FEMS Microbiology Reviews 21 (1997) 179^211

Fermentation in cyanobacteria 1
Lucas J. Stal a *, Roy Moezelaar b

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

;

a
Netherlands Institute of Ecology, Centre for Estuarine and Coastal Ecology, P.O. Box 140, NL-4400 AC Yerseke, The Netherlands
b
Agrotechnological Research Institute (ATO-DLO), P.O. Box 17, NL-6700 AA Wageningen, The Netherlands
Received 24 April 1997; revised 1 August 1997; accepted 2 August 1997

Abstract
Although cyanobacteria are oxygenic phototrophic organisms, they often thrive in environments that become periodically
anoxic. This is particularly the case in the dark when photosynthetic oxygen evolution does not take place. Whereas
cyanobacteria generally utilize endogenous storage carbohydrate by aerobic respiration, they must use alternative ways for
energy generation under dark anoxic conditions. This aspect of metabolism of cyanobacteria has received little attention but
nevertheless in recent years a steadily increasing number of publications have reported the capacity of fermentation in
cyanobacteria. This review summarizes these reports and gives a critical consideration of the energetics of dark fermentation in
a number of species. There are a variety of different fermentation pathways in cyanobacteria. These include homo- and
heterolactic acid fermentation, mixed acid fermentation and homoacetate fermentation. Products of fermentation include CO2 ,
H2 , formate, acetate, lactate and ethanol. In all species investigated, fermentation is constitutive. All enzymes of the
fermentative pathways are present in photoautotrophically grown cells. Many cyanobacteria are also capable of using
elemental sulfur as electron acceptor. In most cases it seems unlikely that sulfur respiration occurs. The main advantage of
sulfur reduction seems to be the higher yield of ATP which can be achieved during fermentation. Besides oxygen and elemental
sulfur no other electron acceptors for chemotrophic metabolism are known so far in cyanobacteria. Calculations show that the
yield of ATP during fermentation, although it is low relative to aerobic respiration, exceeds the amount that is likely to be
required for maintenance, which appears to be very low in these cyanobacteria. The possibility of a limited amount of
biosynthesis during anaerobic dark metabolism is discussed.
Keywords: Fermentation; Cyanobacteria; Dark metabolism; Embden-Meyerhof-Parnas pathway; Lactate dehydrogenase; Lactate fermen-
tation; Mixed acid fermentation; Sulfur reduction

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
2. Occurrence of dark anoxic conditions in cyanobacterial communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2.1. Anoxic hypolimnia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2.2. Microbial mats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

* Corresponding author. Tel.: +31 (113) 577497; Fax: +31 (113) 573616; e-mail: [email protected]
1
Publication 2274 of the Centre of Estuarine and Coastal Ecology, Yerseke, The Netherlands.

0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 5 6 - 9
180 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

2.3. Lake sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

2.4. Surface waterblooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.5. Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
3. Fermentation in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
3.1. Substrates for fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
3.2. Fermentation products and the diversity of fermentation pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.3. The enzymes involved in fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.4. The Embden-Meyerhof-Parnas pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.5. The capability of fermentation is constitutive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
4. Lactate dehydrogenase and lactate production in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
5. Hydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
6. Electron acceptors and anaerobic respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

7. Energetics of fermentation in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
7.1. Maintenance requirements in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
7.2. Energetics of fermentation in Oscillatoria limnetica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
7.3. Energetics of fermentation in Oscillatoria limosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
7.4. Energetics of fermentation in Microcystis aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
7.5. Energetics of fermentation in Microcoleus chthonoplastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

1. Introduction growth serves as the energy source in the dark [1].

Glucose residues from glycogen are degraded via the
The cyanobacteria constitute one of the largest oxidative pentose phosphate pathway and metabolic
groups of prokaryotes. Encompassing a wide diver- energy is generated by respiration with oxygen as
sity in morphology, physiology, cell division pat- electron acceptor [4]. It was demonstrated that the
terns, cell di¡erentiation, and habitats, the cyanobac- planktonic cyanobacterium Oscillatoria agardhii is
teria are uni¢ed by the ability to carry out a plant- able to maintain growth in the dark at the same
like oxygenic photosynthesis using water as electron rate as in the light when cultivated under a light-
donor and the possession of chlorophyll a and phy- dark regime indicating that part of the glycogen is
cobiliproteins as photosynthetic pigments. In addi- used as carbon source for synthesis of cell constitu-
tion, all cyanobacteria are capable of using CO2 as ents [5^7].
the sole carbon source, employing the reductive pen- In addition to oxygenic photoautotrophy and dark
tose phosphate pathway or Calvin cycle [1]. Many respiration of glycogen, cyanobacteria display alter-
species can ¢x molecular nitrogen [2]. native modes of energy generation and growth. More
In nature, most cyanobacteria face a regular cycle than half of the species tested so far are facultative
of day and night. In addition, darkness may occur as photoheterotrophs [1,8]. Photoheterotrophic cyano-
a result of self-shadowing in dense planktonic and bacteria are capable of taking up a limited number
benthic communities, sedimentation in aquatic sys- of organic compounds and assimilate them but need
tems, and sediment deposition on benthic commun- light as energy source. Only a relatively small num-
ities. Certain symbiotic cyanobacteria that live in the ber of species are able to grow chemoorganotrophi-
rhizosphere of plants seem to thrive permanently in cally in the dark at the expense of a limited number
the dark [3]. In order to meet the energy demands in of organic compounds, predominantly glucose, fruc-
the dark for maintenance and the possibility of some tose, or sucrose (Table 1). In most of these cases
growth, cyanobacteria have to resort to a chemotro- chemoorganotrophic growth was observed only
phic mode of energy generation. In most species, under aerobic conditions. Anaerobic chemoorgano-
glycogen accumulated during photoautotrophic trophic growth was reported in Nostoc sp. [24] and
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 181

Table 1
a
Dark chemoorganotrophic growth of cyanobacteria

Strain Condition Substrate Doubling Ref.

Anabaena sp. aerobic sucrose [9]

Anabaena azollae AaN anaerobic glucose, fructose [10]

Anabaena variabilis aerobic fructose, glucose, sucrose, melizitose, ra¤nose 36 h [11]

Anabaenopsis circularis aerobic glucose, fructose, sucrose, maltose [12]

Aphanocapsa sp. 6702 aerobic glucose [13]

Aphanocapsa sp. 6805 aerobic glucose [13]

Calothrix brevissima aerobic sucrose [14,15]

Calothrix membranacea aerobic sucrose [14,15]

Calothrix marchica aerobic sucrose [9]

Chlorogloeopsis fritschii Chlorogloea

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

( ) aerobic sucrose, acetate, mannitol, glucose, maltose, 144 h [16^19]

glycine, glutamine

Chlorogloeopsis sp. 6912 aerobic sucrose 80 h [20]

Fremyella diplosiphon aerobic glucose [21]

Microcystis aeruginosa 7806 anaerobic glucose [22]

Nostoc commune aerobic sucrose [14,15]

Nostoc punctiforme aerobic [23]

Nostoc sp. (an)aerobic glucose, fructose, sucrose 48^103 h [24]

Nostoc MAC aerobic glucose, fructose, sucrose [25^27]

Nostoc sp. Al2 anaerobic glucose, fructose [10]

Oscillatoria agardhii aerobic endogenous glycogen [6,7]

Oscillatoria terebriformis anaerobic glucose, fructose 10 d [28]

Phormidium luridum aerobic sucrose [29]

Plectonema boryanum aerobic glucose, fructose, sucrose, ribose, maltose, 49 h^13 d [29^32]

mannitol

Plectonema calothrioides aerobic sucrose [14,15]

Scytonema schmidlei aerobic sucrose [9]

Spirulina platensis aerobic [33,34]

Synechocystis sp. 6714 aerobic glucose 50^60 h [13,18,20,27]

(Aphanocapsa sp.)

Synechocystis sp. 6803 aerobic (blue-light) [35]

Tolypothrix tenuis aerobic glucose, fructose [36]

Westelliopsis proli¢ca aerobic sucrose [9]

a
Adapted and extended from [28].

Oscillatoria terebriformis [28]. Moezelaar and Stal during the dark, anoxygenic phototrophic bacteria

[22] reported anaerobic decomposition of exogenous generally face anoxic conditions. In order to be

glucose in Microcystis aeruginosa and recently ob- able to generate energy in the dark these bacteria

tained evidence for the occurrence of some growth must be able to carry out fermentation. This has

[37]. With a few exceptions chemoorganotrophic been shown for instance in the anoxygenic non-sul-

growth of cyanobacteria on external substrates is fur purple bacterium Rhodospirillum rubrum [38,39].

much slower than under photoautotrophic condi- Other species can not grow unless an electron accept-

tions. This is probably because the uptake of the or such as dimethylsulfoxide [40] or trimethylamine-

substrate is limiting. As mentioned above, O. agard- N -oxide [41] are present. A very e¤cient mode of

hii is able to maintain its growth rate in the dark at anaerobic dark metabolism has been demonstrated

the same value as in the light, but only at the expense in the anoxygenic phototrophic bacterium Chromati-
of endogenous storage carbohydrate which will last um vinosum . This species has been shown to convert

for a limited period [6,7]. glycogen into poly- L-hydroxybutyrate, using elemen-
Whereas cyanobacteria and eukaryotic microalgae tal sulfur as electron acceptor [42]. This metabolism

normally display aerobic respiratory metabolism results only in a minor loss of storage carbon but
182 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

allows substrate level phosphorylation. In the light, 2.1. Anoxic hypolimnia

sul¢de is oxidized photosynthetically to elemental
sulfur which is stored intracellularly in these bacteria One example of an anoxic hypolimnion environ-
and may subsequently serve as electron acceptor dur- ment inhabited by cyanobacteria is Solar Lake, a
ing the dark. Theoretically this sulfur reduction hypersaline pond on the shore of the Sinai desert.
could be associated with an electron transport chain This lake displays a typical annual cycle of mixing.
and yield additional energy. It is not known whether After a short period of holomixis in summer, strat-
this organism is capable of growth anaerobically in i¢cation builds up in September and lasts until July
the dark at the expense of endogenous carbohydrate. [47]. During the period of strati¢cation, a cyanobac-
Cyanobacteria can also be found in environments terial bloom consisting of Oscillatoria sp. and Micro-
which are periodically anoxic. In the light when sul- coleus sp. develops in the anoxic sul¢de-rich hypo-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

¢de is present, several species may switch to anoxy- limnion which merges into a £occulant mat [48]. The
genic mode of photosynthesis using sul¢de as elec- dominant organism of this bloom, O. limnetica, is
tron donor [43] while in the dark fermentation of capable of anoxygenic photosynthesis, using sul¢de
endogenous glycogen storage and reduction of ele- as the electron donor, oxidizing it to elemental sulfur
mental sulfur occurs in order to sustain the energy which accumulates extracellularly [49]. In the dark,
requirements of these cyanobacteria [44]. energy is generated by anaerobic respiration of gly-
Fermentation of endogenous storage material has cogen using sulfur as electron acceptor [44]. Alterna-
also been observed in green microalgae such as tively, this organism may ferment glycogen to lac-
Chlorella fusca, Chlamydomonas reinhardii and tate.
Chlorogonium elongatum, which produce formate,
acetate and ethanol as fermentation products 2.2. Microbial mats
[45,46]. Not much information is available on the
pathways and regulation of fermentation in these Microbial mats are a typical example of an envi-
eukaryotic algae, which is in part due to the complex ronment which experiences periodically anoxic con-
interactions of di¡erent compartmentalized pathways ditions. The majority of microbial mats are com-
in these organisms. posed of cyanobacteria as the dominant group of
Dark anaerobic metabolism in cyanobacteria has microorganisms [50]. These laminated sediment eco-
received little attention. There is a steadily increasing systems are ubiquitous in a variety of di¡erent envi-
number of publications that report the capacity of ronments such as hot spring e¥uents, intertidal
fermentation in cyanobacteria and this review at- coastal sediments, and hypersaline ponds. Microbial
tempts to summarize these reports and give a critical mats are characterized by marked daily £uctuations
evaluation of fermentative energy generation. of oxygen concentration that can be attributed to the
physiology of the cyanobacteria. During the daytime
oxygenic photosynthesis by these organisms results
2. Occurrence of dark anoxic conditions in in oxygen supersaturation. In the dark cyanobacteria
cyanobacterial communities will switch to respiration, but due to the high oxygen
demand, di¡usion of oxygen into the mat is usually
Mainly because of their oxygen-evolving photo- insu¤cient to cover the demands and as a result the
synthesis, cyanobacteria are usually associated with mat will turn anoxic [51].
aerobic environments, and, consequently, research of
dark energy generation has focused on aerobic me- 2.3. Lake sediments
tabolism. However, this has not recognized the fact
that many cyanobacteria are found in environments The annual life cycle of planktonic cyanobacteria
that are permanently anoxic or become anoxic in the in lakes at temperate climate zones involves a phase
dark. The following sections give some examples of of perennation in the sediment, where the organisms
such anoxic environments in which cyanobacteria accumulate during and after bloom formation. Spe-
thrive. cies belonging to the order of the Nostocales such as
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 183

Anabaena and Aphanizomenon survive as akinetes, di¡usion of oxygen into the soil is reduced by stag-
resting stages that di¡erentiate from vegetative cells nant water.
during blooming [52]. Species of the genus Microcys-
tis, however, do not form such morphologically dis-
tinct resting stages, but survive as colonies of vege- 3. Fermentation in cyanobacteria
tative cells in the sediment. In most cases the bottom
sediments of lakes are permanently in darkness and The occurrence and survival of cyanobacteria in
anoxic. Under these conditions Microcystis is able to environments that are permanently anoxic or be-
maintain cellular integrity and retains the capacity of come anoxic at night implies the capability of anae-
photosynthesis [53,54]. Although the cells also retain robic dark energy generation. Species from such en-
their gas vacuoles, the colonies are not buoyant. The vironments have been shown to be capable of

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

population in the sediment serves as viable stock for fermentation at the expense of intracellular carbohy-
re-establishment of a planktonic population the fol- drates [59]. Table 2 gives a list of cyanobacteria that
lowing year. are capable of fermentation.

2.4. Surface waterblooms 3.1. Substrates for fermentation

The eutrophic state of many lakes and water res- Most of the studies on dark anaerobic energy gen-
ervoirs often results in the mass development of eration in cyanobacteria have only considered the
planktonic cyanobacteria, very often species belong- use of endogenous carbohydrates as substrate. O.
ing to the genera Anabaena, Aphanizomenon, Micro- limnetica is not capable of using exogenous glucose
cystis, or Nodularia. These genera are characterized as substrate for fermentation [44]. Thus far, fermen-
by a colonial organization and the possession of gas tation at the expense of exogenous substrates has
vacuoles, hollow proteinaceous vesicles that provide been described for a few species only. These include
the cells with buoyancy. Thus, when the water col- Nostoc sp. [24], O. terebriformis [28], M. aeruginosa
umn is stable, the colonies will accumulate at the [22] and a number of symbiotic species [10]. In addi-
water surface and form surface waterblooms [55]. tion to endogenous carbohydrates, the Cycad sym-
The wind blowing across the water surface may con- biont Nostoc sp. strain Cc also degrades exogenous
centrate the colonies into dense scums on the leeward glucose according to a homoacetic fermentation [62].
shore. Like microbial mats, such scums become an- The use of glucose as substrate for fermentation al-
oxic at night [56]. The attenuation of light may be so lows the organism to prolong dark anaerobic surviv-
high that even in the daytime cells in the deeper al considerably. The chemoorganotrophic capacities
layers of thick scums experience dark anoxic condi- of cyanobacteria are limited and seem to be predom-
tions. inantly restricted to species occurring symbiotically.
The concentrations of substrate necessary to support
2.5. Soil anaerobic chemoorganotrophic growth in cyanobac-
teria are high (5^30 mM) and are not likely to be
Several species of the N2 -¢xing genus Nostoc de- encountered by free-living organisms.
velop in symbiotic association with cycads, allowing The majority of the cyanobacteria is regarded as
them to use molecular nitrogen as the N source [57]. obligately photoautotrophic [1]. In the light, these
They are found in a mucilage-¢lled space in the outer species accumulate glycogen which serves as energy
cortex of the coralloid roots where they live in per- source in the dark. In addition, marine cyanobacte-
manent darkness up to 50 cm below the soil surface. ria may use their osmoprotectant as substrate during
As a consequence, photosynthesis is not possible and fermentation, as has been shown for O. limosa [63]
the cyanobacteria grow chemoorganotrophically at and Microcoleus chthonoplastes [61]. Remarkably,
the expense of an organic substrate as carbon and M. chthonoplastes, which accumulates glucosylglycer-
energy source supplied by the host [58]. In the cor- ol as osmoprotectant [66], ferments only the glucose
alloid roots anoxia may occur after heavy rains when residue, whereas the glycerol residue is excreted.
184 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

Table 2
Cyanobacteria capable of fermentation
Organism Strain, origin Fermentation pathway Productsa Ref.
Anabaena azollae AaL symbiont from Azolla caroliniana homoacetate acetate (lactate, CO2 , H2 ) [10]
Anabaena azollae AaN symbiont from Azolla caroliniana homoacetate acetate (lactate, CO2 , H2 ) [10]
Anabaena azollae AaS symbiont from Azolla ¢liculoides homoacetate acetate (lactate, CO2 , H2 ) [10]
Anabaena siamensis As1 paddy ¢eld homoacetate acetate (CO2 , H2 ) [10]
Cyanothece PCC 7822 (Inst. Pasteur) mixed acid H2 , ethanol, lactate, formate, acetate [60]
Microcoleus chthonoplastes microbial mat mixed acid H2 , ethanol, lactate, formate, acetate [61]
Microcystis aeruginosa PCC 7806 (Inst. Pasteur) mixed acid H2 , ethanol, acetate [22]
Nostoc sp. Cc symbiont from Cycas circinalis homoacetate acetate (lactate, CO2 , H2 ) [10,62]
Nostoc sp. Al2 symbiont from Anthoceros laevis homoacetate acetate (lactate, CO2 , H2 ) [10]

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Nostoc sp. Ef1 symbiont from Encephalartos ferox homoacetate acetate (lactate, CO2 , H2 ) [10]
Nostoc sp. MAC symbiont from Macrozamia lucida homoacetate acetate (lactate, CO2 , H2 ) [10]
Nostoc sp. Mm1 symbiont from Macrozamia moorei homoacetate acetate (lactate, CO2 , H2 ) [10]
Nostoc sp. M1 symbiont from Macrozamia sp. homoacetate acetate (CO2 , H2 ) [10]
Nostoc sp. Gm symbiont from Gunnera manicata homoacetate acetate (lactate) [10]
Nostoc sp. T1 paddy ¢eld homoacetate acetate (formate, CO2 , H2 ) [10]
Nostoc sp. Bali paddy ¢eld homoacetate acetate (CO2 , H2 ) [10]
Oscillatoria limnetica hypolimnion Solar Lake homolactate lactate [44]
Oscillatoria limosa microbial mat heterolactate homoacetate lactate, ethanol, acetate [63]
Oscillatoria sp. microbial mat not known lactate, ethanol, acetate, formate [64]
Oscillatoria terebriformis hot spring microbial mat homolactate? ? [28]
Spirulina platensis not known mixed acid H2 , ethanol, acetate, formate, lactate [65]
Spirulina minosa not known not known lactate, acetate [64]
a
Compounds in parentheses are produced in minor quantities.

Degradation of the osmoprotectant raises the ques- gen reserve [69]. It has been proposed that cyanobac-
tion if and how the cells will maintain the osmotic teria may degrade arginine to ornithine via the dihy-
pressure of the cytoplasm. It is conceivable that in- drolase route, which would allow the production of
organic ions such as K‡ and Cl3 may temporarily ATP by substrate-level phosphorylation, even under
serve to maintain osmotic pressure [67], and that the anaerobic conditions in the dark [1]. However, this
pool of organic osmolytes will be replenished in the has not been demonstrated and Stal et al. [70] con-
subsequent light period. cluded that this mode of energy generation did not
A few cyanobacteria are capable of accumulating occur in O. limosa.
poly-L-hydroxybutyrate (PHB) [68] but there is no
evidence that this storage compound is used in 3.2. Fermentation products and the diversity of
dark energy metabolism. Decomposition would re- fermentation pathways
quire the tricarboxylic acid (TCA) cycle which is
absent in all of the cyanobacteria investigated. Stal The ¢rst cyanobacterium reported to be capable of
[68] proposed a role as C reserve for PHB, providing fermentative energy generation was O. limnetica [44].
intermediates for biosynthesis. A role of PHB in cy- This organism carries out a homolactic fermentation,
anobacteria similar to that found in the purple sulfur and produces about 1.4^1.8 mol of lactate per mol of
bacterium Chromatium vinosum [42] was also consid- glucose degraded. Although the pathway involved
ered. However, in O. limosa PHB was not formed as was not examined it is likely that conversion of glu-
a product of fermentation even when sulfur as elec- cose to lactate, as in lactic acid bacteria, involves the
tron acceptor was present (L.J. Stal, unpublished Embden-Meyerhof-Parnas glycolytic pathway. In
results). contrast, the marine benthic cyanobacterium O. li-
Some cyanobacteria contain cyanophycin (multi-L- mosa degrades glycogen via the heterolactic fermen-
arginine poly-L-aspartic acid) which serves as a nitro- tation pathway, which shares some sequences with
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 185

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 1. Pathway of heterolactic acid fermentation in Oscillatoria limosa. The products of fermentation are shown in boxes. The numbers

refer to the enzymes involved : 1, enzymes of the oxidative pentose phosphate pathway ; 2, acetaldehyde dehydrogenase ; 3, alcohol dehy-

drogenase ; 4, enzymes of the Embden-Meyerhof-Parnas pathway ; 5, pyruvate kinase ; 6, L-lactate dehydrogenase.

the oxidative pentose phosphate pathway (Fig. 1) quite rare among chemoheterotrophic bacteria, it
[63]. The freshwater unicellular species Cyanothece has been reported to occur in several cyanobacterial
PCC7822 performs a mixed acid fermentation with species. The production of three mol of acetate from
formate as characteristic fermentation product [60]. one mol of glucose by the symbiotic, diazotrophic
Based on the ratios of glucose utilization and prod- cyanobacterium Nostoc sp. strain Cc and the absence
uct formation it was calculated that both the glyco- of other products strongly suggested a homoacetic
lytic and the oxidative pentose phosphate pathway fermentation, but no enzymatic evidence was given
were operative during fermentation (Figs. 1, 3 and for this [62]. Also in O. limosa this type of fermen-
4). However, the enzymes that were demonstrated in tation was reported to occur but curiously not with
cell-free extracts did not include the key enzymes of glycogen as the substrate [63]. These authors noticed
the glycolytic pathway (6-phosphofructokinase) and that the production of acetate did not correlate with
the phosphoketolase pathway (phosphoketolase) glycogen degradation. Moreover, the degradation of
[71]. Whereas homoacetic fermentation is already glycogen was fully accounted for by the fermentation
186 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 2. Pathway of homoacetate fermentation in Oscillatoria limosa. The products in boxes are the fermentation products that are pro-
duced. The broken lines indicate reactions of relatively minor importance. The fermentation of trehalose yields 5 acetate instead of 6. The
balance is made by H2 and CO2 . Although the ATP balance of acetate formation from CO2 is zero, the energy liberated by this pathway
must be conserved by other mechanisms. It is likely that this is achieved electrochemically, e.g. by the generation of a Na‡ gradient [72].
The numbers refer to the enzymes involved: 1, trehalase; 2, hexokinase; 3, enzymes of the Embden-Meyerhof-Parnas pathway; 4, pyruva-
te:ferredoxin oxidoreductase; 5, phosphotransacetylase; 6, acetate kinase; 7, hydrogenase; 8, formate dehydrogenase; 9, carbon monoxide
dehydrogenase. THF, tetrahydrofolic acid.

products lactate and ethanol. Instead the production occurrence of the homoacetic fermentation pathway
of acetate was found to correlate with the degrada- in O. limosa was supported by the demonstration of
tion of trehalose, which serves as osmoprotectant in the key enzymes in cell-free extracts [63,73] (i.e. for-
O. limosa. For each mol of trehalose degraded 5^6 mate dehydrogenase, carbon monoxide dehydrogen-
mol of acetate was recovered (Fig. 2). The use of ase, pyruvate:ferredoxin oxidoreductase and acetate
osmoprotectant as substrate for fermentative energy kinase). Also the presence and activity of trehalase
generation is surprising and it is unknown why this was demonstrated in cell-free extracts of O. limosa.
compound is used for this purpose and how osmotic The source of the nitrogenase-independent produc-
equilibrium of the cell cytoplasm is maintained. The tion of H2 by this organism is a reversible hydro-
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 187

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 3. Pathway of glycogen fermentation in the unicellular cyanobacterium Microcystis PCC7806. Compounds in boxes are possible fer-
mentation products. Broken line: reaction only occurs in case of over£ow metabolism but is not a regular fermentation product. The
numbers refer to the enzymes involved: 1, enzymes of the Embden-Meyerhof-Parnas pathway; 2, CoA-linked pyruvate:ferredoxin oxidore-
ductase; 3, hydrogenase; 4, CoA-linked aldehyde dehydrogenase; 5, alcohol dehydrogenase; 6, phosphotransacetylase; 7, acetate kinase ;
8, ferredoxin:NADP oxidoreductase; 9, NAD-dependent lactate dehydrogenase. This pathway has also been proposed to occur in the uni-
cellular cyanobacterium Cyanothece PCC7822.

genase [70,74]. Homoacetic fermentation in O. limosa that were grown under a light-dark regime and that
usually yielded a little less than the 6 acetate that contained relatively low amounts of glycogen more
should be expected from the degradation of treha- than four times more ethanol was produced than
lose, and the balance was made up by some CO2 and acetate. This phenomenon was attributed to the ac-
H2 . It was proposed that the source of hydrogen was tivity of ferredoxin:NADP oxidoreductase. In con-
the reduced ferredoxin produced from the decarbox- trast, cultures grown under continuous light and
ylation of pyruvate by pyruvate:ferredoxin oxidore- containing a large amount of glycogen formed about
ductase (Fig. 2). equimolar amounts of ethanol and acetate and, in
More recently, Moezelaar and Stal [22] reported a addition, produced some lactate [37,75] (Fig. 3).
mixed acid fermentation in the unicellular cyanobac- Moezelaar et al. [61] reported a mixed-acid fer-
terium Microcystis aeruginosa PCC 7806, a fresh- mentation in the marine benthic cyanobacterium
water species known to produce nuisance water M. chthonoplastes, a cosmopolitan microbial mat-
blooms. This organism degraded glycogen via the forming organism. As was the case in O. limosa,
Embden-Meyerhof-Parnas pathway, producing M. chthonoplastes not only fermented glycogen but
CO2 , ethanol, acetate and some H2 (Fig. 3). In cells also part of its osmoprotectant. The heteroside O-K-
188 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 4. Pathways of anaerobic energy generation in the mat-forming cyanobacterium Microcoleus chthonoplastes. A: Fermentation of gly-
cogen and the osmoprotectant glucosyl-glycerol. B: Fermentation in the presence of elemental sulfur. C: Fermentation in the presence of
ferric iron and/or elemental sulfur. The products in boxes are fermentation products excreted. The numbers refer to the enzymes involved:
1, enzymes of the Embden-Meyerhof-Parnas pathway; 2, pyruvate formate-lyase; 3, formate hydrogen-lyase; 4, CoA-linked aldehyde de-
hydrogenase; 5, alcohol dehydrogenase; 6, phosphotransacetylase; 7, acetate kinase; 8, NAD-dependent lactate dehydrogenase. The en-
zymes pyruvate formate-lyase and formate hydrogen-lyase have been suggested to play a role in fermentation in the unicellular cyanobac-
terium Cyanothece PCC7822.

D-glucopyranosyl-(1,2)-glycerol (glucosyl-glycerol) ceased and formate and ethanol were produced in

serves as osmoprotectant in M. chthonoplastes. This small quantities (Fig. 4B). Formate could also be
was especially the case when the intracellular amount oxidized when ferric iron was present (Fig. 4C) [76].
of glycogen was low. The organism produced equi-
molar amounts of ethanol, acetate and formate in 3.3. The enzymes involved in fermentation
addition to some H2 . When M. chthonoplastes con-
tained a large amount of glycogen, glucosyl-glycerol The pathways that cyanobacteria employ during
was not used. Such cultures produced some lactate in fermentation have been deduced from the nature of
addition to the fermentation products mentioned fermentation products and the ratios in which they
above (Fig. 4A). Of glucosyl-glycerol only the glu- are formed, but in only four cyanobacteria, O. limo-
cose part was fermented while glycerol was excreted sa [63], Cyanothece PCC7822 [60], M. aeruginosa [22]
in the medium. When elemental sulfur was present and M. chthonoplastes [61] has the assumption con-
sul¢de was produced and acetate and CO2 were the cerning the pathway been supported by the presence
main fermentation products. The production of H2 of the key enzymes in cell-free extracts (Table 3).
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 189

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 4 (continued).

Likewise, the occurrence of certain enzymes might [78,79]. Since a catabolic role for the enzyme in a
indicate the ability of fermentative energy genera- fermentative metabolism was not considered, the
tion. Such enzymes have indeed been reported to search for a function of pyruvate:ferredoxin oxido-
occur in cyanobacteria, but a role for these enzymes reductase in cyanobacteria focused on a role in N2 -
in fermentative metabolism was not considered. In- ¢xation. Leach and Carr [78] suggested that in the
stead, they were supposed to have other physiolog- heterocystous Anabaena variabilis the ferredoxin re-
ical functions. duced by pyruvate:ferredoxin oxidoreductase could
The enzyme pyruvate:ferredoxin oxidoreductase is be used as electron donor for nitrogenase. This idea
found in many obligately and facultatively anaerobic is supported by the observation of Neuer and Bothe
bacteria in which it is involved in fermentative deg- [80] that in Anabaena cylindrica activity of pyru-
radation of pyruvate [77]: vate:ferredoxin oxidoreductase was almost exclu-
sively con¢ned to heterocysts. However, the nitro-
pyruvate ‡ CoA ‡ 2Fdox ! genase-independent production of H2 under dark
anoxic conditions by A. variabilis [81] and Anabaena
acetyl3CoA ‡ CO2 ‡ 2Fdred PCC7120 [82] might involve pyruvate:ferredoxin ox-
idoreductase for the supply of reductant for hydro-
Among cyanobacteria, pyruvate:ferredoxin oxidore- genase. In O. limosa [63] and Cyanothece PCC7822
ductase was ¢rst found in two N2 -¢xing species [60], pyruvate:ferredoxin oxidoreductase indeed ap-
190 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Fig. 4 (continued).

pears to serve both processes. When grown in a me- enzymes with a possible function in fermentation in
dium devoid of combined nitrogen, both organisms O. limosa, M. chthonoplastes, M. aeruginosa and Cy-
are capable of dark N2 ¢xation, whereas in nitrate- anothece sp. are given. In all cases the speci¢c activ-
grown cells the enzyme is presumably involved in ities measured were su¤cient to explain the in vivo
fermentative H2 production. observed rates of fermentation. The enzymes de-
Sanchez et al. [83] reported the presence of NAD- tected were used as con¢rmation for the supposed
dependent lactate dehydrogenases in a number of fermentation pathway as deduced from the nature
unicellular cyanobacteria. Under in vivo conditions and ratios of the fermentation products formed.
these enzymes catalyze the conversion of pyruvate When comparisons between the four cyanobacteria
into lactate rather than the reverse reaction [84] were possible it was noticeable that large di¡erences
(see also Section 4). in speci¢c activities existed, except for acetate kinase
The enzymes acetate kinase and phosphotransace- which was in the same order of magnitude in all
tylase in A. variabilis were assumed to be involved in organisms.
the conversion of exogenous acetate to acetyl-CoA
[85]. Acetyl-CoA synthetase, which is involved in 3.4. The Embden-Meyerhof-Parnas pathway
many other bacteria in the activation of acetate,
was not found in A. variabilis. In fermenting bacte- All cyanobacteria examined thus far seem to em-
ria, acetate kinase and phosphotransacetylase oper- ploy the Embden-Meyerhof-Parnas (EMP) pathway
ate in the opposite direction and thus provide a path- during fermentation for degradation of glucose resi-
way for synthesis of ATP [77]. dues to pyruvate. Involvement of the EMP pathway
In Table 3 the speci¢c activities of a number of has been assumed on the basis of similarity of the
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 191

Table 3

Comparison of speci¢c activities of enzymes involved in fermentation in the cyanobacteria Oscillatoria limosa (O. lim.), Microcoleus

chthonoplastes (M. chthon.), Microcystis aeruginosa (M. aerug.) (PCC7806) and Cyanothece sp. (PCC7822).
Enzyme O. lim. M. chthon. M. aerug. Cyanothece
Fermentation Heterolactic (glycogen) Mixed acid

Homolactic (trehalose)

Hydrogenase 0.4 52 28 3.8

Acetate kinase 24 76 51 30.2

a a
Lactate dehydrogenase 4 41 160 4.2
b
Alcohol dehydrogenase (NADH) 4 0 0 0 nd

Alcohol dehydrogenase (NADPH) 10 42 0.2

CO dehydrogenase 0.6 0 nd nd

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Formate dehydrogenase 4 0 nd nd

Pyruvate :Ferredoxin oxidoreductase 5.4 nd 30 4.2

Formate :H2 lyase nd nd nd 0.3

Pyruvate :Formate lyase nd nd nd 1.8

Pyruvate kinase nd 37 63 nd

6-Phosphofructokinase 0.005 8 23 nd
c
Fructose-1,6-bisphosphate aldolase 115 nd 19 9.2
c
Glyceraldehyde-3-phosphate dehydrogenase 0.252 16 92 nd
c
Glucose-6-phosphate dehydrogenase nd 118 67 nd
c
6-Phosphogluconate dehydrogenase nd 85 40 nd

Speci¢c activities in nmol (mg protein)

1 3
min
1 3
; nd : not determined.
a
Not analyzed under optimal conditions : in the presence of 5 mM pyruvate and 10 mM fructose-1,6-bisphosphate [75] and therefore these

activities may be much higher.

b
Measured colorimetrically and not known whether the activity is NADH- or NADPH-dependent. Data of O. limosa from [63,70,73,74], of
M. chthonoplastes from [61], of M. aeruginosa from [22] and of Cyanothece PCC7822 from [71].
c
These activities were measured in cultures grown under an alternating light-dark cycle (16-8 h), whereas all other activites were measured in

cultures grown under continuous light.

fermentation pattern to those of other bacteria tion was not even conceived (Table 4). However,
[22,44,60,61,63], but for only three species, O. limosa there is evidence that failure to detect signi¢cant ac-
[73], Microcystis PCC7806 [22] and M. chthono- tivities of this enzyme may be due to absence of
plastes [61], has this assumption been con¢rmed by stabilizing compounds during preparation of the
the presence of the key enzyme of the EMP pathway,
6-phosphofructokinase, in cell-free extracts of axenic
cultures (Table 3). In O. limosa the activity of 6-
Table 4

6-Phosphofructokinase in cell-free extracts of cyanobacteria

phosphofructokinase was very low but in the other
Organism Spec. activity Ref.
two organisms the speci¢c activity of this enzyme
was su¤ciently high to account for the rate of glu- Aphanocapsa PCC6308 6 0.1 [86]

cose degradation by cell suspensions. As far as we

Aphanocapsa PCC6714 6 0.1 [86]

Anabaena cylindrica 1.8 [80]

are aware these reports were the ¢rst that associated
Anabaena variabilis 17 [87]
the presence of 6-phosphofructokinase in cyanobac- 8.1 [88]

teria with a physiological function. Anacystis nidulans 13 [87]

5.8 [89]
The occurrence of 6-phosphofructokinase and the
physiological signi¢cance of the EMP pathway in
Nostoc muscorum 25 [87]

Microcystis PCC7806 18 [22]

cyanobacteria as a route for glucose degradation
Microcoleus chthonoplastes 8 [61]
has been a matter of uncertainty for a long time. Oscillatoria limosa 0.005 [63]

While signi¢cant speci¢c activities of 6-phosphofruc- Synechococcus PCC6301 6 0.1 [86]

tokinase were found in several species, the activity Synechococcus PCC6716 1.3 [83]

detected in others was so low that a metabolic func- The speci¢c activities are given in nmol min
31 (mg protein)
31 .
192 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211
cell-free extract. In cell-free extracts ofM. chthono- In O. limosa [63] and Cyanothece PCC7822 [60],
plastes , no 6-phosphofructokinase is detected unless the OPP pathway is also operative during fermenta-
its substrate, fructose-6-phosphate, is added to the tion. Remarkably, O. limosa
employs the OPP path-
cell suspension prior to cell breakage [61]. Omission way for degradation of glycogen, whereas the osmo-
leads to a complete loss of activity which cannot be protectant trehalose is degraded via the glycolysis.
restored by adding it to the assay mixture. Similarly, Stal et al. [70] have proposed that the heterolactic
Fewson et al. [87] reported that in Anabaena acid and homoacetate fermentation in this organism
variabilis Anacystis nidulans
, , andNostoc muscorum must be con¢ned to di¡erent compartments in the
no activity of 6-phosphofructokinase was detected cell. In their model the EMP pathway (involved in
unless extracts were prepared with cysteine present. homoacetate fermentation) (Fig. 2) is in the cyto-
This may also have been the reason for the very low plasm which contains the substrate trehalose, where-
O. limosa

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

activity observed in [73] (Table 3). Thus, as the OPP pathway (partly involved in heterolactic
this enzyme may be more widely distributed among acid fermentation) (Fig. 1) is in the thylakoid space
cyanobacteria than has been assumed so far. where glycogen is stored (L.J. Stal, unpublished re-
The presence of signi¢cant speci¢c activities of 6- sults). However, no conclusive evidence for this com-
phosphofructokinase in several strains raised the partmentalization of these fermentation pathways in
question of what purpose this enzyme served in cy- O. limosa is available.
anobacteria. A role in photoautotrophic metabolism
is di¤cult to imagine. During photoautotrophic 3.5. The capability of fermentation is constitutive
growth, CO2 ¢xed in the Calvin cycle enters the me-
tabolism as 3-phosphoglycerate. Conversion of 3- All cyanobacteria examined thus far switch imme-
phosphoglycerate to fructose-6-phosphate, which is diately from photoautotrophy to fermentation when
part of the Calvin cycle, involves some of the sequen- exposed to dark anoxic conditions, suggesting that
ces of the EMP pathway in the reverse direction. the ability for fermentation is constitutive, and that
This series of reactions, however, does not include induction of new enzymes is not required. This has
6-phosphofructokinase, since the reaction catalyzed been con¢rmed for O. limnetica Microcystis
[44],
by this enzyme, the phosphorylation of fructose-6- PCC7806 [22], and M. chthonoplastes [61], in which
phosphate to fructose-1,6-bisphosphate, is virtually fermentation is not a¡ected by the presence of anti-
irreversible and thus serves the EMP pathway only biotics that inhibit protein synthesis. All enzymes are
in the direction of pyruvate formation. A role for 6- readily detected in photoautotrophically grown cells
phosphofructokinase in dark aerobic energy genera- and anaerobic incubation did not induce higher en-
tion is not very likely either. Degradation of glucose zyme activities in any of the cyanobacteria tested for
residues via glycolysis would only be conceivable in this. Fermentation in these cyanobacteria is therefore
combination with the TCA cycle. However, cyano- not regulated at the level of expression of genes.
bacteria lack the enzyme K-ketoglutarate dehydro- Onset of fermentation does not require strictly an-
genase and thus do not possess a complete TCA oxic conditions, but occurs at reduced oxygen partial
cycle. Moreover, changes in the size of metabolite pressures [63]. In Nostoc sp. strain Cc fermentation
pools upon transfer from light to dark and the pres- occurs with 3.4% oxygen in the gas phase [62]. In O.
ence of the enzymes glucose-6-phosphate dehydro- limnetica fermentation occurs even under atmospher-
genase and 6-phosphogluconate dehydrogenase ic oxygen levels when respiration is inhibited by the
have identi¢ed the oxidative pentose phosphate addition of cyanide [44]. Fermentation in cyanobac-
(OPP) pathway as the most likely route of aerobic teria may be under control of a particular metabolite
glycogen degradation (reviewed by Smith [1]). It is which may either inhibit or activate certain enzymes.
therefore conceivable that in cyanobacteria 6-phos- Lactate dehydrogenase in M. aeruginosa is subject to
phofructokinase serves primarily, if not exclusively, such regulation [75] (see Section 4) but other exam-
the fermentative metabolism, and that its presence in ples are lacking. Nevertheless, a metabolic control of
a cyanobacterium indicates the capability of fermen- the pentose phosphate pathway must be conceived.
tation. In the light this pathway should operate in the re-
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 193

ductive mode and allow oxidative processes only in Moezelaar et al. [75] found NAD-dependent lac-
the dark. Part of the OPP pathway is involved in tate dehydrogenase (LDH) (EC 1.1.1.27) in the uni-
heterolactic fermentation, which occurs in O. limosa cellular cyanobacterium Microcystis aeruginosa PCC
[63]. In the majority of cyanobacteria fermentation 7806, although they were initially unable to detect
involves the EMP pathway which does not seem to any lactate production during fermentation. This
play a role in phototrophic metabolism. It is there- was remarkable since the speci¢c activity of LDH
fore also possible that fermentation pathways in in Microcystis PCC7806 was 0.14^0.16 U (mg
these cyanobacteria lack a good regulation and protein)31 the highest reported of cyanobacterial
give occasion to suppose that fermentation occurs cell-free extracts. Activity of LDH from Microcystis
regardless of the prevailing conditions. On the other PCC7806 was like other NAD-dependent LDHs in-
hand, the activities of enzymes of fermentative path- hibited by ATP and ADP [83,84]. However, the en-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

ways are so much lower than those involved in aero- zyme of Microcystis was not inhibited by inorganic
bic or phototrophic metabolism that fermentation phosphate which is known as a general inhibitor of
pales into insigni¢cance beside it. The advantage fructose-1,6-bisphosphate-dependent lactate dehy-
for the organism of possessing a constitutive anaero- drogenases [84]. The signi¢cance of these regulations
bic metabolism is its ability to quickly react to of LDH in Microcystis are not clear. Recently, using
changes of environmental conditions. cultures with high levels of glycogen Moezelaar and
Stal could show also small amounts of L-lactate
among the fermentation products [37]. Lactate dehy-
4. Lactate dehydrogenase and lactate production in drogenase activity appeared to be tightly regulated in
cyanobacteria M. aeruginosa. The enzyme required the EMP path-
way intermediate fructose-1,6-bisphosphate for activ-
In a screening of 27 unicellular cyanobacteria ity and displayed positive cooperativity towards pyr-
(Synechococcus and Aphanocapsa spp.) for NAD-de- uvate [75]. Moezelaar and Stal [37] concluded that
pendent lactate dehydrogenases, eight strains were the role of NAD-dependent lactate dehydrogenase in
found to possess both D- and L-lactate dehydrogen- this organism is probably over£ow metabolism as it
ases whereas 12 strains were found to contain only is in certain other bacteria [84]. However, in these
D-lactate dehydrogenase [83]. Initially it was assumed organisms this type over£ow metabolism depends
that these were involved in the incorporation of on the amount of extracellular substrate o¡ered. In
exogenous lactate into biomass. However, it is now this respect the observation of De Philippis et al. [10]
generally accepted that in vivo NAD-dependent lac- is of interest. These authors studied a large number
tate dehydrogenases function in the conversion of of di¡erent strains of symbiotic and free-living het-
pyruvate to lactate rather than in the opposite direc- erocystous cyanobacteria of the genera Nostoc and
tion [84]. Excretion of D-lactate under dark anoxic Anabaena. These strains were all able to utilize exog-
conditions as an end product of endogenous carbo- enous sugars and ferment them under anoxic condi-
hydrate catabolism has been reported for Synecho- tions in the dark probably via the homoacetic acid
coccus PCC6716 [83]. No attempts were made to pathway. Most of these strains produced variable
determine other fermentation products but, accord- amounts of lactate. These results also hint to a role
ing to the authors, the amount of lactate produced in over£ow metabolism.
``corresponded fairly well'' with the decrease in car- In other strains lactate is among the normal fer-
bohydrate during such incubations. Conversion of mentation products. In O. limnetica glucose is fer-
glycogen to lactate in this organism may involve mented via the homolactic acid pathway and lactate
the EMP pathway, since most of the enzymes of is the only product [44]. These authors did not meas-
this route, including the key enzyme 6-phosphofruc- ure LDH activity and therefore the characteristics of
tokinase and NAD-linked D-lactate dehydrogenase, this enzyme are not known. The analytical procedure
were demonstrated in cell-free extracts [83]. Synecho- also did not allow conclusions about whether L- or
coccus PCC6716 is not capable of fermenting exoge- D-lactate was produced. O. limosa ferments glycogen
nous glucose. via the heterofermentative lactic acid pathway, pro-
194 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211
ducing L-lactate as fermentation product in addition cess reductant [77]. Hydrogenase-dependent H2 evo-
to ethanol [63]. NAD-dependent LDH was deter- lution under dark anoxic conditions at the expense
mined and amounted to 0.004 U (mg cell protein)31 . of endogenous substrate has been observed with
One unit (U) of enzyme activity is de¢ned as the cyanobacteria of various genera [81,91^96]. In Ana-
amount of enzyme catalyzing the transformation of baena cylindrica, hydrogenase is activated after 1^5 h
1 Wmol of substrate or the formation of 1 Wmol of of dark anaerobic incubation [81]. Additional syn-
product in 1 min. Also in Microcoleus chthonoplastes thesis of hydrogenase has been observed during
NAD-dependent LDH was present (0.041 U (mg anaerobic incubation in the light [82,96] or upon
protein)31 ) but small amounts of lactate were pro- depletion of NH‡4 [95].
duced only in cultures that contained a large amount
of glycogen [61] and it is probable therefore that this
Micro-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

enzyme is regulated in the same manner as in 6. Electron acceptors and anaerobic respiration
cystis. Van der Oost et al. [60] found lactate as a
normal fermentation product in the unicellular cya- In addition to lactate fermentation, O. limnetica
nobacterium Cyanothece PCC7822. Van der Oost exhibits a second mode of anaerobic glucose catab-
[71] also measured NAD-dependent LDH but his olism in the dark [44]. In the presence of elemental
analyses did not allow the distinction between D- sulfur a considerable part of the endogenous carbo-
or L-lactate as the fermentation product. O. terebri- hydrates is oxidized completely to CO2 and concom-
formis produced small amounts of lactate when in- itantly elemental sulfur is reduced to sul¢de. The
cubated anaerobically in the dark with a large remaining part of the glucose is fermented to lactate.
amount (30 mM) of fructose (or glucose) as substrate Other sulfur compounds like thiosulfate or sulfate
[28]. were not used as electron acceptors. It was assumed
In summary it can be concluded that lactate pro- that the use of elemental sulfur as electron acceptor
duction in cyanobacteria is either a main fermenta- represented a true sulfur respiration but this was not
tion product or is only produced as a product of convincingly demonstrated. As we argue in Section
over£ow metabolism when alternative fermentation 7, sulfur respiration would yield only an insigni¢-
pathways are saturated. Cyanobacteria that produce cantly larger amount of ATP in this organism.
lactate as main fermentation product may either lack O. limosa is also capable of reducing elemental
a tight regulation of LDH or produce lactate because sulfur to sul¢de under dark anoxic conditions [63].
of the absence of other fermentation pathways. For this organism elemental sulfur acts as a sink for
electrons that are otherwise released as H2 and does
not a¡ect the formation of the other fermentation
5. Hydrogenases products. Synechococcus lividus strain Y52, isolated
from a hot spring microbial mat, reduces thiosulfate
The capability of cyanobacteria to evolve molecu- and sulfate to sul¢de when incubated anaerobically
lar hydrogen has been known for a long time. Of the in the dark [97,98]. The physiological status of this
three enzymes involved in H2 metabolism in cyano- process is not clear since production of sul¢de from
bacteria (reviewed by Houchins [90]), two are known (thio)sulfate occurs at even higher rates in the light
to catalyze the evolution of H2 in vivo: nitrogenase, when CO2 is absent.
which obligately produces H2 as a by-product of N2 The mat-forming cyanobacterium M. chthono-
¢xation, and reversible or soluble hydrogenase. Ni- plastes reduced elemental sulfur during anaerobic
trogenase-linked production of H2 is not considered dark metabolism [59,61]. As can be seen from Table
here since it is an inherent property of the enzyme 5 the addition of elemental sulfur had the following
and hence does not seem to serve a particular func- e¡ects. The amount of acetate produced almost
tion in fermentation. In contrast, the reversible hy- doubled while the production of ethanol decreased
drogenase resembles the enzyme that in many to the same extent. This is an important aspect since
chemoorganotrophic bacteria is involved in fermen- one additional ATP is generated for each acetate
tative production of H2 as a means of releasing ex- produced (Fig. 4B). Other e¡ects were the much low-
 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211 195

Table 5
creases from 1.34 to 1.46 (nmol min31 (mg cell
Comparison of fermentation in Microcoleus chthonoplastes in the protein)31 ) when compared with a culture in the
presence and absence of elemental sulfur
presence of sulfur. The carbon and redox balances
Product 3S³ +S³
of the latter fermentation indicate that despite the
Ethanol 1.04 0.31 higher energy yield less biosynthesis could have tak-
Acetate 1.00 1.72
en place. Because of this, the energy available for
Formate
H2
0.72
0.09
0.28
0 maintenance purposes increased from m q
ATP 0.88 to
CO2 1.32 1.75 1.20 (nmol min31 (mg cell protein)31 ) when sulfur
Sul¢de 0 2.28 was present. Thus, if the reduction of sulfur itself
Amounts are expressed as mol per mol of glucose fermented. Data were associated with energy generation, it could be
from [61]. questioned for what purpose, since it did not in-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

crease biosynthesis.
An interesting di¡erence between sulfur reduction
er production of formate and the complete cessation in M. chthonoplastes and O. limnetica
is that in the
of hydrogen evolution, while sul¢de was formed. By latter electrons apparently are generated via the OPP
comparing the fermentation of M. chthonoplastes pathway, which is clearly not the case in Microco-
with and without elemental sulfur (Table 5) it can leus . Because cyanobacteria lack the TCA cycle [1]
be concluded that elemental sulfur serves as an elec- and O. limnetica
oxidizes glycogen almost completely
tron sink in this organism. In the absence of elemen- to CO2 in the presence of sul¢de, it is inevitable that
tal sulfur the cleavage of formate seems to be limited degradation is via the OPP pathway, which is also
by the accumulation of H2 , which makes this reac- the route when glycogen is metabolized aerobically
tion thermodynamically less favorable [77]. When [1]. Apparently the OPP pathway is blocked in M.
sulfur is present much more formate is cleaved, be- chthonoplastes under anoxic conditions, even when
cause instead of H2 the thermodynamically more fa- sulfur is present as electron acceptor. If, as we be-
vorable sul¢de is produced. Unless sulfur serves as lieve, sulfur does not serve as a terminal acceptor in
terminal electron acceptor in a respiratory electron a respiratory electron transport chain in this organ-
transport system, the only advantage of this reaction ism, oxidation of glucose via the OPP pathway
may be the removal of the toxic formate. In its nat- would not yield any energy at all. In O. limnetica
,
ural environment, microbial mats, the sul¢de pro- on the other hand, sulfur could play a role as termi-
duced will normally precipitate as FeS which will nal electron acceptor in anaerobic respiration but as
eliminate toxic e¡ects of sul¢de. On the other hand Oren and Shilo [44] calculated the energy yield of
other microorganisms in the ecosystem may use H2 this process would be only slightly higher than in
or formate (e.g. sulfate-reducing bacteria) and there- the case of fermentation.
fore it is uncertain whether this sul¢de production The reduction of sulfur is widely distributed in the
will take place under natural conditions. More im- microbial world but in only few cases it is associated
portantly, sulfur reduction could also regenerate with an electron transport chain [99]. Virtually all
NAD(P) reduced during glucose oxidation in the cyanobacteria we have tested, appeared to be capa-
EMP pathway. In the absence of elemental sulfur ble of reducing elemental sulfur (Table 6). However,
the reduction of acetyl-CoA to ethanol serves the further investigations are required in order to prove
regeneration of NAD(P). The obvious advantage of whether cyanobacteria are capable of true sulfur res-
the presence of sulfur is that more acetyl-CoA can be piration.
converted into acetate, allowing the production of Oren and Shilo [44] have tested the possibility of
ATP. Theoretically, when sulfur serves as terminal sulfate and thiosulfate serving as electron acceptors
electron acceptor in a respiratory electron transport in anaerobic dark metabolism in O. limneticawith a
chain, its reduction could also yield energy. A higher negative result. We have done the same for M. chtho-
energy yield should be translated in a larger amount noplastes and also concluded that sulfate, sul¢te and
of biosynthesis. This was not the case. The ATP of q thiosulfate could not serve as electron acceptors in
the culture incubated without elemental sulfur in- anaerobic dark metabolism in this organism (L.J.
196 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

Stal, unpublished results). The utilization of sulfate acceptor in anaerobic dark metabolism in M. chtho-
and thiosulfate as electron acceptors in dark anaero- noplastes. They indeed showed that this organism
bic metabolism has been reported for the unicellular reduced DMSO to dimethylsul¢de (DMS) but were
cyanobacterium S. lividus Y52-s [97,98]. This organ- unable to associate this process with fermentative
ism reduces sulfate to sul¢de and thiosulfate to sul- metabolism. Unlike elemental sulfur the presence of
¢te and sul¢de while endogenous carbohydrate is DMSO did not alter the fermentation pattern. More-
oxidized to CO2 . Exogenous carbohydrates were over, as was the case with ferric iron, the rate of
not utilized. In the absence of CO2 , sulfate and thi- reduction was much too slow to be important as
osulfate were also reduced in the light. As far as we electron acceptor during fermentation. DMSO as
are aware, S. lividus is the only organism known with well as trimethylamine-N-oxide (TMAO) have been
this type of anaerobic metabolism, which could shown to serve as electron acceptors in anaerobic

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

present a mode of anaerobic respiration, or a de- dark metabolism in anoxygenic phototrophic bacte-
regulated assimilatory sulfate reduction [101]. ria [40,41].
Moezelaar et al. [61] considered the possibility that In the ¢lamentous non-heterocystous nitrogen-¢x-
ferric iron could serve as an electron acceptor in ing cyanobacterium O. limosa acetylene could serve
anaerobic dark metabolism in M. chthonoplastes. It as an electron acceptor [59]. Under a helium atmos-
was already known that this organism is capable of phere, nitrogen-¢xing O. limosa produced hardly de-
accumulating and reducing ferric iron [102]. Schaub tectable amounts of lactate and no sul¢de when ace-
and Stal [76] demonstrated that M. chthonoplastes is tylene (C2 H2 ) was present. Nitrogenase which
capable of reducing ferric iron mediated through the normal function is the reduction of N2 in nitrogen-
oxidation of the fermentation product formate, but ¢xing organisms is also capable of reducing acetylene
they also showed that the rate at which this occurred to ethylene, a property widely used for the assay of
was much too slow to be signi¢cant as electron ac- nitrogenase activity [104]. In O. limosa nitrogenase
ceptor during fermentation. These authors suggested activity under anaerobic conditions in the dark as
that formate mediated iron reduction rather plays a measured by the acetylene reduction technique is
role in iron acquisition. However, iron may indi- 1.3 nmol C2 H4 min31 (mg protein)31 [105]. Com-
rectly serve as electron acceptor when sulfur is pared with the rate of glycogen utilization (1.1
present [102]. The sul¢de formed from the reduction nmol glucose min31 (mg cell protein)31 , Table 7)
of elemental sulfur will reduce ferric iron according and the rate of trehalose degradation (0.2 nmol tre-
to the following reaction: halose min31 (mg cell protein)31 [63]), it is obvious
that a considerable amount of the electrons pro-
2 3‡
‡ S23 !2Fe2‡ ‡ S …1† duced are transported via nitrogenase. Acetylene re-
duction followed precisely the kinetics of glycogen
Van Bergeijk and Stal [103] investigated the possibil- degradation [59]. In stead of yielding energy, nitro-
ity of dimethylsulfoxide (DMSO) serving as electron genase mediated electron transport will be only at

Table 6
Cyanobacteria capable of sulfur reduction
Strain Origin Ref.
Oscillatoria limosa microbial mat, North Sea [63]
Microcoleus chthonoplastes microbial mat, North Sea [61]
Merismopedia punctata microbial mat, North Sea [100]
Chroococcus turgidus microbial mat, North Sea [100]
Anabaena variabilis microbial mat, North Sea [100]
Spirulina subsalsa microbial mat, North Sea [100]
Oscillatoria limnetica Solar Lake, Sinai [44]
Aphanothece halophytica saltern [44]
Microcystis aeruginosa freshwater lake, PCC7806 Moezelaar and Stal, unpublished
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 197

Table 7

q q
Glycogen degradation ( glucose ) and ATP production (expected when glycogen is totally fermented) ( ATP ) in cyanobacteria during fermen-

tation

Organism qglucose ATP/glucose qATP Ref.

Oscillatoria limnetica 1.7 3 5.1 [44]

Oscillatoria limosa [63]

nitrate-grown 0.8 2 1.6

N2 -grown 1.1 2 2.2

Cyanothece PCC7822 0.8 3.2 2.6 [60]

Nostoc sp. strain Cc. 1.7 5 8.5 [62]

Microcystis PCC7806 0.4^0.9 4 1.6^3.6 [22,37]

Microcoleus chthonoplastes 0.2^0.4 4 0.8^1.6 [61]

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Rates are expressed in nmol min
31 (mg cell protein)31 . In order to convert published data from chlorophyll a to protein the ratio 26 :1
(protein :chlorophyll a) was used [61]. The rates refer only to glycogen degradation and not to extracellularly added glucose or degradation of
osmoprotectant (see text). In case multiple pathways were assumed, the average ATP yield was calculated. The range of the rate of glycogen

degradation is given when this varies with glycogen content.

the expense of a considerable amount of energy (2 energy yield of fermentation is so low that at best it
133
ATP (e ) ) (see Section 7). The fermentation ex- can sustain maintenance [60]. However, very little is
periments with O. limosa were carried out under an known about maintenance energy requirements in
atmosphere of either helium [59] or argon [63]. Un- cyanobacteria [106].
fortunately, no experiments were carried out under a In all cyanobacteria investigated thus far, degra-
nitrogen atmosphere, but the fact that acetylene re- dation of glycogen during fermentation occurs at low
duction occurred anaerobically in the dark in nitro- rates ranging from 0.2 to 1.7 nmol min
1
(mg cell
3
gen-¢xing cultures makes it likely that molecular ni- protein)
1 3
(Table 7). