University of Groningen

Thermophilic methanol utilization by sulfate reducing bacteria Goorissen, Helene Petronel

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General Introduction

Parts of this Chapter have been published previously (Weijma, 2000) Chapter 1

General Introduction

Contents

1.1 Biological desulfurization 3 1.1.1 Sulfur dioxide emission 3 1.1.2 Biological desulfurization with methanol 4 1.1.3 Sulfate- and sulfite reduction 5 1.1.4 Selection of methanol as electron donor 6 1.1.5 Selection of thermophilic conditions 7 1.2 Microbiology of thermophilic anaerobic methanol conversion 7 1.2.1 Anaerobic degradation of methanol 7 1.2.2 Thermophilic sulfate reducing bacteria 9 1.2.2.1 The genus Desulfotomaculum 14 1.2.2.2 Spore-formation and heat resistance 18 1.2.3 Thermophilic methanogenic archaea 18

1.2.4 Thermophilic homoacetogens growing on C1-compounds 19 1.3 Biochemistry of anaerobic methanol degradation 23 1.3.1 Enzymes involved in degradation of primary alcohols 23 1.3.2 Methanol metabolism in homoacetogens and methanogenic archaea 24 1.3.3 Methanol metabolism in sulfate reducing bacteria 27 1.4 Methanol conversion by mixed communities 28 1.4.1 Substrate competition 28 1.4.2 Direct competition for methanol 29 1.4.3 Indirect competition 30 1.4.4 Syntrophic methanol conversion 31 1.5 Factors affecting the fate of methanol in biological desulfurization 31 1.5.1 Sulfur compounds 32 1.5.2 Methanol and acetate toxicity 33 1.5.3 Environmental conditions 34 1.6 Outline of this thesis 34 References 35

2 Introduction

1.1 Biological desulfurization In sub-section 1.1.1, sulfur dioxide emission and general aspects of the flue gas desulfurization process are discussed. In sub-section 1.1.2 the biological desulfurization process is described in more detail. Sulfate- and sulfite reduction (sub-section 1.1.3); the selection of methanol as electron donor for reduction of sulfur oxyanions (sub-section 1.1.4), and the selection of thermophilic conditions for the biological desulfurization process are discussed (sub-section 1.1.5).

1.1.1 Sulfur dioxide emission Sulfur dioxide (SO2) represents the main fraction of anthropogenic sulfur emissions worldwide. According to the U.S. Environmental Protection Agency (EPA), roughly 23 million tonnes of SO2 are emitted annually in the United States. In the member states of the European Union, about 16.5 million tonnes of SO2 were emitted in 1990. Anthropogenic sulfur dioxide emission is mainly caused by combustion of sulfur-containing fossil fuels such as coal and oil. Electricity generating plants account for nearly 70% of all SO2 emissions (The 1994 Protocol on Further Reduction of Sulfur Emissions. United Nations Economic Commission for Europe). The second major source of sulfur dioxide is industrial combustion processes, such as boilers, process heaters, metallurgical operations, roasting and sintering, coke oven plants, processing of titanium dioxide, pulp production, and the thermal treatment of municipal and industrial wastes. Also some non-combustion processes contribute to sulfur dioxide emission, such as sulfuric acid production, specific organic synthesis processes, treatment of metallic surfaces and oil refining processes. Overall data (1990) for North America, Western, Central, and Eastern Europe, and Central Asia show that 88% of total sulfur emissions originate from combustion processing, 5% from production processing and 7% from oil refining. In terms of contribution by fuel type, coal-fired industrial and electricity-generating plants account for more than 90% of all SO2 emitted by stationary fuel- combustion sources. Sulfur dioxide (along with NOx) has a number of unwanted environmental effects. First, acid rain is formed when sulfur dioxide mixes with and dissolves in the water in clouds, eventually forming dilute sulfuric acid. Acid rain causes lake and soil acidification, forest die-off, and corrosion of stone and metalwork. Furthermore, SO2 contributes to the formation of acidic aerosols, which can cause haze to form over large regions. It is believed that such haziness can substantially reduce average temperatures in affected areas (Charlson et al., 1992). Through this mechanism, SO2 could affect the earth's climate. SO2 and related pollutants have also been linked to a number of human diseases (Amdur & Underhill, 1968). Therefore, the need for sulfur dioxide removal from flue-gases is evident and acknowledged by many countries in treaties such as ‘The protocol to the 1979 convention on long-range transboundary air pollution on further reduction of sulfur emissions’ of the United Nations Economic Commission for Europe and the ‘1990 Clean Air Act Amendments’ of the United States government. General options for reduction of sulfur emissions include energy management measures, increasing the proportion of non-combustion renewable energy sources (i.e. hydro, wind, etc.) of the total energy supply, fuel switching (e.g. from high- to low-sulfur coals and/or liquid fuels), or from coal to gas, fuel desulfurization and advanced combustion technologies (e.g. coal gasification combined with gas desulfurization). Another category of processes aims at removing already formed sulfur oxides, referred to as Flue-Gas Desulfurization (FGD) processes. FGD was already applied in the Battersea power plant in London in 1926, where it consisted of scrubbing the flue- gas with alkaline water (Pfeiffer, 1975). The state-of-the-art technologies for flue-gas treatment processes are all based on the removal of sulfur dioxide by wet, dry or semi-dry (also referred to as wet and dry absorption processes) and catalytic chemical processes. In some cases, techniques for reducing sulfur emissions may also result in the reduction of CO2 and NOx emissions and other pollutants. Flue-gas treatment processes currently applied include: lime/limestone wet scrubbing (LWS); spray dry absorption (SDA); Wellman Lord process (flue-gas scrubbing using sulfite); ammonia scrubbing; and combined NOx/SOx removal processes (activated carbon process and

3 Chapter 1 combined catalytic removal). In the power generation sector, LWS and SDA cover 85% and 10% respectively of the installed flue-gas desulfurization capacity. In LWS, aqueous lime or limestone slurries are brought inot contact with the flue-gas in a scrubber. The sulfur dioxide dissolves in the - aqueous phase and then reacts with hydroxide ions to form bisulfite (HSO3 ), which subsequently 2+ reacts with Ca to form the poorly soluble CaSO3. CaSO4 (gypsum) is another product, as part of the bisulfite is oxidized to sulfate, due to the presence of oxygen in flue-gas. The resulting CaSO3 and CaSO4 mixture is used in construction materials. However, impurities - such as fly-ash and dust - originating from the flue-gas may limit this commercial application. Disposal as a waste then becomes the only alternative, resulting in additional costs and environmental pollution. Over the last decade, efforts have been made to develop a biotechnological alternative to conventional physico-chemical processes for the removal of sulfur dioxide from flue-gases. This process is called Biotechnological Flue-Gas Desulfurization (Bio-FGD). In Bio-FGD, bacteria are used to fix SO2 as elemental sulfur.

1.1.2 Biological desulfurization with methanol

Figure 1.1. Flow sheet of the BIO –FGD process

Biotechnological Flue-Gas Desulfurization makes use of the following conversions of the sulfur cycle:

- + SO2 +H2O ⇒ HSO3 + H 1 - 2- + HSO3 + ½ O2 ⇒ SO4 + H 2 - - HSO3 + 6 {H} ⇒ HS + 3 H2O3a 2- - - SO4 + 8 {H} ⇒ HS + 3 H2O + OH 3b - - HS + ½ O2 ⇒ S + OH 4

Figure 1.1 shows the flow sheet for Bio-FGD. In the first step of biological flue-gas desulfurization, sulfur dioxide is scrubbed from the flue-gas using a bicarbonate solution (reaction 1). the presence of oxygen in the flue-gas results in oxidation of part of the sulfite into sulfate (2). In the subsequent step, sulfite and sulfate are reduced under anaerobic conditions with an added electron donor ({H}) to sulfide by sulfate-reducing bacteria (3a and b). In a micro-aerobic reactor, the sulfide produced is partially oxidized to elemental sulfur by autotrophic sulfur bacteria like Thiobacillus spp. (4) with concomitant production of hydroxide. Separation of the solid sulfur particles from the medium enables the recovery of elemental sulfur as a valuable product. The

4 Introduction remaining alkaline solution, with a pH of about 9, can be reused for the scrubbing of sulfur dioxide. Because - along with SO2 - heat is also transferred from the flue-gas to the scrubbing solution, it is economically attractive to operate the desulfurization process at thermophilic conditions (50-65°C). In biological desulfurization processes, methanogenesis should be avoided as this decreases the selectivity of sulfate reduction with the added electron donor.

1.1.3 Sulfate- and sulfite reduction Sulfite reduction is energetically more favorable than sulfate reduction. At a biochemical level, this is manifested by the ATP demanding activation of sulfate to adenosine-5’- phosphosulfate (APS) by ATP-sulfurylase, which is followed by APS reduction to form sulfite and AMP. Sulfite is directly suitable as an electron acceptor for the SRB. A review of the biochemistry of sulfate reduction can be found elsewhere (Widdel & Hansen, 1992). Sulfite addition to sulfidogenic bioreactors may lead to the chemical formation of thiosulfate (Krämer & Cypionka, 1989):

- - 2- o 4 HSO3 + 2 HS ⇒ 3 S2O3 + 3 H2O(∆G ’= -167 kJ/mol )

Van Houten et al (1997) detected about 15 mg.L-1 (0.13 mM) thiosulfate in a thermophilic bioreactor fed with H2/CO2, sulfite, and sulfate. The rather low thiosulfate concentration led to the conclusion that the rate of sulfite reduction was higher than the chemical conversion rate of sulfite plus sulfide to thiosulfate. An alternative explanation could be that the rates of thiosulfate formation and reduction are about as high as each other. The use of thiosulfate as terminal electron acceptor is energetically also more favorable than the use of sulfate, as - like sulfite reduction - thiosulfate reduction requires, no ATP-dependent activation (Krämer & Cypionka, 1989). This may explain the preferential use of thiosulfate over sulfate as the electron acceptor in fresh water sediment (Jørgensen & Bak, 1991). Thiosulfate reduction by SRB may lead to higher cell yields compared to sulfate reduction (Badziong & Thauer, 1978).

Disproportionation Bak and Pfennig (1987) were the first to describe the disproportionation of sulfite and thiosulfate to sulfide and sulfate by the sulfate-reducing bacterium Desulfovibrio sulfodismutans according to the following stoichiometry:

2- 2- - + o 2- S2O3 + H2O ⇒ SO4 + HS + H ∆G ’= -21.9 kJ/mol S2O3

2- + 2- - o 2- 4 SO3 + H ⇒ 3 SO4 + HS ∆G ’ = -58.9 kJ/mol SO3 Later it was found that many SRB are able to disproportionate sulfite and thiosulfate (Krämer & Cypionka, 1989). In batch experiments using fresh sludge from a thermophilic methanol-fed, sulfidogenic bioreactor, sulfite disproportionation activity was observed, although rates were 4 times lower than sulfate reduction rates with methanol (Weijma, 2000). Jørgensen and Bak (1991) demonstrated that disproportionation and thiosulfate reduction may occur simultaneously. Even disproportionation of elemental sulfur by SRB has recently been demonstrated (Finster et al., 1998). For growth, most disproportionating SRB known thus far, require acetate as a carbon source. Recently it was shown that Dm. thermobenzoicum can disproportionate thiosulfate in the absence of any added carbon source (Jackson & McInerney, 2000). Thus far, only strain NTA 3 is known to grow autotrophically by thiosulfate disproportionation (Bak & Cypionka, 1987). The finding that Dm. thermobenzoicum effectively disproportionates thiosulfate shows that disproportionation metabolism is not exclusively restricted to mesophilic gram-negative SRB.

5 Chapter 1

1.1.4 Selection of methanol as electron donor An important factor determining the economic feasibility of biological desulfurization is the cost of the electron donor needed for sulfate reduction in the anaerobic step. Formation of undesirable side-products, such as methane and acetate, needs to be minimized. Possible electron donors include organic waste materials, such as primary sewage sludge, spent yeast from breweries, dairy whey, molasses, and bulk chemicals like H2, synthesis gas (a mixture of H2, CO2 and CO), ethanol, and methanol (Table 1.1). Organic waste has the advantage of low costs, but because of its complex composition, adequate control of the process may be difficult. For instance, intermediates formed during degradation of organic waste may promote undesirable growth of methanogens. Also, incomplete degradation of organic compounds may decrease the performance of the sulfide-oxidizing bioreactor of the desulfurization process (Janssen et al., 1997). The applicability of pure chemicals such as lactate, ethanol, and acetate for sulfate reduction has been demonstrated in mesophilic laboratory-scale reactors (Table 1.1), but use of these chemicals on an industrial scale will probably be prohibitively expensive. Relatively cheap bulk chemicals as synthesis gas or H2/CO2 are better options in this respect. Moreover, reasonable to good sulfate elimination rates can be achieved using these substrates in mesophilic gas-lift reactors (Table 1.1). However, under thermophilic conditions (preferable for Bio-FGD) elimination rates with H2/CO2 are lower, while it has been found that about half of the added hydrogen is used for methanogenesis, presumably due to the good kinetic growth properties of thermophilic methanogens (Van Houten, et al., 1997).

Table 1.1. Sulfate and sulfite elimination rates found in biological desulfurization processes with various electron donors.

2- 2- Electron T Bioreactor SO4 removal SO3 removal COD to Reference donor type -1 -1 -1 -1 H2S/CH4 (ºC) (g.L .day ) (g.L .day ) (%/%) a b molasses 31 packed bed 6.5 na nr Maree & Strydom, 1987 c m.s.d. 30 packed bed na 46 100/0 Selvaraj et al., 1997 d lactate RT plugflow 0.41 na nr Hammack et al., 1994 acetate 35 packed bed 65 na 100/0 Stucki et al., 1993 e acetate 33 EGSB 9.4 na nr Dries et al., 1998 f ethanol 35 UASB 6 na nr Kalyuzhnyi et al., 1997 syngas 30 gas-lift 10 na 100/0 Van Houten et al., 1995

H2/CO2 30 packed bed 1.2 na 100/0 duPreez & Maree, 1994

H2/CO2 30 gas-lift 30 na 100/0 Van Houten et al., 1994

H2/CO2 55 gas-lift 7.5 9.3 50/50 Kaufman et al., 1996 CO 30 packed bed 2.4 na 100/0 duPreez & Maree, 1994 a) na: no sulfate or sulfite added; b) nr : not reported; c) m.s.d.: municipal sewage digest; d) RT: room temperature; e) EGSB : expanded granular sludge bed; f) UASB: upflow anaerobic sludge bed.

In the present study, the use of methanol as the electron donor for thermophilic sulfate reduction was investigated. Methanol is a relatively cheap bulk chemical and therefore an attractive substrate for use in biotechnological processes (Dijkhuizen et al., 1985). Methanol is successfully used as an electron donor in denitrification (Germonpre et al., 1991), reductive dehalogenation (Gerritse et al., 1999), and it has also been proposed as an electron donor in other sulfate-reducing processes (Hard & Babel, 1995). Moreover, chemically synthesized methanol contains few organic impurities, resulting only in a low extent of undesirable biological side-reactions resulting from these impurities. A low level of impurities also makes additional treatment of biologically

6 Introduction desulfurized wastewater redundant. Hence, methanol was selected as the electron donor for reduction of sulfur oxyanions in our investigation.

1.1.5 Selection of thermophilic conditions Most of the biological desulfurization processes have been operated at mesophilic temperatures (Table 1.1). The feasibility of the process will be determined partly by the energy requirements of the operational system. A thermophilic process in the temperature range of the flue-gas wastestream, can decrease the energy demand of the process. As well as producing SO2 heat is transferred from the flue-gas to the scrubbing solution, so it is attractive to operate the desulfurization process at (moderate) thermophilic conditions, so that no cooling is required. Moreover, thermophilic conditions might benefit the process in other ways. First, temperatures above 65 ºC might rule out undesirable side reactions, for example methanogenesis with methanol, since no methanogens growing with methanol above 65 ºC have yet been discovered. Second, due to the relatively high temperature, turnover rates might be higher. In the present work, sulfate reduction with methanol was studied at temperatures in the range 60-65ºC. At the start of this research project, only a few thermophilic methanol-utilizing SRB were known.

1.2 Microbiology of thermophilic anaerobic methanol degradation In this section, the possible biological degradation routes of methanol are presented (sub- section 1.2.1). An overview of sulfate reducers, methanogenic archaea and homoacetogens possibly involved in thermophilic methanol degradation is given in sub-sections 1.2.2, 1.2.3, and 1.2.4, respectively.

1.2.1 Anaerobic degradation of methanol Possible degradation pathways for methanol under anaerobic conditions are shown in Figure 1.2. Reaction stoichiometries and Gibbs free energy changes are shown in Table 1.2. Three groups of microorganisms are involved in anaerobic methanol degradation, namely sulfate- reducing bacteria (SRB), methanogenic archaea (MA), and homoacetogenic bacteria (AB). Methanol can be used directly as a carbon and energy source by SRB (conversion 1) (Nazina, et al., 1987), MA (conversion 5a) (Touzel et al., 1985) and AB (conversion 9) (Savage & Drake, 1986). In addition, MA may reduce methanol to methane using H2 (conversion 5b). Therefore, SRB, MA and AB will compete for the available methanol in mixed cultures. Thermophilic SRB e.g. Desulfotomaculum thermoacetoxidans (Min & Zinder, 1990) and MA (Nozhevnikova & Chudina, 1984) may also compete for acetate (conversions 2 and 6), the product of methanol catabolism by AB (conversion 9). It has also been demonstrated that acetate can be oxidized to H2/CO2 (conversion 11) under mesophilic conditions (Schnürer et al., 1996), as well as under thermophilic conditions (Zinder & Koch, 1984). Therefore, degradation of methanol to methane by a triculture consisting of a methylotrophic acetogen, acetate oxidizing species, and a hydrogenotrophic methanogen is theoretically possible. Furthermore, anaerobic bacteria may partially oxidize methanol to H2/CO2 (conversion 10), when the H2 concentration is kept low by hydrogenotrophic sulfate reducers (conversion 3) or methanogens (conversion 7) (Davidova & Stams, 1996). In the mesophilic temperature range, even methanogens have been shown to produce H2/CO2 from methanol when grown in the presence of SRB (Phelps et al., 1985). Thus, competition for H2 may also take place. At a high hydrogen partial pressure, H2 may be consumed by homoacetogens (Wiegel et al., 1981). As methanol oxidation to hydrogen is thermodynamically unfavorable at a high hydrogen partial pressure, methanol oxidation followed by acetogenesis from H2/CO2 is unlikely to occur. Therefore, this conversion is not included in Figure 1.2 and Table 1.2. Methanol conversion to formate (conversion 12) is thermodynamically unfavorable under standard conditions. To our knowledge, formate formation from methanol has not been reported in literature. However, besides hydrogen, formate can be important in methanogenic environments (Boone et al., 1989; Dong & Stams, 1995). SRB (conversion 4) (Nazina et al., 1987) and MA (conversion 8) (Zhilina & Ilarianov,

7 Chapter 1

1984) can subsequently use formate. Methanol degradation can be even more complex because formate conversion to hydrogen and acetate (Guyot, 1986) and methanol degradation to butyrate (Cato et al., 1986), or alanine (Balk et al., 2002) (not shown in Figure 1.2) may also occur. The above illustrates that mixed cultures may mineralize the relatively simple C1-compound methanol in a complex way. As a consequence, SRB and MA may not only compete for methanol, but also for hydrogen, acetate, and formate.

2- SO4 Sulfide 4 Formate 8 Methane

2- SO4 Sulfide 1 Methanol 5 Methane

9 10 2- SO4 Sulfide 2 7 Acetate H/CO22 Methane 2- 3 SO4

Methane Sulfide

Figure 1.2. Anaerobic methanol mineralization. Conversion numbers correspond to the numbers of reaction equations in Table 1.2

Table 1.2. Stoichiometry and Gibbs free energy changes at standard conditions and pH 7 of reactions possibly involved in anaerobic methanol degradation. Calculated from Thauer et al. (1977). Reaction ∆G°' (kJ/reaction) 2- - - + 1) 4 CH3OH + 3 SO4 ⇒ 4 HCO3 + 3 HS + 4 H2O + H -364 - 2- - - 2) CH3COO + SO4 ⇒ 2 HCO3 + HS -48 2- + - 3) 4 H2 + SO4 +H ⇒ HS + 4 H2O -152 - 2- + - - 4) 4 HCOO + SO4 + H ⇒ HS + 4 HCO3 -172 - + 5a) 4 CH3OH ⇒ 3 CH4 + HCO3 + H2O + H -316 5b) CH3OH + H2 ⇒ CH4 + H2O -113 - - 6) CH3COO + H2O ⇒ CH4 + HCO3 -31 - + 7) 4 H2 + HCO3 + H ⇒ CH4 + 3 H2O -136 - + - 8) 4 CHOO + H2O + H ⇒ CH4 +3 HCO3 -132 - - + 9) 4 CH3OH + 2 HCO3 ⇒ 3 CH3COO + H + 4 H2O -220 - + 10) CH3OH + 2 H2O ⇒ 3 H2 + HCO3 + H +23 - - + 11) CH3COO + 4 H2O ⇒ 4H2 + 2 HCO3 + H +104 - - + 12) CH3OH + 2 HCO3 ⇒ 3 HCOO + H2O + H +19

8 Introduction

1.2.2 Thermophilic sulfate reducing bacteria Sulfate reduction has been reported as occurring at maximum temperatures between 100- 110 ºC, although no organisms with such temperature optima have yet been isolated (Jørgensen et al., 1992). Sulfate reduction with C1 or C2 compounds as substrate may occur at temperatures up to 90 ºC, but is only known for hyperthermophilic archaea (Stetter, 1988; Burggraf et al., 1990; Huber et al., 1997; Stetter et al., 1993). No eubacterial sulfate reducers with an upper temperature limit above 85 ºC have yet been described (Nazina, et al., 1987; Rozanova & Khudyakova, 1974). SRB constitute a diverse group of prokaryotes that contribute to a wide variety of essential functions in many anaerobic environments. On the basis of phylogenetic and physiological characteristics, SRB are classified into four distinct groups: Gram-negative mesophilic SRB; Gram- positive spore-forming SRB; Gram-negative thermophilic SRB; and thermophilic archaeal SRB (Castro et al., 2000). All of these groups are characterized by their use of sulfur oxyanions such as sulfate, sulfite, or thiosulfate as the terminal electron acceptor for anaerobic respiration. Thermophilic sulfate-reducers are spread among different genera, an overview of all described thermophilic sulfate-reducers is given in Table 1.3. Phylogenetic classification of the thermophilic sulfate-reducers within the bacterial domain combined with the archaeal sulfate-reducers, is given in Fig. 1.3. The optimum pH for growth of the known thermophilic SRB lies in the range of 6.5-7.5 and the optimum temperature in the range of 54-70 ºC (Table 1.3., p.12). Most of the thermophilic SRB are able to grow at moderately or high salt concentrations of up to 70 g.L-1. The dominant genus in this overview of thermophilic SRB is the genus Desulfotomaculum, which comprises at least 14 thermophilic species. Desulfotomaculum species play a central role in the work described in this thesis and will be discussed in more detail in sub-section 1.2.2.1. Genera consisting of thermophilic species only are Thermodesulfobacterium, Thermodesulforhabdus, Thermodesulfovibrio, and Desulfacinum, although Desulfacinum hydrothermale can grow at mesophilic temperatures. All species, except Thermodesulforhabdus norvegicus, can use hydrogen as an electron donor. Some species - i.e. Dsm. alkaliphilum, all Thermodesulfobacterium species, and all Thermodesulfovibrio species - need additional acetate as a carbon source for growth on hydrogen. Dsm. thermoacetoxidans produces acetate and sulfide if H2/CO2 is added in excess. Growth on formate with acetate as the carbon source is common, while growth on acetate is rare (Table 1.3). Methanol utilization is even more rare and is restricted only to a few Desulfotomaculum species, although utilization of methanol was not tested for some species (Table 1.3). Use of sulfite and thiosulfate as an alternative electron acceptor is common among SRB, although some species additionally use elemental sulfur (Desulfacinum subterraneum and Dsm. thermoacetoxidans) or nitrate (Dsm. thermobenzoicum and Tvb. islandicus). Under sulfate-limiting conditions, some thermophilic SRB ferment pyruvate (e.g. Desulfacinum species, most Desulfotomaculum species, Tdv. yellowstonii, Tdb. commune, and Tdb. mobile) or lactate (several Desulfotomaculum species). Furthermore, during sulfate limitation some thermophilic SRB (Dsm. thermobenzoicum subsp. thermosyntrophicum, Dsm. thermocisternum) can produce hydrogen growing in a syntrophic association with thermophilic hydrogen scavengers such as Methan-othermobacter thermoautotrophicus, although reports on syntrophic growth of Dsm. thermocisternum are contradictory (Nilsen et al., 1996; Imachi et al., 2000; Plugge et al., 2002). Thermodesulforhabdus norvegicus, isolated from Norwegian oil platforms, was the first thermophilic SRB, that showed good growth on acetate. This strain belongs to the delta subdivision of the proteobacteria together with Desulfacinum infernum, a strain of similar origin. Thermodesulforhabdus norvegicus and the genus Desulfacinum are classified in the group of gram-negative mesophilic SRB and cluster phylogenetically in the family of Desulfobacteriaceae. The majority of members of this family are mesophilic (Castro et al., 2000). The genera Thermodesulfobacterium and Thermodesulfovibrio share similar physiological and phenotypical characteristics but are phylogenetically only distantly related. They both belong to the group of gram-negative thermophilic bacteria and they branch deeply in the Bacteria domain. Their optimal temperatures are higher than those described for gram-positive spore-forming thermophilic SRB and Desulfacinum species, but lower than those of the archaeal SRB.

9 Chapter 1

.

Figure 1.3. Phylogenetic relationships between all sulfate-reducers compared with some reference genera. The genus Archaeoglobus was used as outgroup. Bootstrap values (50 replicants) are expressed as percentages at the branch points. Classification of sulfate-reducers according to Castro et al. (2000); A, thermophilic gram-negative SRB; B, mesophilic gram- negative SRB; C, gram-positive SRB; D, thermophilic archaeal sulfate-reducers.

10 Introduction

The genus Thermodesulfovibrio consists of two species - Tdv. yellowstonii and Tdv. islandicus - both isolated from geothermal vents. In contrast with Tdv. yellowstonii, Tdv. islandicus can reduce nitrate but not sulfite. Four Thermodesulfobacterium species have been described to date: Tdb. commune and Tdb. hveragerdense, both originating from terrestrial hot springs, Tdb. mobile, isolated from warm oilfield water, and Tdb. hydrogeniphilum from marine hydrothermal chimneys and sediments. All four species have a narrow substrate range: in Tdb. commune and Tdb. hveragerdense apart from H2/CO2, only lactate and pyruvate can serve as an electron donor for sulfate reduction. In addition, Tdb. mobile can also use formate as a substrate for sulfate reduction. Growth of Tdb. hydrogeniphilum depends upon the presence of hydrogen, and some additional substrates like acetate and fumarate stimulate growth, but could not be used as sole energy source. Except for Tdb. hydrogeniphilum, all Thermodesulfobacterium species need additional acetate as a carbon source for growth. Four archaeal sulfate-reducers have been isolated yet, all from marine hydrothermal areas. These species, with a lower temperature limit of 60 ºC and optimum growth temperatures above 80 ºC, are members of the genus Archaeaoglobus. Except for A. profundis, all Archaeoglobus strains are facultative chemolithoautotrophs.

11 Table 1.3. Selected physiological characteristics of thermophilic bacterial and archaeal sulfate-reducers

o Organism Origin Gram- Growth substrates Electron acceptors T-opt ( C) Specific Reference o stain T-range ( C) feature Archaeoglobus fulgidus Hydrothermal system - H2/CO2, for, lac, fum, Sox 83 Stetter, 1988 60-95 lithotrophicus Oil field reservoir - H2/CO2, for, ac, fum, etoh Stetter et al., 1993 0 profundis Hydrothermal system - H2 +ac, +lac, +pyr sulfate, thiosulfate 82 S inhibits Burggraf et al., 1990 65-90 0 veneficus Black smoker - H2/CO2, for, ac, fum, etoh sulfite, thiosulfate 80 S inhibits Huber et al., 1997 65-85 Desulfacinum nt infernum Oil reservoir - H2/CO2, for, ac, metoh Sox 60 Rees et al., 1995 nt hydrothermale Marine hydrothermal vent - H2/CO2, for, ac, metoh Sox 60 halophilic Sievert & Kuever, 2000 37-64 nt subterraneum High temperature oil field -H2/CO2, for, ac, metoh Sox, sulfur 60 ferments YE Rozanova et al., 2001 Vietnam 45-65 Desulfotomaculum alkaliphilum Cow/pig manure + H2 + ac, for, lac, etoh, pyr Sox 50-55 alkaliphilic Pikuta et al., 2000 30-58 australicum Geothermal ground water + H2/CO2, ac, lac, etoh nt 68 Love et al., 1993 40-74 geothermicum Saline geothermal +H2/CO2, lac, etoh, fruc sulfate, sulfite, 54 halophilic Daumas et al., 1988 nt ground water thiosulfate 30-57 kuznetsovii Hot spring + H2/CO2, for, ac, metoh, Sox 60 Nazina, et al., 1987 etoh 50-85 luciae Hot spring sediments + H2/CO2, for, lac, etoh, pyr sulfate, thiosulfate nr Liu et al., 1997 50-70 nigrificans Freshwater + H2/CO2, lac, etoh Sox 55 Klemps et al., 1985 30-70 putei Deep terrestrial rock + H2/CO2, for, lac, etoh Sox nr Liu et al., 1997 50-65 thermoacetoxidans Anaerobic digester + H2/CO2, for, ac, lac sulfate, thiosulfate, 55-60 Min & Zinder, 1990 Table continued

Organism Origin Gram- Growth substrates Electron acceptors T-opt (°C) Specific Reference stain T-range (°C) feature thermobenzoicum Deep submarine +H2/CO2, for, lac Sox, nitrate 62 Tasaki et al., 1991 groundwater 40-70 thermocisternum North sea oil reservoir + H2/CO2, lac, etoh Sox 62 halophilic Nilsen et al., 1996 41-75 thermosapovorans Rice compost + H2/CO2, for, lac Sox 50 Fardeau et al., 1995 35-60 thermobenzoicum subsp. Thermophilic granular +H2/CO2, lac, pyr, prop sulfate 55 syntrophic Plugge et al., 2002 thermosyntrophicum methanogenic sludge 45-62 growth strain TPOSR Sulfidogenic +H2/CO2, for, ac, metoh, Sox 60 Stams, unpublished thermophilic bioreactor etoh nr strain WW1 Sulfidogenic +H2/CO2, metoh, etoh, for Sox 62-68 Weijma, 2000 thermophilic bioreactor 45-75 strain T93B Statfjord oil field + H2/CO2, metoh, etoh, for Sox 65 halophilic Rosnes et al., 1991 43-78 Thermodesulfobacterium commune Volcanic hot water - H2+ac, lac, pyr, sulfate, thiosulfate 70 unusual Rozanova & Khudyakova, 45-85 morphology 1974;Rozanova & Pivovarova, 1988 hydrogeniphilum Marine hydrothermal -H2 sulfate 75 Jeanthon et al., 2002 chimney 50-80 hveragerdense Icelandic hot spring - H2+ac, lac, pyr, Sox 70-74 Sonne-Hansen & Ahring, 55-74 1999;Sonne-Hansen et al., 1999 mobile (formerly Oil field water - H2+ac, lac, pyr, for Sox 65 different cell Rozanova & Pivovarova, 1988, Desulfovibrio sizes Rozanova & Khudyakova, 1974 thermophilus)

