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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol. 14 no. 4 São Paulo Dec. 1997 



L. Florencio1,*, J. A. Field2 and G. Lettinga2

1Department of Civil Engineering - Federal University of Pernambuco
Av. Acadêmico Hélio Ramos, s/n, 50570-530 Recife PE, Brazil - Phone: 081 271-8228/8220
Fax: 081 271-8219/8205 - e-mail: FLORENCIO@NPD.UFPE.BR
2Dept. of Environmental Technology - Wageningen Agricultural University
Bomenweg 2, 6702 HD Wageningen, The Netherlands


(Received: June 11, 1997; Accepted: October 30, 1997)


Abstract - Modern high-rate anaerobic wastewater treatment processes are rapidly becoming popular for industrial wastewater treatment. However, until recently stable process conditions could not be guaranteed for alcoholic wastewaters containing higher concentrations of methanol. Although methanol can be directly converted into methane by methanogens, under specific conditions it can also be converted into acetate and butyrate by acetogens. The accumulation of volatile fatty acids can lead to reactor instability in a weakly buffered reactor. Since this process was insufficiently understood, the application of high-rate anaerobic reactors was highly questionable. This research investigated the environmental factors that are of importance in the predominance of methylotrophic methanogens over acetogens in a natural mixed culture during anaerobic wastewater treatment in upflow anaerobic sludge bed reactors. Technological and microbiological aspects were investigated. Additionally, the route by which methanol is converted into methane is also presented.
Acetogenesis, anaerobic degradation, cobalt, inorganic carbon, methanogenesis, methanol, substrate competition, UASB.




Modern high-rate anaerobic wastewater treatment (AWWT) processes are rapidly becoming popular for industrial wastewater treatment. The practical feasibility of AWWT has already been demonstrated for a large variety of industrial effluents, including those in food industries and paper mills and also increasingly those in chemical industries (Lettinga and Hulshoff Pol, 1991). Brewery and distillery wastewaters are examples of well-known and successfully treated wastewaters, where ethanol and other long chain alcohols represent an important fraction of the organic pollution. However, until recently stable process conditions could not be guaranteed for anaerobic reactors treating alcoholic wastewaters containing higher concentrations of methanol, such as kraft evaporator condensates in pulping mills (Minami et al., 1991).

Methanol, the simplest alcohol, contains only one carbon. Under anaerobic conditions, methanol potentially supports a complex food chain composed of a variety of trophic groups (Figure 1 and Table 1). Methylotrophic methanogens can directly convert methanol into methane (Jarrel and Kalmokoff, 1988). Methylotrophic acetogens produce acetate and butyrate but this conversion is limited by the availability of inorganic carbon (Ljungdahl, 1986). While the conversion of methanol to H2/CO2 is usually thermodynamically unfavourable, acetogens are also able to generate H2/CO2 from methanol in a syntrophic partnership with hydrogen consumers, e.g. sulfate-reducing bacteria (Cord-Ruwisch and Ollivier, 1986; DiStefano et al., 1992; Heijthuijsen and Hansen, 1986) and tetrachloroethene-reducing bacteria (Freedman and Hashsham, 1993). Thus, methanol can indirectly support hydrogenotrophic and acetoclastic methanogens via acetate or H2/CO2 generation by acetogenic bacteria.

The chemical oxygen demand (COD) removal efficiency and stability of anaerobic reactors treating methanolic wastewaters depend on which route methanol degradation follows. Methane is the target end-product responsible for an effective COD removal, whereas with the production of volatile fatty acids (VFA) little COD removal is achieved. Thus, it is necessary that the acetogenesis is prevented or that occasional VFA accumulation is converted into methane as well. Moreover, VFA accumulation may lead to reactor instability in a weakly buffered system.

Since the route of methanol degradation in anaerobic environments was not sufficiently understood, this research investigated the environmental factors that are of importance in the predominance of methylotrophic methanogens over acetogens in a natural mixed culture during anaerobic wastewater treatment in bioreactors. Technological and microbiological aspects were investigated. Additionally, the route by which methanol is converted into methane is also presented.



