Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Microbial Ecophysiology of Whey Biomethanation: Comparison of Carbon Transformation Parameters, Species Composition, and Starter Culture Performance in Continuous Culture

Changes in lactose concentration and feed rate altered bacterial growth and population levels in a whey-processing chemostat. The bacterial population and methane production levels increased in relation to increased lactose concentrations comparable to those in raw whey (6%) and converted over 96% of the substrate to methane, carbon dioxide, and cells. Sequential increases in the chemostat dilution rate demonstrated excellent biomethanation performance at retention times as low as 25 h. Retention times shorter than 25 h caused prevalent bacterial populations and methane production to decrease, and intermediary carbon metabolites accumulated in the following order: acetate, butyrate, propionate, lactate, ethanol, and lactose. Bacterial species dominated in the chemostat as a function of their enhanced substrate uptake and growth kinetic properties. The substrate uptake kinetic properties displayed by the mixed chemostat population were equivalent to those of individual species measured in pure culture, whereas the growth kinetic properties of species in mixed culture were better than those measured in pure culture. A designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum displayed biomethanation performance, which was similar to that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Preserved starter cultures were developed and used as inocula for the start-up of a continuous anaerobic digestion process that was effective for biomethanation of raw whey at a retention time of 100 h.     

Gas Metabolism Evidence in Support of the Juxtaposition of Hydrogen-Producing and Methanogenic Bacteria in Sewage Sludge and Lake Sediments

We developed new techniques to measure dissolved H₂ and H₂ consumption kinetics in anoxic ecosystems that were not dependent on headspace measurements or gas transfer-limited experimentation. These H₂ metabolism parameters were then compared with measured methane production rates, and estimates of H₂ production and interspecies H₂ transfer were made. The H₂ pool sizes were 205 and 31 nM in sewage sludge from an anaerobic digestor and in sediments (24 m) from Lake Mendota, respectively. The H₂ turnover rate constants, as determined by using in situ pool sizes and temperatures, were 103 and 31 h⁻¹ for sludge and sediment, respectively. The observed H₂ turnover rate accounted for only 5 to 6% of the expected H₂-CO₂-dependent methanogenesis in these ecosystems. Our results are in general agreement with the results reported previously and are used to support the conclusion that most of the H₂-dependent methanogenesis in these ecosystems occurs as a consequence of direct interspecies H₂ transfer between juxtapositioned microbial associations within flocs or consortia.     

Growth of Desulfovibrio in Lactate or Ethanol Media Low in Sulfate in Association with H2-Utilizing Methanogenic Bacteria

In the analysis of an ethanol-CO₂ enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H₂-utilizing methanogen resembling Methanobacterium formicicum were isolated, and they were shown to represent a synergistic association of two bacterial species similar to that previously found between S organism and Methanobacterium strain MOH isolated from Methanobacillus omelianskii. In lowsulfate media, the desulfovibrio produced acetate and H₂ from ethanol and acetate, H₂, and, presumably, CO₂ from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H₂-utilizing methanogenic bacterium. The type strains of D. vulgaris and Desulfovibrio desulfuricans carried out the same type of synergistic growth with methanogens. In mixtures of desulfovibrio and strain MOH growing on ethanol, lactate, or pyruvate, diminution of methane produced was stoichiometric with the moles of sulfate added, and the desulfovibrios grew better with sulfate addition. The energetics of the synergistic associations and of the competition between the methanogenic system and sulfate-reducing system as sinks for electrons generated in the oxidation of organic materials such as ethanol, lactate, and acetate are discussed. It is suggested that lack of availability of H₂ for growth of methanogens is a major factor in suppression of methanogenesis by sulfate in natural ecosystems. The results with these known mixtures of bacteria suggest that hydrogenase-forming, sulfate-reducing bacteria could be active in some methanogenic ecosystems that are low in sulfate.     

Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer

A mixed culture from an anaerobic biowaste digester was enriched on propionate and used to investigate interspecies hydrogen transfer in dependence of spatial distances between propionate degraders and methanogens. From 20.3 mM propionate, 20.8 mM acetate and 15.5 mM methane were formed. Maximum specific propionate oxidation and methane formation rates were 49 and 23 mmol?mg^?1?day^?1, respectively. Propionate oxidation was inhibited by only 20 mM acetate by about 50 %. Intermediate formate formation during inhibited methanogensis was observed. The spatial distribution and the biovolume fraction of propionate degraders and of methanogens in relation to the total population during aggregate formation were determined. Measurements of interbacterial distances were conducted with fluorescence in situ hybridization by application of group-specific 16S rRNA-targeted probes and 3D image analyses. With increasing incubation time, floc formation and growth up to 54 μm were observed. Propionate degraders and methanogens were distributed randomly in the flocs. The methanogenic biovolume fraction was high at the beginning and remained constant over 42 days, whereas the fraction of propionate degraders increased with time during propionate feeding. Interbacterial distances between propionate degraders and methanogens decreased with time from 5.30 to 0.29 μm, causing an increase of the maximum possible hydrogen flux from 1.1 to 10.3 nmol?ml^?1?min^?1. The maximum possible hydrogen flux was always higher than the hydrogen formation and consumption rate, indicating that reducing the interspecies distance by aggregation is advantageous in complex ecosystems.     

Control of interspecies electron transfer flow during anaerobic digestion: dynamic diffusion reaction models for hydrogen gas transfer in microbial flocs

Dynamic reaction diffusion models were used to analyze the consequences of aggregation for syntrophic reactions in methanogenic ecosystems. Flocs from a whey digestor were used to measure all model parameters under the in situ conditions of a particular defined biological system. Fermentation simulations without adjustable parameters could precisely predict the kinetics of H(2) gas production of digestor flocs during syntrophic methanogenesis from ethanol. The results demonstrated a kinetic compartmentalization of H(2) metabolism inside the flocs. The interspecies electron transfer reaction was mildly diffusion controlled. The H(2) gas profiles across the flocs showed high H (2) concentrations inside the flocs at any time. Simulations of the syntrophic metabolism at low substrate concentrations such as in digestors or sediments showed that it is impossible to achieve high H(2) gas turnovers at simultaneously low steady-state H(2) concentrations. This showed a mechanistic contradiction in the concept of postulated low H(2) microenvironments for the anaerobic digestion process. The results of the computer experiments support the conclusion that syntrophic H(2) production may only be a side reaction of H(2) independent interspecies electron transfer in methanogenic ecosystems.     

Methanobacillus omelianskii,a symbiotic association of two species of bacteria

Summary Two bacterial species were isolated from cultures of Methanobacillus omelianskii grown on media, containing ethanol as oxidizable substrate. One of these, the S organism, is a gram negative, motile, anaerobic rod which ferments ethanol with production of H₂ and acetate but is inhibited by inclusion of 0.5 atm of H₂ in the gas phase of the medium. The other organism is a gram variable, nonmotile, anaerobic rod which utilizes H₂ but not ethanol for growth and methane formation. The results indicate that M. omelianskii maintained in ethanol media is actually a symbiotic association of the two species.Prof. C. B. van Niel has been interested in the biological formation of methane for many years. According to Barker (1936) he was the first to suggest the carbon dioxide reduction theory of methane formation which has subsequently been shown to be generally applicable to biological methane formation from organic compounds other than acetate and methanol (Barker, 1956). Much of the knowledge presently available on methanogenic bacteria has been accumulated by his students or Scientific grandchildren. It is a great pleasure for the authors to contribute in his honor an account of the association of two bacterial species in the formation of methane from ethanol and CO₂.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

Propionate-Degrading Bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from Methanogenic Ecosystems

A new genus and species of a nonmotile gram-negative rod, Syntrophobacter wolinii, is the first bacterium described which degrades propionate only in coculture with an H₂-using organism and in the absence of light or exogenous electron acceptors such as O₂, sulfate, or nitrate. It was isolated from methanogenic enrichments from an anaerobic municipal sewage digestor, using anaerobic roll tubes containing a medium with propionate as the energy source in association with an H₂-using, sulfate-reducing Desulfovibrio sp. which cannot utilize fatty acids other than formate. S. wolinii produced acetate and, presumably, CO₂ and H₂ (or formate) from propionate. In media without sulfate and with Methanospirillum hungatei, a methanogen that uses only H₂-CO₂ or formate as an energy source, acetate, methane, and, presumably, CO₂ were produced from propionate and only small amounts of Desulfovibrio sp. were present. Isolation in coculture with the methanogen was not successful. S. wolinii does not use other saturated fatty acids as energy sources.     

The influence of sulphate on substrate utilization in a thermophilic sewage sludge digestor

Summary Bacterial sulphate reduction and the interaction between sulphate reduction and methane production was studied in an unadapted and sulphate-adapted thermophilic anaerobic sludge digestor. Addition of sulphate to a concentration of 5 mm (100 times the background level) did not influence gas production or volatile fatty acid concentration compared to the control digestor. When sulphate reduction was not limited by the sulphate concentration, the sulphate-adapted digestor had a sulphate reduction rate of 910 mol l^–1 day compared with 17 mol l^–1 day in the control digestor. The results indicate that the potential for sulphate reduction is low in a thermophilic sewage sludge digestor receiving a low sulphate concentration. Counts of sulphate-reducing bacteria and methanogens showed that sulphate-reducing bacteria were found only in significant numbers in the sulphate-adapted digestor and only with H₂/CO₂ as substrate. Only low numbers of acetate-utilizing sulphate-reducing bacteria were found in both digestors. When using radio-labelled acetate, the relative percentage of 2-labelled acetate converted to CO₂ was two to four times higher in the sulphate-adapted digestor compared to the control digestor. These results suggest that oxidation of acetate seems to play a larger role in the sulphate-adapted digestor.Offprint requests to: B. K. Ahring     

Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H₂-CO₂, formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO₂, 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH₄ was derived from CO₂. Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO₂ transformed to methane but did not alter the amount of ¹⁴CO₂ produced from [2-¹⁴C]acetate (~24%). Analysis of both carbon transformation parameters with ¹⁴C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

Syntrophic Association by Cocultures of the Methanol- and CO2-H2-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H₂-CO₂, and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO₂, and H₂ from pectin. The coculture produced one-third more acetate, and butyrate and CO₂ were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

Terminal Reactions in the Anaerobic Digestion of Animal Waste

An anaerobic mesophilic digestor was operated using beef cattle waste (diluted to 5.75% volatile solids) as substrate; retention time was 10 days with daily batch feed. Volatile solids destruction was 36%. Daily gas production rate was 1.8 liters of gas (standard temperature and pressure) per liter of digestor contents (0.99 liters of CH₄ per liter of digestor contents). Acetate turnover was measured, and it was calculated that 68% of the CH₄ was derived from the methyl group of acetate. When the methanogenic substrates acetic acid or H₂/CO₂ were added to the digestor on a continuous basis, the microflora were able to adapt and convert them to terminal products while continuing to degrade animal waste to the same extent as without additions. The methanogenic substrates were added at a rate at least 1.5 times the microbial production rate which was measured in the absence of added substrates. Added acetate was converted directly to CH₄ by acetoclastic methanogens; H₂ addition greatly stimulated acetate production in the digestor. A method is described for the measurement of acetate turnover in batch-fed digestors.     

Mixed-Culture Fermentor for Simulating Methanogenic Digestors

Propionate degradation in an anaerobic digestor degrading animal waste (10-day retention time, 5.75 g liter⁻¹ day⁻¹ volatile solids loading rate, 40°C) was 0.304 mM h⁻¹, measured with [2-¹⁴C]propionate; this value indicated that CH₄ produced from propionate accounted for 14.8% of the CH₄ produced in the digestor (34.5%, including acetate produced from propionate). The mean propionate concentration was 0.67 mM, giving a propionate turnover rate of 0.46 h⁻¹. A continuous-, mixed-culture fermentor was developed to mimic the digestor. When degradation rates of methanogenic precursors (H₂, CO₂, and acetate) equalled those measured in the digestor, propionate degradation was inhibited. When the H₂ turnover rate was lowered by decreasing addition of H₂-generating substrates or by allowing a portion of the H₂ degradation to occur in an isolated compartment, propionate degradation in the fermentor resumed. The possibility is discussed that in digestors, much of the H₂ is produced and degraded within microenvironments associated with particles. Thus, the gross turnover rate of H₂ measured in digestors is an average, and specific microenvironments within the digestor may have different rates of turnover.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Metabolic activity of subterranean microbial communities in deep granitic groundwater supplemented with methane and H2

It was previously concluded that opposing gradients of sulphate and methane, observations of 16S ribosomal DNA sequences displaying great similarity to those of anaerobic methane-oxidizing Archaea and a peak in sulphide concentration in groundwater from a depth of 250–350 m in Olkiluoto, Finland, indicated proper conditions for methane oxidation with sulphate. In the present research, pressure-resistant, gas-tight circulating systems were constructed to enable the investigation of attached and unattached anaerobic microbial populations from a depth of 327 m in Olkiluoto under in situ pressure (2.4 MPa), diversity, dissolved gas and chemistry conditions. Three parallel flow cell cabinets were configured to allow observation of the influence on microbial metabolic activity of 11 mℳ methane, 11 mℳ methane plus 10 mℳ H₂ or 2.1 mℳ O₂ plus 7.9 mℳ N₂ (that is, air). The concentrations of these gases and of organic acids and carbon, sulphur chemistry, pH and E_h, ATP, numbers of cultivable micro-organisms, and total numbers of cells and bacteriophages were subsequently recorded under batch conditions for 105 days. The system containing H₂ and methane displayed microbial reduction of 0.7 mℳ sulphate to sulphide, whereas the system containing only methane resulted in 0.2 mℳ reduced sulphate. The system containing added air became inhibited and displayed no signs of microbial activity. Added H₂ and methane induced increasing numbers of lysogenic bacteriophages per cell. It appears likely that a microbial anaerobic methane-oxidizing process coupled to acetate formation and sulphate reduction may be ongoing in aquifers at a depth of 250–350 m in Olkiluoto.     

Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation

Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chlamydomonas MGA 161, was unusual in having high H₂ but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH₄Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations; starch degradation, and formation of ethanol, H₂, and CO₂ increased with the addition of NH₄Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO₂ as its main products, with H₂, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H₂ evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.