Thermodesulforhabdus Oil field reservoir - Ac, lac, etoh sulfate, sulfite 60 Beeder et al., 1995 norvegicus Thermodesulfovibrio yellowstonii Thermal vent water - H2+ac, lac, pyr, for+ac Sox 65 Henry et al., 1994 40-70 islandicus Icelandic hot Spring - Lac, pyr, H2, for Sulfate, thiosulfate, 65 Sonne-Hansen & Ahring, 1999 nitrate 45-70 metoh: methanol, etoh: ethanol, glu: glucose, ac: acetate, for: formate, fruc: fructose, lac: lactate, pyr: pyruvate, prop: propionate, YE: yeast extract, ; Sox: sulfate, sulfite, thiosulfate, nt: not tested Chapter 1

1.2.2.1 The genus Desulfotomaculum Thermophilic methanol utilization by SRB has been described only for Desulfotomaculum species. Several Desulfotomaculum strains were the subjects of the present study, but we focussed mainly on the extremely heat-resistant spore producer Dsm. kuznetsovii. SRB that form heat-resistant endospores share this trait with many Bacillus and Clostridium species and were classified in one genus, namely Desulfotomaculum. Recently another spore-producing genus has been proposed, namely the genus Desulfosporosinus, however, this genus comprises only mesophilic species. Due to endospore formation, thermophilic Desulfotomaculum species are widespread in nature and large numbers of spores may be found even in permanently cold sediments at temperatures insufficient to support growth of these species (Isaksen et al., 1994; Goorissen, unpublished results). The first isolate and type strain of this genus, is the moderate thermophile, Dsm. nigrificans. This strain was isolated in the 1920s as Clostridium nigrificans and finally named Dsm. nigrificans in 1965 by Campbell and Postgate (1965). Subsequently more mesophilic and thermophilic species were isolated and this genus now comprises 20 validly described species. This genus exhibits a great versatility in the types of electron donors used for growth, including H2/CO2, alcohols, fatty acids, other aliphatic monocarboxylic and dicarboxylic acids, alanine, catechol, indole, nicotinate, phenol, acetone, and others. Depending on the species, substrates are incompletely oxidized to acetate, or completely to CO2. Sulfite or thiosulfate can replace sulfate as the electron acceptor and some species show the ability to use terminal electron acceptors as Fe (III) (Dsm. reducens) or arsenate (Dsm. auripigmentum) for growth. These properties are unusual, but also have not been tested for many species. Although several Desulfotomaculum species show negative gram-stain, they are true gram- positives as is evident from their cell wall architecture. Phylogenetically, they are separate from other sulfate-reducing bacteria, and the genus is placed within the low G+C%-gram-positive branch of the Clostridium-Bacillus subphylum (Fig. 1.4). However, recently published phylogenetic trees questioned the coherence of the family and therefore changes have been proposed (Stackebrandt et al., 1997; Kuever et al., 1999; Stubner & Meuser, 2000). In contrast to the majority of the species, Dsm. orientis and Dsm. auripigmentum branch together with a Desulfosporosinus cluster and Dsm. orientis has therefore been classified as Desulfosporosinus orientis (Stackebrandt et al., 1997). Dsm. guttoideum is closely related to a Clostridium cluster, and appears on a separate branch of the Desulfotomaculum main cluster, which justifies a reclassification (Stackebrandt et al., 1997; Castro et al., 2000). Sporotomaculum hydroxybenzoicum is closely affiliated to Dsm. thermosapovorans (>95%), although this species is mesophilic and can grow on 3-hydroxybenzoate as the sole carbon- and energy source. Furthermore, some phylogenetic analyses provide grounds for the separation of mesophilic and thermophilic species in two distinct genera with the name “Thermodesulfotomaculum” proposed for the thermophilic species (Nazina et al., 1999). An overview of the 14 thermophilic species of the genus Desulfotomaculum is given in Table 1.4. (p 16). On the basis of 16S rRNA sequence analyses, thermophilic Desulfotomaculum species are scattered among five subclusters of the gram-positive spore-forming SRB phylogenetic tree (Fig. 1.4). Species are separated by similarity values of 83 to 99%, which illustrates the heterogeneity of the Desulfotomaculum thermophiles. Common phenotypic and physiological characteristics are shared by all thermophilic Desulfotomaculum species, but some distinguishing features can be mentioned. Desulfotomaculum species differ in the percentage G+C, ranging from 41 % for Dsm. alkaliphilum to 56% for Dsm. thermocisternum. NaCl tolerance differs significantly between species, varying from 10 to 70 g/L. Dsm. alkaliphilum, Dsm. geothermicum, and Dsm. thermocisternum can be regarded as halotolerant. All species can use hydrogen as an electron donor, but some species require acetate as an additional carbon source. Growth with acetate as the carbon- and energy source is only reported for Dsm. australicum, Dsm. kuznetsovii, Dsm. thermoacetoxidans, Dsm. thermobenzoicum, and strain TPOSR. However, growth on acetate is weak, except for Dsm thermoacetoxidans. Growth on methanol is observed for Dsm. kuznetsovii, strain TPOSR, strain WW1, Dsm. putei, and Dsm. thermosapovorans, although in the latter two

14 Introduction organisms, growth is weak. Most of the thermophilic Desulfotomaculum species can use sulfite and thiosulfate next to sulfate as electron donor. Exceptions are Dsm. thermobenzoicum subsp. thermosyntrophicum, which can only use sulfate, and strains Dsm. luciae and Dsm. thermo- acetoxidans, which both cannot use sulfite.

Figure 1.4. Phylogenetic tree of the genus Desulfotomaculum combined with some reference genera, based on 16S rDNA sequence comparisons. Bacillus methanolicus was used as outgroup. Cluster names in roman numerals are according to Stackebrandt et al. (1997). Thermophilic species are represented in bold. * = subspecies thermosyntrophicum

15 Table 1.4. Selected properties of moderately thermophilic Desulfotomaculum species

Desulfotomaculum alkaliphilum australicum geothermicum kuznetsovii luciae nigrificans putei Origin Cow/pig manure Geothermal Geothermal Hot spring Hot spring Freshwater Hot spring groundwater groundwater sediments sediments T optimum (ºC) 50-55 68 54 60-65 60-65 55 64 T range (ºC) 30-58 40-74 37-56 50-85 50-70 40-62 35-65 NaCl tolerance < 7% nr >5% 0-2% 0-1% 1-2% < 2 % Spores + spherical, central + spherical, spherical, central subterminal paracentral, central oval Closest relative halophilum thermocisternum thermosapovorans luciae strain TPOSR putei nigrificans (Desulfotomaculum) 16S rDNA % 92.7 99 92 96 98 94 94 Phyl. Cluster If Ic Ib Ic Ic Ia Ia G+C mol% 40.9 48 50.4 49 51.4 44.5 47.1 Substrate utilization Methanol ---+--+ H2/CO2 + ac + nr + + + ac + Acetate -+-+--- Fermentation pyr pyr, lac nr nr pyr, lac pyr, fruc Electron acceptors thiosulfate, sulfite nr nr thiosulfate, thiosulfate thiosulfate, thiosulfate, next to sulfate sulfite sulfite sulfite Reference Pikuta et al., 2000 Love et al., 1993 Daumas et al., 1988 Nazina, et al., Karnauchow et al., Klemps et al., Liu et al., 1997 1987 1992 1985 nr: not reported, ben: benzoate, fruc: fructose, fum: fumarate, gly: glycine, lac: lactate, pyr: pyruvate Table continued

Desulfotomaculum thermoacetoxidans thermobenzoicum thermocisternum thermosapovorans thermobenzoicum strain strain WW1 subsp. thermosyn- TPOSR trophicum Origin Anaerobic digester Anaerobic digester Deep submarine Rice paddy soil Methanogenic Anaerobic Thermophilic groundwater granular sludge digester bioreactor T optimum (ºC) 55-60 62 62 50 55 60 62-68 T range (ºC) 45-65 40-70 41-75 35-60 45-62 nr 45-75 0 NaCl tolerance <1.5% nr 0.1 /00-4.7% 0-3.5 % nr nr nr Spores spherical, central + spherical, central central oval, central Sperical, Sperical, central central Closest relative thermobenzoicum thermobenzoicum strain WW1 sapomandens thermoacetoxidans luciae thermocisternum (Desulfotomaculum) subsp. thermosyn- subsp. thermosyn- trophicum trophicum 16S rDNA % 98 96 99 97 98 98 99 Phyl. Cluster Id Id Ic Ib Id Ic Ic G+C mol% 49.7 52.8 56 51.2 53.7 55 58.9 Substrate utilization ++ Methanol nr - - + - + + H2/CO2 + ++++nrnr Acetate + +----- Fermentation lac-, pyr- pyr, lac pyr pyr, lac pyr, lac, fum, gly, nr nr ben Electron acceptors thiosulfate, sulfur thiosulfate, sulfite, thiosulfate, sulfite thiosulfate, sulfite - thiosulfate, thiosulfate, next to sulfate nitrate sulfite sulfite Reference Min & Zinder, 1990 Tasaki et al., 1991 Nilsen et al., 1996 Fardeau et al., 1995 Plugge et al., 2002 Stams, un- Weijma, 2000 published nr = not reported, ben: benzoate, fruc: fructose, fum: fumarate, gly: glycine, lac: lactate, pyr: pyruvate Chapter 1

1.2.2.2 Spore-formation and heat resistance The production of endospores as an effective reproducing and survival strategy is a phenomenon observed in prokaryotic as well as in eukaryotic species. Especially in food industrial processess and in the manufactory of sterile medical devices, there is great awareness of the control of sporulation by pathogenic and non-pathogenic bacteria. Dormant spores are not hazardous, but spore germination, outgrowth and proliferation could result in food spoilage and toxin production, in turn causing food poisoning. The exceptional resistance properties of bacterial endospores are the subject of many studies. Heat resistance of endospores has been studied extensively in Bacilli (Gonzalez et al., 1999; Mazas et al., 1999; Casadei et al., 2001; Fernandez et al., 2001), Clostridia (Adams, 1973; Nakamura et al., 1985; Payot et al., 1999) and Moorella (Van Rijssel et al., 1992; Byrer et al., 2000) species. Sporulation, spore dormancy, and spore germination are relatively well understood (see for instance Serrano et al., 1999; Atrih & Foster, 1999; Boland et al., 2000; Henriques & Moran, 2000), in contrast with the mechanism of heat resistance of bacterial spores which remains enigmatic. Spore heat resistance is both complex and multifactorial, and it is unquestionable that the spore cortex peptidoglycan, the dehydration, mineralization, and dipicolinic acid content of the spore core are involved in spore heat resistance (Beaman & Gerhardt, 1986; Atrih & Foster, 2001). The apparent heat resistance of spores can be influenced dramatically by changing the growth conditions during sporulation (Leguerinel et al., 2000). Medium composition, for example, affects the mineralization state of the spore cortex and the peptidoglycan structure and thereby the heat resistance of the spores. Growth temperature is another parameter likely to affect spore heat resistance: spores of the same strain produced at a high temperature are more heat resistant than spores produced at a lower temperature (Warth, 1978; Setlow, 1994; Palop et al., 1999; Gonzalez et al., 1999). Within the heterogeneous group of sulfate-reducing prokaryotes, Desulfotomacula are unique with respect to their spore producing properties. The production of spores by Desulfotomaculum species undoubtedly has been reponsible for the widespread occurrence of these organisms in nature. The extreme heat resistance of spores of Desulfotomaculum strains was first recognized several decades ago. Donelly and Busta (1980) reported a decimal reduction value of 5 min for Dsm. nigrificans, a moderate thermophile. However, no other extreme heat resistant spores of Desulfotomaculum strains have been described since then.

1.2.3 Thermophilic methanogenic archaea In general, thermophilic methanogenic archaea display a narrow substrate range, varying from strict autotrophic growth with H2/CO2 to the use of several C1-compounds. However, methanol oxidation is restricted to moderate thermophiles, i.e. Methanosarcina species, Methanohalobium evestigatum, and Methanosalsum zhilinae. The upper temperature limit for growth of these organisms is equal to or below 60 ºC (Table 1.5). The Methanosarcina species have very similar physiological properties and are probably all M. thermophila strains (Clarens & Moletta, 1990; Zinder, 1990). M. thermophila TM-1 is the only strain which can grow on H2/CO2 and all Methanosarcina strains can grow on acetate. Other acetate-utilizing methanogenic species are found in the genus Methanothrix, for which the upper temperature for growth is 70ºC. Methanothrix species have been isolated mainly from anaerobic digesters. Thermophilic methanogens are dominated by the genus Methanothermobacter. This genus was recently proposed to include several thermophilic Methanobacterium species (Wasserfallen et al., 2000) based on three independent data sets: analysis of 16S rRNA sequences, antigenic fingerprinting, and data pertaining to extrachromosomal elements, plasmids, and phages. As a result of the proposal mentioned above, mesophilic and thermophilic Methanobacterium species are separated into two different genera, although the thermophile Methanobacterium thermoaggregans has not been renamed yet. All Methanothermobacter and Methanobacterium species can grow autotrophically and some are also capable of utilizing formate for growth.

18 Introduction

Methanothermobacter species are ubiquitous in a number of environments such as anaerobic digesters, hot springs and digested sludge. The genus Methanogenium comprises a great number of mesophilic species, one true psychrophilic species, and a few subspecies of the species Mg. thermophilum (Jarrell & Kalmokoff, 1988; Franzmann et al., 1997). If H2/CO2 is used as the growth substrate, all Mg. thermophilum strains need additional acetate as a carbon source. Methanothermococcus thermolithotrophicus was originally classified as a member of the genus Methanococcus, which comprises some methylotrophic mesophilic species as well (Blotevogel et al., 1986; Zhilina, 1986). However, recently it was reclassified as a member of the new genus Methanothermococcus, which consists of thermophilic, H2/CO2 en formate utilizing species. Hydrogenotrophic methanogenesis may occur at temperatures as high as 97 ºC (Stetter et al., 1990), although most thermophilic species grow at moderately high temperatures.

1.2.4 Thermophilic homoacetogens growing on C1-compounds Homoacetogenic bacteria - often called ‘acetogenic’ bacteria - are probably the most versatile anaerobes with respect to their substrate profile. Several homoacetogens grow with methyl compounds such as methanol, methoxylated compounds or methylchloride. Well-studied mesophilic methylotrophic utilizing homoacetogens are Sporomusa sp., Eubacterium limosum, and Acetobacterium woodii. The best-characterized thermophilic methanol degrading homoacetogens are the heat-resistant spore producing species Moorella thermoautotrophica and Moorella thermoacetica (Table 1.6, p.22). They are physiologically and phylogenetically closely related to Clostridium species, some of which can also convert methanol to acetate. For homoacetogenic bacteria, the conversion of methanol to acetate is energetically more favorable than its decomposition to CO2 and H2 (Table 1.1). However, in the presence of hydrogen consuming SRB or MA, hydrogen might be produced. For methylotrophic mesophilic acetogens, at low hydrogen partial pressure, syntrophic associations with hydrogenotrophic methanogens have been observed (Cord-Ruwisch & Garcia, 1985; Heijthuijsen & Hansen, 1986). In a thermophilic sulfidogenic enrichment culture, a syntrophic interaction between a methanol degrading homoacetogen and a sulfate reducer was observed (Davidova & Stams, 1996). This culture was partly characterized and the homoacetogen degraded methanol, but was unable to use hydrogen and formate as growth substrate (Table, 1.6, strain AG).

19 Table 1.5. Selected physiological characteristics of moderately thermophilic MA

Organism Origin Growth T-opt (°C) pH opt. Specific Reference Substrates T-range (°C) feature Methanobacterium Mud from cattle H2/CO2 65 7.0-7.5 forms Blotevogel & Fischer, 1985 thermoaggregans pasture 40-75 aggregates

Methanogenium thermophilum CR1 River sediment H2/CO2, formate marine strain Rivard & Smith, 1982 thermophilum LA Kelp digester H2/CO2, formate Zabel et al., 1984 Methanohalobium Saline Lagoon, metoh, 50 7.0-7.5 extreme Zhilina & Zavarzin, 1987 evestigatum Crim methylamines halophilic Methanosalsum Bosa Lake metoh, 45 9.2 moderate Mathrani et al., 1988; Boone et zhilinae (formerly methylamines halophilic al., 1993 Methanohalophilus zhilinae)

Methanosarcina thermophila TM-1 Anaerobic digester metoh, ac, 50 6-7 Zinder & Mah, 1979; Zinder et methylamines, al., 1985 H2/CO2 sp. CHTI55 Anaerobic digester metoh 55 6-8 Zinder et al., 1984a; Touzel et al., 1985 MP Anaerobic digester metoh 55 6.5-7 Ollivier et al., 1984 30-60 MSTA-1 Anaerobic digester metoh, ac, 55 7 Clarens & Moletta, 1990 methylamines 30-60 CALS-1 Anaerobic digester metoh, ac 55-58 6.5 Zinder et al., 1984a 30-60 Methanothrix thermoacetophila Anaerobic digester ac 65 nr Nozhevnikova & Chudina, <70 1984 thermophila (formerly strain Anaerobic ac 60 6.5 Zinder et al., 1987; Kamagata CALS-1, and Methanosaeta sp. bioreactor 37-70 & Mikami, 1991; Kamagata et T strain P ) al., 1992 Table continued

Organism Origin Growth T-opt (°C) pH opt. Specific Reference Substrates T-range (°C) feature Methanothermobacter thermoautotrophicus (formerly Anaerobic sewage H2/CO2 65-70 7.2-7.6 Zeikus & Wolfe, 1972; Touzel Methanobacterium thermoauto- sludge digester 40-75 et al., 1992; Zhilina & Ilarianov, T trophicum ∆H and Methano- 1984 bacterium thermoformicicum sp.)

defluvii (formerly Methanobacterium Anaerobic digester H2/CO2 60 7.0 halophilic Kotelnikova et al., 1993 defluvii)

marburgensis (formerly Mesophilic sewage H2/CO2 65 6.8-7.4 Schönheit et al., 1980; Methanobacterium thermo- sludge 45-70 Wasserfallen et al., 2000 T autotrophicum strain Marburg )

thermoflexus (formerly Digester sludge H2/CO2 55 7.9-8.2 halophilic Kotelnikova et al., 1993; Methanobacterium thermoflexum) Wasserfallen et al., 2000

thermophilus (formerly Thermophilic H2/CO2 57 7.5 requires Laurinavichius et al., 1988; Methanobacterium thermophilum) methane tank 45-75 6.5-8.5 coenzym M Wasserfallen et al., 2000

wolfeii (formerly Methanobacterium Sewage H2/CO2, formate 55-65 7.0-7.5 Winter et al., 1984; wolfeii and Methanobacterium sludge/river 37-74 Wasserfallen et al., 2000; thermoformicicum sp.) sediment Zhao et al., 1986

Methanothermococcus okinawensis Deep sea H2/CO2, formate 60-65 6-7 Takai et al., 2002 hydrothermal vent + CO2 40-75 4.5-8.5 thermolithotrophicus Hot oil field H2/CO2, formate 60 5.1-5.9 Nilsen et al., 1996; Belyaev et ST22 (formerly Methanococcus reservoir 17-62 al., 1991; Whitman, 2001 thermolitotrophicus) Table 1.6. Selected physiological characteristics of thermophilic methanol- and H2/CO2- utilizing homoacetogens

Organism Origin Growth Substrates T-opt (°C) Specific feature Reference T-range (°C) Clostridium Horse manure, human metoh, formate 60-64 McBee, 1954 thermocellum feces, soil, marine mud nr Moorella thermoacetica metoh, H2/CO2, 55-60 Kerby & Zeikus, 1983;Wiegel & formate, CO nr Garrison, 1985 thermoautotrophica Hot spring metoh, H2/CO2, 56-60 production of heat Ljungdahl, 1986;Wiegel et al., formate, CO 36-70 resistant spores 1981 Strain AG Granular sludge metoh 70 Davidova & Stams, 1996 55-75 Thermoacetogenium Anaerobic metoh, H2/CO2, 58 reduction of sulfate, Hattori et al., 2000 phaeum methanogenic reactor formate, ac thiosulfate with ac syntrophic growth on acetate Thermoanaerobacter Freshwater lake H2/CO2, CO, 66 Leigh, et al., 1981; kivui (formerly sediment formate 50-73 Collins et al., 1994 Acetogenium kivui) nr. : not reported, metoh: methanol, ac: acetate Introduction

1.3 Biochemistry of methanol oxidation In this section the possible enzymes involved in thermophilic anaerobic methanol conversion are presented. An overview is given of the enzymes possibly involved in methanol degradation in methylotrophic bacteria (sub-section 1.3.1). Enzymes found in methanogenic archaea and homoacetogens (sub-section 1.3.2), and SRB (sub-section 1.3.3) are discussed.

1.3.1 Enzymes involved in primary alcohol degradation Enzymes involved in primary alcohol degradation under anaerobic conditions can be divided into three different groups depending on the cofactor or coenzyme used:

I NAD(P)-independent alcohol dehydrogenases Gram-negative bacteria employ NAD(P)-independent alcohol dehydrogenases (ADHs) (Anthony, 1982). These enzymes use pyrroloquinoline quinone (PQQ), haem and/or F420 instead of NAD(P) as cofactor. PQQ is a typical cofactor for the periplasmic methanol dehydrogenase (MDH) in methylotrophic gram-negative bacteria (Duine & Frank, 1980). In the methylotrophic homoacetogen Moorella thermoautotrophica, for example, a PQQ containing methanol dehydrogenase is present mediating the oxidation of methanol via formaldehyde to formate (Winters & Ljungdahl, 1989).

II NAD(P)- dependent alcohol dehydrogenases Three families of NADP dependent ADHs have been described (Table 1.7). Members of Family I show no (or low) activities with methanol; Family II members involved in methanol oxidation have not been described up to now, but several Familiy III enzymes from aerobic methylotrophic gram-positive bactera are active with methanol (for a review see, for instance, Hektor et al., 2000). ADHs have been identified in a few gram-positive bacteria but, in contrast with their gram- negative opposites, studies on this subject are limited. Most ADHs have been characterized from some Clostridium species. C. acetobutylicum and C. beijerinckii both contain several NADPH- dependent ADHs but none of these were active with methanol. The only ADHs found in thermophilic species (i.e. in Thermoanerobacter brockii, Thermoanaerobacter ethanolicus, and Clostridium thermocellum) are involved in the production of ethanol from carbohydrates. However, these NADP-linked ADHs exhibit no activity with methanol (Ben-Bassat & Zeikus, 1981).

Table 1.7. Classification of NAD(P)-dependent ADHs Family Metal Subunit Quaternary Activity Specific features dependence size structure with methanol I zinc 43 kDa di-or Not or very Long-chain alcohol tetrameric low dehydrogenases

II none nr nr nr Short primary structure; Short-chain alcohol broad substrate specifity dehydrogenases

III Iron, Various di-, tetra- or yes Ubiquitous in gram-, gram+, Iron-dependent magnesium, 50-66 decameric anaerobic, aerobic bacteria, alcohol zinc kDa yeasts, and amoeba dehydrogenases nr: not reported

III Methyltransferases In the degradation of methanol, corrinoid proteins (corrinoids are cobalt-containing co- factors) play a role in methyl transfer processes. Corrinoid-dependent methyltransferases are

23 Chapter 1 mainly found in homoacetogens and methanogenic archaea, and they catalyze the initial step in methanol conversion.

1.3.2 Methanol metabolism in homoacetogens and methanogenic archaea The metabolic pathways involved in the conversion of methanol to methane and acetate by mesophilic methanogenic archaea and acetogens respectively have been partially resolved in the recent years. In the elucidation of these pathways, several enzymes, coenzymes, and cofactors (most of them containing transition metals) have been discovered. The initial step in methanol conversion in mesophilic methanogens and homoacetogens is catalyzed by a corrinoid-dependent methyltransferase (Van der Meijden et al., 1984a; Van der Meijden et al., 1984b; Stüpperich & Konle, 1993; Daas et al., 1996).

Methanol reduction to methane The results of investigations into the biochemistry of methanogenesis have been extensively reviewed during recent decades (Thauer, 1990; Ferry, 1992; Blaut, 1994; Deppenmeier et al., 1996). In methanogenesis, three basic metabolic pathways can be distinguished: the pathway from H2/CO2, from methanol, and from acetate to methane. Among methanogenic archaea, Methanosarcina barkeri is the best-studied organism regarding the biochemistry of methylated compounds. The reactions involved in the degradation of methanol to methane in this organism proceed via methyl-coenzym M. Besides, two methyltransferases are involved: MT1, of which the corrinoid is methylated by methanol, and MT2, which transfers the methylgroup to coenzym M. A detailed reaction mechanism is shown in Fig. 1.5.

Methanol oxidation to CO2 Part of the methanol is oxidized to CO2 to generate the reducing equivalents needed for the reduction of the remainder of the methanol to methane. Part of the oxidation route of methanol remains to be established and a number of possible alternatives have been postulated (see Fig. 1.5): (1) is the alternative oxidation of methanol by a methanol dehydrogenase and an unknown one-carbon carrier (Blaut & Gottschalk, 1984). Other alternatives are the MT1-dependent activation of methanol (3), or CH3-S-CoM synthesis (2), followed by the methylgroup transfer to tetrahydrosarcinapterin (H4SPT), or a direct methylation of H4SPT by methanol catalyzed by a novel methyltransferase (4) (Keltjens & Vogels, 1993).

Methanol conversion to acetate In AB, several enzymes are involved in acetate synthesis, such as formate dehydrogenase, a corrinoid protein, and carbon monoxide dehydrogenase. The formation of acetate from methanol is only possible if other carbon containing compounds more oxidized than methanol - such as formate, CO, or CO2 - are present (Zeikus et al., 1984). In Eubacterium limosum, the first step of methanol conversion is catalyzed by a methyltransferase containing corrinoid (Van der Meijden et al., 1984a). Tetrahydrofolate served as methylacceptor in this reaction, which is also the case for a number of other AB (Jansen, 2000). The synthesis of acetate has been most thoroughly studied in Clostridium thermoaceticum (Ragsdale, 1991). This organism is the only thermophilic methanol utilizing acetogen for which methyltransferase activities were described (Kasmi et al., 1994). A detailed reaction mechanism for the conversion of methanol to acetate is shown in Fig. 1.6 (p. 26).

24 Introduction

Figure 1.5. Scheme of methanogenesis and alternative pathways for the methanol methylgroup oxidation. Abbreviations: H4SPT, 5,6,7,8-tetrahydrosarcinapterin; F420, a 8-hydroxy-5- deazaflavin derivative; X, unknown one carbon carrier suggested by Blaut and Gottschalk (1984); MT1, methanol:5-hydroxybenzimidazolylcobamide methyltransferase; [Co(III)], [Co(II)], and [Co(I)] represent the various oxidation states of the cobalt of the corrinoid prosthetic groups of MT1; MT2, Co-methyl-5-hydroxybenzimidazolylcobamide:coenzyme M methyltransferase; HS-CoM, coenzyme M; HS-HTP, 7-mercaptoheptanoylthreonine phosphate. Numbers refer to reactions mentioned in the text (from Daas, 1996).

25 Chapter 1

Figure 1.6. Pathway of acetate synthesis from methanol in homoacetogens. Abbreviations: THF, tetrahydrofolate; CoA, coenzyme A; CH3-E[Co], methyl corrinoid (bound). Numbers indicate the following enzymes: 1, methyltransferase; 2, methylene-THF reductase; 3, methylene- THF-dehydrogenase; 4, methenyl-THF-cyclohydrolase; 5, formyl-THF synthetase; 6, formate dehydrogenase; 7a, carbon monoxide dehydrogenase; 7b, acetyl-CoA synthase (7a en 7b are probably one enzyme); 8, phosphotransacetylase and acetate kinase (from Heijthuijsen & Hansen, 1986)

26 Introduction

1.3.3 Methanol metabolism in sulfate reducing bacteria Methanol utilization by SRB is reported for a limited number of strains, i.e. Desulfovibrio carbinolicus (Nanninga & Gottschal, 1987), Desulfovibrio alcoholovorans (Qatibi et al., 1991), Desulfobacterium anilini (Schnell et al., 1989), Desulfobacterium catecholicum (Szewzyk & Pfennig, 1987), Desulfosporosinus orientis (Klemps et al., 1985, Strain UFZ B378 (Hard & Babel, 1995), and several thermophilic Desulfotomaculum strains (Table 1.4). Although the mechanism of methanol oxidation in acetogens and methanogenic archaea is well studied, this mechanism is unresolved for SRB. Ethanol is a common electron donor and carbon source for SRB and all methanol utilizing thermophilic Desulfotomaculum strains can also use ethanol. Alcohol dehydrogenase activities have been reported for some Desulfovibrio strains (Kremer et al., 1988) and the first NAD-linked alcohol dehydrogenase was purified from Desulfovibrio gigas (Hensgens et al., 1993). This alcohol dehydrogenase is an oxygen-labile, decameric enzyme with barely any activity towards methanol (2%). The thermotolerant, gram-positive Bacillus methanolicus C1 contains a methanol dehydrogenase with a similar decameric structure as the ADH from D. gigas. This enzyme performs a a key role in the oxidation of methanol in B. methanolicus (Vonck et al., 1991). For Desulfotomaculum strains, no reports have been published regarding the biochemistry of alcohol metabolism. A possible route involving an ADH for the degradation of methanol coupled to sulfate reduction is shown in Fig. 1.7. The involvement of methyltransferases in methanol conversion by thermophilic SRB remains unclear.

Figure 1.7. Possible pathway for methanol metabolism coupled to sulfate reduction. Numbers indicate the following enzymes: 1. methanoldehydrogenase; 2, formaldehydehydrogenase; 3, formate-dehydrogenase. APS, adenosine-5’-phosphosulfate.

27 Chapter 1

1.4 Methanol conversion by mixed communities This section deals with general aspects of competition between different trophic groups involved in anaerobic methanol degradation. In sub-section 1.4.1 substrate competition is described, followed by sub-sections on direct competition for methanol (sub-section 1.4.2) and indirect competition for acetate, hydrogen, and formate (sub-section 1.4.3). Syntrophic interactions between SRB, MA, and AB are the subject of sub-section 1.4.4.

1 4.1 Substrate competition A simple method of predicting the outcome of competition between bacterial species for a common substrate is to calculate the Gibbs free energy change of the conversion of the substrate. Organisms performing the conversion with the highest Gibbs free energy change presumably outcompete other bacteria. Based on this assumption, SRB should outcompete MA for substrates such as methanol, acetate, hydrogen and formate Table 1.2. However, this does not always correspond with findings from literature. For instance, Gupta (1994) found that methanol was solely used by methanogens in mesophilic chemostats.

Growth kinetics The rate at which bacteria grow can be described by the classical Monod equation: S

µ = µmax*  S + Ks in which: µ : specific growth rate S: substrate concentration

µmax: maximum specific growth rate

KS: affinity constant for substrate.

For sulfate-reducing bacteria, the Monod-equation can be extended to:

2- S SO4 µ = µmax* *  2- 2- S + Ks SO4 + KSO4

2- in which: SO4 : sulfate concentration 2- KSO4 : affinity constant for sulfate

According to Monod growth kinetics, growth only stops when all substrate is depleted. However, many bacteria, including MA and SRB, stop growing below a certain substrate ‘threshold’ concentration (Lovley, 1985; Conrad & Wetter, 1990). In addition, sulfate reducers may encounter a threshold concentration for sulfate as well (Sonne-Hansen et al., 1999). The Monod equation can be adapted to account for threshold concentrations (Pavlostathis & Giraldo-Gomez, 1991):

S - St µ = µmax*  (S – St ) + Ks in which: St = substrate threshold concentration.

28 Introduction

For SRB, the equation becomes:

2- 2- S- St SO4 - SO4 t µ = µmax* -*  2- 2- 2- (S - St + Ks) (SO4 - SO4 t)+ KSO4

2- in which: SO4 t: sulfate threshold concentration

The kinetic parameters of the Monod equation are conditional constants: they depend on environmental conditions such as pH and temperature. Growth kinetics may be used to explain the outcome of competition between microbial species in high-rate anaerobic reactors. For instance, Methanothrix species will dominate in thermophilic anaerobic sludge cultivated at low acetate concentrations because of their higher acetate affinity as compared to that of Methanosarcina (Zinder et al., 1984b). However, it should be kept in mind that most reported values for kinetic growth properties were determined at optimal growth conditions in pure culture, and such optimal and well-defined conditions obviously do not prevail in bioreactors. The ratio µmax/KS is a useful parameter for comparing growth properties of bacteria on a common substrate. At substrate concentrations around or below the KS, bacteria with a high µmax/KS-ratio have better growth properties than bacteria with a low µmax/KS-ratio. At low sulfate concentrations, growth of the sulfate reducing bacteria will be sulfate-limited. Under such conditions, sulfate reducers have to compete with other sulfate reducers for the available sulfate. The affinity for sulfate will play a role in this competition; however, no affinity constants for sulfate for thermophilic sulfate reducers are known. Competition between mesophilic MA and SRB has been studied quite extensively. Reviews on this subject have been presented elsewhere (Hulshoff-Pol et al., 1998; Colleran et al., 1995; Oude Elferink et al., 1994). Competition between methanogens and sulfate reducers in high-rate anaerobic reactors is not merely determined by growth kinetics, but also by immobilization properties of the various microorganisms, substrate diffusion limitations inside biofilms, environmental conditions such as hydrogen sulfide concentration, and the composition of the medium, temperature and pH. In addition, the bacterial composition of the seed sludge and the applied hydraulic retention time (Alphenaar et al., 1993) may also be important.

1.4.2 Direct competition for methanol

Table 1.8. Selected growth properties of thermophilic SRB and AB on methanol

Methanol degrading culture µmax yield Reference -1 (h ) (g dry wt/mol acetate) Sulfate-reducing a Desulfotomaculum kuznetsovii 0.03 nr Chapter 2 Coculture acetogen AG and sulfate Davidova & Stams, 0.011 nr reducer SR 1996 Homoacetogenic Davidova & Stams, Strain AG 0.07 nr 1996 Savage & Drake, Moorella thermoautotrophica 0.077 6-9 1986 a) nr: not reported

Common substrates for which MA and SRB may compete in the anaerobic degradation of methanol are methanol and methanol degradation products such as hydrogen, formate, and acetate. AB may also compete with the MA and SRB for methanol. Growth kinetic data for thermophilic methanol-degrading SRB and AB are summarized Table 1.8. No data are available for

29 Chapter 1

MA. The limited amount of data does not allow a conclusion to be drawn on the outcome of the competition for methanol.

1.4.3 Indirect competition The kinetics of acetate and hydrogen degradation by mesophilic MA and SRB has been studied extensively (Colleran et al., 1995; Oude Elferink et al., 1994). Some relevant information about the growth kinetics of hydrogen and acetate utilizing thermophilic MA is also available ( shown in Tables 1.9 and 1.10), but so far this is hardly the case for thermophilic SRB. Unfortunately, to date not for a single thermophilic sulfate reducer both the Ks- and µmax- are known. In general it can be stated that the Ks-values for hydrogen are about 40 times lower for SRB than for MA, while the values for µmax of MA are maximally about 10 times higher compared to those of SRB. Therefore, it looks reasonable to expect a higher µmax/KS-ratio for hydrogen for thermophilic SRB than for MA. Consequently, SRB will outcompete MA at low hydrogen concentrations. At a high hydrogen concentration, the situation is reversed due to the high maximum specific growth rates of MA. For acetate, the situation is much less clear, as no KS values of thermophilic SRB have been reported to date. Because no growth kinetic data are available for growth on formate of thermophilic SRB and MA, a comparison of growth properties of these groups of bacteria is not yet possible.

Table 1.9. Selected growth kinetic properties of thermophilic MA and SRB on acetate.

a Acetate degrading culture µmax KS Treshold Yield µmax/KS Reference -1 –1 -1 (h ) (mM) (mM) (h .mM ) Methanogenic b Methanosarcina thermophila TM-1 0.058 4.8 1 nr 0.012 Zinder & Mah, 1979 Min & Zinder, Methanosarcina CALS-1 0.058 nr 0.8-2.5 nr nr 1989;Zinder et al., 1984b Methanosarcina MP nr nr nr nr nr Ollivier et al., 1984 Clarens & Moletta, Methanosarcina MSTA-1 0.052 11.4 4.1 3.1-4.6 0.0046 1990 Methanosarcina CHTI 55 0.085 10 nr 1.4 0.0085 Touzel et al., 1985 Nozhevnikova & Methanothrix thermoacetophila nr nr nr nr nr Chudina, 1984 Kamagata & Methanosaeta sp. PT 0.020 nr nr nr nr Mikami, 1991 0.012- Methanothrix sp. CALS-1 0.028 <1.1 nr >0.025 Zinder et al., 1987 0.021 0.025- Ahring & TAM 0.012 0.85 nr 0.014 0.075 Westermann, 1985

Sulfate-reducing Desulfotomaculum 0.022 nr nr nr nr Min & Zinder, 1990 thermoacetoxidans a) yield expressed in g dry cells/mol acetate; b) nr: not reported.

30 Introduction

Table 1.10. Selected growth properties of thermophilic MA, SRB and AB on hydrogen.

a Hydrogen degrading culture µmax KS threshold yield µmax/KS Reference –1 –1 -1 (h ) (µM) (Pa) (h .mM ) Methanogenic Schönheit et al., 1980; Taylor & Pirt, Methanothermobacter 0.14- 80- 0.6-1.6 0.0018- 5 1977; Zeikus & Wolfe, 0.69 120 b 0.004 thermoautotrophicus 3 1972; Schmidt & Ahring, 1993 c Methanobacterium Strain THF nr nr 14 nr nr Lovley, 1985 Sulfate-reducing Desulfotomaculum 0.077 nr nr nr nr Min & Zinder, 1990 thermoacetoxidans Schmidt & Ahring, Desulfotomaculum spp. nr 2 0.01 nr nr 1993 Davidova & Stams, Strain SR 0.052 nr nr nr nr 1996

Thermodesulfobacterium d Sonne-Hansen et al., nr 2.4 1.2 nr nr Strain JSP 1999

Thermodesulfovibrio d Sonne-Hansen et al., nr 1.9 0.5 nr nr Strain R1Ha3 1999 Homoacetogenic Savage & Drake, Moorella thermoautotrophica 0.021 nr nr nr nr 1986 Leigh, et al., 1981; Acetogenium kivui 0.35 nr 1000 nr nr Conrad & Wetter, 1990 a) yield expressed in g dry cells/mol end product; b) under hydrogen limitation; c) nr: not reported; d) Km.

1.4.4 Syntrophic methanol conversion Interspecies hydrogen transfer in cocultures with methanol as a substrate has been observed between methanol-utilizing homoacetogens and SRB or MA (Heijthuijsen & Hansen, 1986), and between methanol-utilizing methanogens and SRB (Phelps et al., 1985). These observations of hydrogen transfer with methanol as a substrate were both done under mesophilic conditions. Moderately thermophilic SRB, MA and AC might be involved in hydrogen transfer reactions, but methanol was never used as a substrate. Examples of cocultures in which hydrogen transfer is observed are: (1) a combined culture of Dsm. nigrificans and Methanothermobacter thermoautotrophicus which grew and produced methane from lactate or ethanol (Klemps et al., 1985); and (2) Dsm. thermobenzoicum subsp. thermosyntrophicum, which was able to grow in coculture with Methanothermobacter thermoautotrophicus Z245 converting a variety of substrates, including propionate, and lactate. However, neither methanol nor ethanol were used by this coculture. The only thermophilic sulfate-reducing consortium growing at 65 oC in which hydrogen transfer with methanol was observed is described by Davidova and Stams (1996). The nature of the hydrogenotrophic sulfate reducer in this culture is still unclear.

1.5 Factors affecting the fate of methanol in biological desulfurization In cultures growing at 65 oC with methanol, sulfite, and sulfate, inhibition may result from high concentrations of substrates or possible intermediates and products such as acetate and sulfide. A different susceptibility of SRB and MA towards these compounds may act as a selection criterion in bioreactors. Also the pH, temperature NaCl concentration, and the presence of trace elements may affect the competition. All these factors are discussed below.

31 Chapter 1

1.5.1 Sulfur compounds Sulfide toxicity. Sulfate reduction results in the production of hydrogen sulfide (H2S) which, at high concentrations, can become quite inhibitory for microbial growth. H2S is a very weak acid (pKa of 7.0 at 30°C) and therefore at neutral values (the optimal pH range for most anaerobic - microorganisms) sulfide is mainly present as H2S (hydrogen sulfide or free sulfide) and HS (bisulfide). The sulfide ion (S2-) only occurs (>1% of total sulfide) as important sulfide species at - pH > 10, because the pKa of HS is about 12 (Sillén & Martell, 1964). Hydrogen sulfide is considered to be the most toxic form of sulfide (Reis et al., 1992b). The neutrality of the H2S- molecule allows its easy diffusion through the lipid cell membrane into cytoplasm, where it reacts with cell components. The reversibility of sulfide inhibition, as observed by Okabe et al. (1992) and Reis et al. (1992a), seems contradictory as it may be expected that chemical reactions with cell components are irreversible. As sulfide is a characteristic end product of SRB, it may be speculated that SRB have developed a high tolerance towards sulfide in order to prevent self- poisoning. However, this is not necessarily the case; hydrogen sulfide concentrations as low as 60 mgS.L-1 are already inhibitory for a thermophilic Desulfotomaculum species (Min & Zinder, 1990). Moreover, bacteria not capable of dissimilatory sulfate reduction (such as methanogens) may have a higher tolerance to H2S than SRB (McCartney & Oleszkiewicz, 1991; Uberoi & Bhattacharya, 1995). The presence of sulfide may affect SRB in several ways. For the mesophilic Desulfovibrio desulfuricans it was demonstrated that 250 mgS.L-1 of total sulfide lowers the growth rate and growth yield by 50% (Okabe et al., 1995). By contrast, the substrate utilization rate increased at higher sulfide concentration, showing that growth and activity were uncoupled. Uncoupling of growth and activity at higher sulfide concentrations was also observed for anaerobic sulfate reducing and methanogenic sludge granules (Visser, 1995). By increasing the total sulfide concentration, the cell size may decrease, as was shown for Desulfovibrio desulfuricans (Okabe et al., 1992). This may partly explain the decreased cell yield at increasing sulfide concentrations. Literature regarding H2S inhibition levels at mesophilic conditions has been reviewed elsewhere (Hao et al., 1996; Colleran et al., 1995; Oude Elferink et al., 1994). The free H2S levels which are inhibitory for mesophilic methanogenesis vary from 50-400 mg.L-1. Complete inhibition of -1 growth of mesophilic SRB has been observed at a H2S concentration of 85 mg.L (Widdel, 1988) to 547 mg.L-1 (Reis et al., 1992a). No data are available on sulfide inhibition of methanogens under thermophilic conditions. Complete inhibition of growth of thermophilic SRB may occur at total sulfide levels as low as 60 mgS.L-1 (Min & Zinder, 1990) or as high as 400 mgS.L-1 (Nazina et al., 1987). The variation in published data regarding H2S-toxicity reflects the influence of several factors, such as the type of bacterial species studied, growth substrate (Maillacheruvu et al., 1993) and time of exposure to sulfide. For undefined cultures, the discrepancies may also be a result of interference with competitive and mutualistic microbial interactions between individual species. Another cause of the discrepancies in literature may originate from neglecting pH and sulfide concentration gradients in biofilms (Lens et al., 1993). The lack of uniformity in methods for quantifying sulfide inhibition, the many factors that affect sulfide inhibition and the possible interference with bacterial interactions and diffusion hardly justify comparison of literature data. Published data cannot lead to a reliable prediction as to whether SRB or MA will be more affected by sulfide in a specific situation.

Sulfate and sulfite toxicity. Sulfate is generally not toxic for anaerobic bacteria at concentrations up to 10 g.L-1 (Minami et al., 1988; Karhadkar et al., 1987). For most wastewaters, as well as for the scrubbing solution from a Bio-FGD plant, sulfate toxicity is not relevant, as the concentration generally remains below this value. On the other hand, sulfite is very toxic for microorganisms and for that reason it is used as anti-bacterial agent, for example in wine processing. The mechanism of sulfite inhibition is not known precisely (Chang et al., 1997). In pure cultures of SRB, complete inhibition of growth at concentrations as low as 40 mg.L-1) 0.5 mM sulfite was observed (Widdel & Bak, 1992). On the other hand, the sulfate reducer Archaeaoglobus veneficus can tolerate sulfite concentrations of 1.5 g.L-1 (Huber et al., 1997).

32 Introduction

Methane production by Methanobacterium ruminantium decreased by a factor of 2 at 100 mg.L-1 sulfite (Prins et al., 1972). Sulfite may have various effects on the activity of methanogenic sludge. Puhakka et al. (1985) found that sulfite toxicity leads to a prolonged lag phase in methane production by anaerobic sludge in batch reactors at concentrations exceeding 250 mg.L-1. In addition, the rate of methane production decreased linearly to very low values in the range of 150 to 2500 mg.L-1 sulfite. However, after repeated sulfite addition to sludge, the toxicity effect may decrease due to the growth of SRB or due to adaptation of the biomass.

1.5.2 Methanol and acetate toxicity. Methanol. Alcohols are toxic for microorganisms at high concentrations, presumably due to the fact that they damage the cell membrane and due to end product inhibition of glycolytic enzymes (Dürre et al., 1988). Most bacteria are able to withstand ethanol concentrations of at least 10 g.L-1. As alcohol toxicity towards bacteria decreases with decreasing chain length, it may be speculated that methanol toxicity will not occur at concentrations below 10 g.L-1 (0.3 M). This was confirmed for Moorella thermoautotrophica and Moorella thermoacetica as these species tolerate methanol concentrations up to 16 g.L-1 (0.5 M) (Wiegel & Garrison, 1985; Wiegel, 1986). With 10 g.L-1 methanol, 22 g.L-1 sulfate can be reduced to sulfide. As sulfate concentrations normally will be less than 6 g.L-1 in biodesulfurization of flue-gases, added methanol concentrations will normally not exceed 3 g.L-1, which probably does not result in toxicity effects.

Acetate. Methanol degradation by AB may result in the accumulation of acetate, which is toxic for microorganisms at higher concentrations. As with sulfide, unionized acetate (acetic acid) is considered the most toxic form (Morvai et al., 1992. Lier et al., 1995) found 50% inhibition of methane formation by thermophilic sludge occurred at an acetic acid concentration of about 1 mM, while they observed a 10 times lower susceptibility of mesophilic methanogenic sludge towards acetic acid. For thermophilic methylotrophic Methanosarcina spp., complete inhibition of growth was found at 9 mM acetic acid (Yamaguchi et al., 1989). Inhibition by acetic acid may manifest in weakly buffered bioreactors producing acetate. At pH 6 and a temperature of 55°C, a concentration of 1 mM of undissociated acetate already is present at a total acetate concentration of 17 mM. This can be calculated using a pKa for acetic acid of 4.8 at 55°C (Yamaguchi et al., 1989).

1.5.3 Environmental conditions pH. SRB and MA may have different pH-optima or pH ranges for growth on common substrates. + As the speciation of compounds such as acetate, H2S and NH4 is affected by the pH, the effect of a pH change on the growth of SRB and MA may partially also result from a change in the concentration of these compounds (Visser et al., 1992) found for anaerobic sludge that thermophilic (55°C) SRB outcompete methanogens for acetate at pH 8.3-8.6, while the rates of methanogenesis and sulfate reduction at pH 7.6-7-9 were about equal. Minami et al. (1988) suggested that pH may have a large effect on the occurrence of methanogenesis or sulfate reduction from methanol. They found that sulfate reduction prevailed at pH 7.0-7.5 in a moderate thermophilic (53°C) methanol-fed bioreactor. At pH values between 6.2 and 6.8, sulfate reduction was suppressed and methanogenesis prevailed. However, inhibition of SRB in the lower pH range may also have resulted from elevated H2S concentrations.

Salt requirement and salt tolerance. Sulfate reducers from marine environments often require salt for growth and may be damaged in fresh water. Where marine species are moderate halophiles requiring 1-3% NaCl for optimum growth (Cord-Ruwisch et al., 1985), fresh water species may be inhibited at these concentrations. Most SRB are rather adaptable with respect to salt concentrations, and grow without NaCl as well as with 5-6% NaCl (Rees et al., 1995). The highest tolerance and optimum (10%) NaCl concentration among SRB is reported for the mesophilic Desulfohalobium retbaense (Ollivier et al., 1991). SRB are known to outcompete MA in marine environments under sulfate-limiting conditions; H2 and acetate are used mainly for sulfate reduction. However,

33 Chapter 1 methanogenesis is not totally absent in these environments and it is thought to occur from noncompetitive substrates as methylamines (Oremland & Polcin, 1982). On the other hand, halophilic hydrogenotrophic MA have been isolated and the highest tolerated salt concentration reported so far for MA using H2 or formate is 8.3 % (Huber et al., 1982). From marine environments, halophilic moderately thermophilic methylotrophic MA, i.e. Methanosalsum zhiliniae and Methanohalobium evestigatum have been isolated (Table 1.5.). While methanogenic activity from H2 is low or not expressed at salt concentrations above 15%, (Ollivier et al., 1994), Mhb. evestigatum grows optimally at a salt concentration of 24%. Mesophilic homoacetogenic halophiles seem to have a similar upper growth limit as methylotrophic MA, e.g. 25% (Ollivier et al., 1994).

Temperature. Differences in optimal growth temperatures and growth temperature ranges may cause shifts in the microbial composition of mixed cultures as a result of a temperature change. A shift from a methanogenic to a sulfate-reducing population or vice versa also alters the anaerobic mineralization profile, as exemplified by a study conducted by Visser et al. (1993). They found a rapid shift from methanogenesis to sulfate reduction after elevating the temperature of an acetate and sulfate fed UASB reactor from 30 to 55°C. A temperature increase from 37 to 55°C had the same effect (Rintala & Lettinga, 1992). No acetoclastic methanogens have been isolated growing beyond a temperature of 70°C (Zinder, 1990). Therefore, it may be speculated that acetoclastic methanogenesis does not occur in reactors beyond this temperature. As acetotrophic sulfate reduction is still possible up to at least 85°C (Table 1.3), the electron flow in acetate-rich environments may therefore be diverted from methane to sulfide as a result of a temperature increase from below 70°C to 70-85°C. The situation is similar for methanol: no methylotrophic methanogens are known that grow at temperatures above 60°C (Table 1.4), while the methanol- utilizing sulfate reducer Desulfotomaculum kuznetsovii was reported to grow up to 85°C (Nazina et al., 1987). Temperature may also affect SRB and MA indirectly, as temperature decreases the concentration of inhibitory H2S due to a lower pKa of H2S at increasing temperature.

Trace elements. As essential constituents of cell components - in particular proteins - trace elements need to be available to microorganisms in order to facilitate growth. Bacteria compete for trace elements when these are limiting, and it may be expected that species with a low (or no) requirement or a high affinity for limiting trace elements will eventually dominate. Iron, cobalt, nickel, zinc and copper were identified as trace elements that are necessary to maintain maximum growth of MA (Speece, 1996). Growth of mesophilic methylotrophic MA and AB was found to be optimal at an added cobalt concentration of 0.1 mg.L-1 (Florencio et al., 1994). In this case, the cobalt requirement may be explained by the high content of corrinoids of methanol-grown MA and AB (Krzycki & Zeikus, 1980; Stüpperich et al., 1988; Inoue et al., 1992). It is not known if corrinoids are involved in methanol degradation by SRB. As opposed to MA, little is known about the trace element requirements of SRB. Under sulfate-reducing conditions, it may be speculated that trace metals (such as zinc and cobalt) are growth-limiting as the concentration of these metals may be extremely low due to the precipitation of insoluble metal sulfides. However, Parkin et al. (1990) found that the concentration of trace metals in microbial cultures was independent of the sulfide concentration. This independence was explained by the microbial production of chelating agents.

1.6 Outline of this thesis The aim of the research presented in this thesis was to study the microbiology of thermophilic sulfate reduction with methanol as the electron donor in order to apply a thermophilic desulfurization process of flue gas. The thermophilic anaerobic degradation of methanol can proceed either via sulfidogenesis in the presence of sulfate, or via acetogenesis. Moreover, a combination of both processes via intermediates such as hydrogen is possible, and MA might be involved in the hydrogen transfer process. The outcome of the competition for the substrate methanol and possible intermediates is difficult to predict based on current literature and was therefore one of the main questions to be

34 Introduction answered in the present study. The first part of this thesis (Chapter 2) deals with the different aspects of competition, inhibition and syntrophy that can occur in methanol fed thermophilic sulfidogenic bioreactors. In continuous culture experiments defined mixtures of organisms were studied with respect to methanol degradation. The biochemistry of methanol utilization in the thermophilic sulfate-reducer Desulfotomaculum kuznetsovii is described in Chapter 3, while Chapter 4 describes the extremely heat resistant spore formation by this strain. This extremely heat resistance is unprecedented. Next to sulfate reduction, sulfite reduction will be an important biological process in the desulfurization of off-gasses. To investigate whether we could isolate new thermophilic sulfite-reducing strains, we collected samples from solfataric fields in Iceland. The isolation and characterization of a new species from these inocula, Desulfotomaculum solfataricum is described in Chapter 5. The results presented in this thesis are summarized and discussed in Chapter 6. Different aspects of the thermophilic sulfate- and sulfite reduction processes with methanol in Expanded Sludge Bed Reactors have been studied by Jan Weijma in a counterpart thesis project and have been published previously (Weijma, 2000).

References

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35 Chapter 1

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