Batch and continuous (upflow anaerobic sludge bed reactor - UASB) experiments were carried out using a model medium composed of methanol and defined mineral nutrients in a temperature-controlled room at 30 ± 2 °C. The pH was determined potentiometrically. Methanol, VFA and biogas composition were determined by gas chromatography. Experimental details were described elsewhere (Florencio, 1994).



Four important factors were found to contribute to the final fate of methanol in anaerobic bioreactors: the presence of cobalt in the media, the reactor’s methanol concentration, the level of bicarbonate, and high concentrations of undissociated volatile fatty acids.

The Effect of Cobalt

The effect of trace elements on the methanogenesis of methanol and acetate was studied (Table 2). Cobalt was the only trace element tested which greatly enhanced methanogenesis from methanol. In contrast, no significant influence of any trace element was observed when acetate was used as the substrate. In a continuous experiment, less acetate was formed in a cobalt-deprived reactor than in a cobalt-supplemented reactor (Figure 2). These results suggested that methanogens are better scavengers for cobalt than acetogens and that cobalt levels could be used to prevent acetate formation from methanol. It was hypothesized that the cobalt concentration could be used as a parameter for controlling substrate flow during anaerobic treatment of methanolic wastewaters.

The effect of cobalt addition on each individual trophic group potentially involved in the anaerobic conversion of methanol was evaluated (Table 3). For this purpose, a sludge was cultivated with methanol in a mineral medium deprived of cobalt for one year. Activity assays with specific inhibitors indicated that methane was being formed directly from methanol and not via the intermediate formation of acetate or H2/CO2. The addition of cobalt stimulated only those trophic groups which directly utilized methanol, while the other trophic groups utilizing downstream intermediates, H2/CO2 or acetate, were largely unaffected. Consequently, the influence of increasing cobalt concentrations on the growth rate and specific activity was determined for methylotrophic methanogens and acetogens. At low cobalt concentrations, both trophic groups had similar activities and growth rates, whereas at the optimal cobalt concentration, acetogens had slightly higher values for a specific activity and growth. Both trophic groups had similar cobalt optima for growth and activity, with values around 0.05 mg /l.

This higher cobalt requirement by methylotrophic microorganisms, compared to hydrogenotrophic and acetoclastic methanogens, has several technological and microbiological implications. From a technological standpoint, cobalt is an important nutrient that should be considered during the anaerobic treatment of wastewaters with methylotrophic substrates. From a microbiological standpoint, this high requirement for cobalt is presumably due to the production of corrinoids where cobalt is the central ion (Stupperich et al., 1990). Methylotrophic methanogens and acetogens are reported to possess a much higher corrinoid (i.e., vitamin B12) content than the same or other microorganisms consuming other substrates (Krzycki and Zeikus, 1980; Zeikus et al., 1980). This is probably due to the involvement of unique corrinoid-containing methyltransferases in the initial step of methanol conversion in both methanogens and acetogens (van der Meijden, van der Drift and Vogels., 1984; van der Meijden et al., 1984). The metabolism of methanol induces the production of these corrinoid-containing enzymes, such as in the acetogenic bacterium Sporomusa ovata grown on methanol (Stupperich et al., 1992; Stupperich and Konle, 1993). In the mixed culture enriched experiment, cobalt addition resulted in much higher growth rates and specific activities for the methylotrophic microorganisms. Presumably, cobalt enhances these rates by stimulating the production of corrinoids. It has been reported that the addition of vitamin B12 greatly enhanced the biotransformation of tetrachloromethane under anaerobic conditions (Freedman and Hashsham, 1993). Therefore, cobalt addition can potentially be used in practice to speed up both methanol and halomethane biodegradation in anaerobic bioreactors.

Figure 1: Possible pathways of methane formation from methanol.


Figure 2: VFA, Methanol-COD influent, and Methanol-COD effluent during the anaerobic treatment of methanol in UASB reactors. A: without cobalt addition; B: with cobalt addition.



