogenogenic sulphate reducer dtm . thermophilic ( 5055c ) sulphate reduction rates of up to 20  mmol  l  d were obtained at hydraulic retention times ( hrts ) between 6 and 14 hours   showing the potential of sulphate reduction with co as an external electron donor . however , due to predominant methanogenic h2 consumption , sulphate reduction rates were generally low . similar competition effects at 55c leading to methanogenesis have been reported with methanol   and h2/co2 . fast growth rates of the methanogens ( generation time of 4.5 hours ) enabled them to recover from imposed ph and temperature shocks , and they consumed more than 90% of the co - derived h2 . nevertheless , steep increases in sulphide production in periods with low methanogenic activity suggest that once methanogenesis is eliminated , sulphate reduction with co - rich gas as electron donor has a great potential . operation at hrts shorter than the generation time of the methanogens allowed   stable sulphate reduction for prolonged periods , and up to 95% of the co - derived h2 was used for sulphate reduction . the achieved sulphate reduction rates of 17  mmolld were limited by the amount of co supplied and its conversion efficiency ( about 85% ) at higher co loads likely results from biomass limitation . methane production , however , persisted when operating under these conditions and increasing the hrt by returning it to values > 5.5 hours resulted in a dominance of methanogenesis over sulfate reduction . although the operation of a thermophilic co fed gas lift reactor at extremely low hrts   clearly demonstrated the potential of co or synthesis gas as an electron donor for biotechnological sulphate reduction , the low biomass concentrations present limited the applicable sulphate loads and thus its practical application . for high - rate biotechnological sulphate reduction processes , biomass retention is a prerequisite , and thus elimination or efficient suppression of methanogenesis is essential . simple changes in operation conditions , such as ph , temperature , or salinity were ineffective for steering the competition for h2 utilization towards sulphate reduction . suppression of thermophilic hydrogenogenic methanogenesis has been demonstrated with the use of chemical inhibitors   or pretreatment of the sludge prior to inoculation [ 8688 ] . however , the use of chemical inhibitors represents a potential risk as they might be released into the environment . furthermore , adaptation to the inhibitor might result in increased operational costs due to an increasing demand of chemical inhibitors to maintain the same level of inhibition . weijma and coworkers   observed that the application of a continuous dosing of 2  gl 2-bromoethanesulfonate to a bioreactor was ineffective for the suppression of methanogenesis , as methanogenesis resumed after two days . in contrast , sparling et al .   successfully inhibited methane formation in an h2 producing anaerobic digester by the addition of 1% v / v acetylene in the headspace . heat or acid / base treatment does not inhibit methanogens during reactor operation as would be the case with chemical agents , but rather eliminates methanogens before reactor operation is initiated . such a pretreatment poses no environmental risk , because no harmful chemicals will be released , and furthermore , such a pretreatment in principle needs to be performed only once ; whereas , chemical inhibitors need to be applied constantly most likely at an increased demand . nevertheless , for the successful suppression of methanogens their complete elimination is a prerequisite , especially fast growing thermophilic methanogens . in incubations at 55c with dtm . carboxydivorans , sulphide production occurred at a pco exceeding 100  kpa . however , dtm . carboxydivorans has a ph - dependent sensitivity for sulphide inhibition , that is , 9  mm sulfide at ph 7.2 and 5  mm at ph 6.5 cause complete inhibition . thus , most likely not toxicity of co , but of sulphide determines the sulphate reduction capacity of dtm . carboxydivorans , as bulk liquid co concentrations will likely be kept low due to biological conversion . therefore , to develop a high - rate sulphate - reducing bioreactor employing dtm . carboxydivorans , additional features to maintain the sulphide concentration below inhibitory levels are required . to maintain sulphide concentrations sufficiently low , operation at slightly elevated ph - values could be considered or application of an alkaline h2s absorber through which the recycle gas is led . the h2s absorbed from the gas phase could be fed to a second micro - aerobic biological reactor , preferably operated at high ph to minimize consumption of chemicals , in which the sulphide is partially oxidized to elemental sulphur . another attractive option is the use of h2s extractive membranes   placed inside the bioreactor mixed liquor , which could result in direct recovery of elemental sulphur when combining with a fe - containing extraction solution . successful application of co - containing h2-rich synthesis gas in biodesulphurization requires not merely tolerance for the presence of co , but also the use of co within the synthesis gas or its conversion products for sulphate reduction as well . the conversion of co to acetate , although a potential substrate for srb , can be considered a disadvantage as acetate gives rise to elevated effluent cod concentrations and requires the presence of both hydrogenotrophic and acetotrophic srb . for the maximal utilization of syngas as electron donor , the distinct dominating groups of srb should be capable of outcompeting other microorganisms that use h2 and acetate . mesophilic srb have been shown capable of outcompeting hydrogenotrophic methanogens [ 31 , 92 ] . the competition between srb and methanogens for acetate appears less predictable despite the thermodynamic and kinetic advantages ascribed to srb . presently , only about ten sulphate - reducing bacteria and one archaeon are able to use co as an electron donor for sulphate reduction . gram - negative srb of the desulfovibrio genus are more sensitive to co. still very little is known about the dissimilation and assimilation of co by sulphate - reducing bacteria and the toxic effect of co on their metabolism . the genomes of some gram - negative sulphate - reducing bacteria are available as well as that of archaeoglobus fulgidus . the only genome sequence of desulfotomaculum sp .- dtm . the upcoming genomic data will give us information about the presence of the enzymes of co - utilization among srb . with genomes from co - metabolizing gram - positive sulphate reducers , a comparative analysis can be made of the co metabolism of gram - negative and gram - positive sulphate reducers . furthermore , more detailed investigation of acetyl - coa pathway enzymes and hydrogenases of carboxydotrophic srb might give more insight to co - utilization by srb . the occurrence of thermophilic microorganisms capable of a high - rate hydrogenogenic co conversion as well as sulfate reduction in the presence of high levels of co enables the use of co containing synthesis gas in biotechnological desulphurisation . successful application of h2-rich synthesis gas in biodesulphurisation , without the need for prior purification , requires tolerance for the presence of co. furthermore , it would be most beneficial to the overall sulfate reduction process if co within the synthesis gas is used for sulphate reduction as well . thus , both toxicity and potential metabolic use of co as an electron donor are important factors governing the utilization potential of co - rich synthesis gas for biotechnological sulphate reduction . in that respect , the recent discovery of   co tolerant srb , such as dtm . carboxydivorans ,   indicates that co - rich synthesis gas holds promise as a cheap alternative electron donor for biodesulphurisation . carboxydivorans , special attention should be directed to the competition for the electron donor with methanogens as well as inhibitory effects of the hydrogen sulphide formed .
several strains of gram - negative and gram - positive sulphate - reducing bacteria ( srb ) are able to use carbon monoxide ( co ) as a carbon source and electron donor for biological sulphate reduction . 
 these strains exhibit variable resistance to co toxicity . 
 the most resistant srb can grow and use co as an electron donor at concentrations up to 100% , whereas others are already severely inhibited at co concentrations as low as 1 - 2% . here 
 , the utilization , inhibition characteristics , and enzymology of co metabolism as well as the current state of genomics of co - oxidizing srb are reviewed . 
 carboxydotrophic sulphate - reducing bacteria can be applied for biological sulphate reduction with synthesis gas ( a mixture of hydrogen and carbon monoxide ) as an electron donor .