Table 1: Reported reactions involved in the anaerobic degradation of methanol

1. 4 CH3OH   ® 3 CH4 + HCO3- + H+ + H2O
2. CH3OH + H2 ® CH4 + H2O
3. 4 CH3OH + 2 HCO3- ® 3 CH3COO- + H+ + 4 H2O
4. CH3OH + 2 H2O ® 3 H2 + HCO3- + H+
5. 2 HCO3- + 4 H2 + H+ ® CH3COO- + 4 H2O
6. HCO3- + 4 H2 + H+ ® CH4 + 3 H2O
7. CH3COO- + 4 H2O ® 2 HCO3- + 4 H2 + H+
8. CH3COO- + H2O ® CH4 + HCO3-

a from Ljungdahl, 1986; Thauer et al., 1977; Wood et al., 1986; and Zinder and Koch, 1984.


Table 2: Influence of trace elements on methanogenesis of methanol and acetate. Activity is expressed as a percentage of a control where all trace elements were present

Trace element acetatea Substrate Methanola methanolb

a only one trace element is present; b only one trace element is absent


Table 3: Influence of cobalt addition on the activities and growth rate of methanogens and acetogens

Pathwaysa competitive pathway inhibitorb Activities (mgCOD.gVSS-1.d-1) Growth rate (d-1)
-Coc +Cod -Coc +Cod
1 3,4 vancomycin 316 2488 0.293 0.923
2 3,4 (5,6)e vancomycin 377 2434 nd nd
3 1,2,4f BESA 5 15 0.280 1.147
4 1,2,3g BESA nd nd nd nd
5 6 BESA 10 12 2.240 2.440
6 5 vancomycin 32 29 1.000 0.790
7 8 BESA nd nd nd nd
8 9 vancomycin 12 12 0.254 0.209

a see Figure 1; b to block the competitive pathways; c without cobalt; d with cobalt; e pathway 6 not stopped; f BESA stops pathway 4 indirectly because of its effect on methanogens (destroys H2 sink); g pathway 3 not stopped. VSS: volatile suspended solids.
BESA: 2-bromoethenesulfonic acid.


The Effect of Methanol Concentration

Since cobalt alone could not explain the predominance of methanogens in our mixed culture, other factors were investigated. The concentration of the available substrate in the bioreactor is another important factor that was considered to possibly influence the competition between methanogens and acetogens. Therefore, substrate affinity coefficients were determined for both of these trophic groups. The apparent substrate affinity coefficients were 0.25 and 16 mM for methylotrophic methanogens and acetogens, respectively. Substrate affinity together with growth rate are of importance in the evaluation of substrate competition. Methanogens were found to have a 60-fold-higher affinity for methanol than acetogens. As long as the reactor methanol concentration is lower than about 80 mM, methanogens will have a faster growth rate than acetogens and, as such, will be expected to outcompete. This prediction was confirmed in continuous experiments where the formation of VFA was directly related to periods when high levels of methanol (> 1000 mg COD/l) occurred in the bioreactors during occasional organic overloadings (Figure 3). At low cobalt concentrations (< 0.0001 mg Co/l), however, both populations had similar growth rates, indicating that methanogens will easily predominate over acetogens in a much wider methanol concentration range. Therefore, the role of cobalt deprivation is to enhance the competitive edge of methanogens over acetogens.

The Effect of Exogenous Addition of Bicarbonate and Undissociated Volatile Fatty Acids

Bicarbonate plays an important role in the anaerobic conversion of methanol, not only as a weak acid in the pH buffering system but also as a required cosubstrate in the acetogenic breakdown of methanol. Bicarbonate is produced when methanol is converted into methane (Table 1). According to stoichiometry, up to one third of the methanol can potentially be consumed by acetogens from the endogenous methanogenic supplied bicarbonate. For the complete conversion of methanol to acetate, exogenous bicarbonate must be added. For this conversion, the total bicarbonate requirement (endogenous plus exogenous) is 0.64 g HCO3- / g COD.

Figure 4 illustrates the percentage of COD-methanol conversion into methane, acetate, and cells when different amounts of exogenous bicarbonate were applied. Without exogenous addition of bicarbonate, methanogens are the predominant trophic group because the meagre endogenous resources of bicarbonate generated by methanogenesis cannot support significant acetogenesis. When exogenous bicarbonate is applied, more favourable conditions are created for the development of acetogens. If insufficient buffering capacity is present, the eventual production of CO2 and VFA decreases the pH. Low pH itself is not toxic to methylotrophic methanogens (Florencio et al., 1993). However, the fraction of undissociated VFA increases when the pH decreases due to VFA production by acetogens. Undissociated VFA is toxic to methanogens (Andrews, 1969; Duarte and Anderson, 1982). When the concentration of undissociated VFA remains high for prolonged periods, methanogens are slowly wiped out and acetogens predominate in the biorectors. If sufficient buffering capacity is present, the eventual production of VFA during occasional overloadings will not decrease the pH and, consequently, the undissociated VFA fraction will be too small to significantly disturb the methanogens.



To date, the anaerobic treatment of methanolic wastewaters has been considered troublesome due to the undesirable accumulation of VFA. Four factors are of importance in the predominance of acetogens or methanogens: the methanol concentration inside the reactor, the cobalt concentration level, the presence of exogenous inorganic carbon and a high concentration of undissociated VFA.

Figure 5 presents a simple conceptual model based on the findings of this study. Methanogens will predominate if either the reactor methanol concentration, the inorganic carbon content or the cobalt concentration is low. Moreover, methanol is converted directly to methane by methylotrophic methanogens and not via the intermediate formation of VFA. On the other hand, significant acetogenesis can only be expected to predominate if the reactor methanol concentration is high, exogenous inorganic carbon is supplied, cobalt is available and methanogens are inhibited, e.g., by undissociated VFA. All four conditions have to be met.

Depending on the target end product, different measures have to be taken for the predominance of methanogens or acetogens. For methane production, some measures are presented below for the complete predominance of methanogens. During the start-up period, low levels of cobalt should be applied. Additionally, the methanol concentration in the reactor should be kept low by underloading the reactor. Once methanogenesis has predominated, a higher organic load rate can be applied by stimulating the methanogens with cobalt supplementation. In order to prevent the accumulation of undissociated VFA, high levels of alkalinity should also be applied. Much care should be taken when NaOH is used to increase the pH. In the absence of a weak acid necessary for the creation of buffering capacity, the addition of NaOH can break the delicate pH balance and accidentally cause severe alkaline pH values. NaHCO3 is preferred since it supplies alkalinity together with a weak acid. A cheap source of NaHCO3 can be obtained by scrubbing the biogas with NaOH. Alternatively, methane production can also be achieved without any increase in alkalinity at low pH values. However, this latter possibility has yet to be improved before practical application.

For the predominance of acetogenesis, cobalt concentration should be applied at optimal conditions, the reactor methanol concentration should be maintained high by organic overloading and an excess of exogenous bicarbonate must be supplied. Additionally, methanogens have to be inhibited by ensuring that undissociated VFA levels are high. Addition of moderate levels of NaHCO3 (approximately 10-20 meq/l) was found to create such conditions if the reactor was overloaded. Since dichloromethane and trichloromethane are specific methanogenic inhibitors, acetogens might be expected to predominate in halomethane contaminated sites subject to anaerobic bioremediation. Sludge pasteurization can also be considered, since many methylotrophic acetogens, such as Sporomusa sp. (Moller et al., 1984), Clostridium CV-AAI (Adamse and Velzeboer, 1982), and Butyribacterium methylotrophicum (Zeikus et al., 1980), are spore formers.


Figure 3: The effect of the bicarbonate level on the efficiencies of methanol conversion in three UASB reactors (Organic loading rate = 20.0 g COD/l.d). A: applied NaHCO3 = none, pH inside reator = 4.2; B: applied NaHCO3 = 15 meq/l, pH inside reactor = 5.5; C: applied NaHCO3 = 50 meq/l, pH inside reactor = 7.2.


Figure 4: Influence of methanol concentration on the formation of VFA.


Figure 5: The final fate of methanol in anaerobic bioareactors.


Broader Applications

The results obtained in this study on the anaerobic degradation of methylotrophic substrates have a broad range of applications. Some examples of applications in environmental biotechnology include: COD removal from methanolic wastewaters, the biodegradation of methoxylated aromatics and halomethanes, and the use of methanol as a cheap cosubstrate. Furthermore, the results of this work provide clues to natural processes occurring in nature such as the formation of biogas in acid peats. Methylotrophic methanogens are active over a broad pH range. Thus, biogas production in acid peats may very well be due to the conversion of methylotrophic substrates derived from methyl ethers/esters in natural plant phenolics, hemicellulose, and pectin.



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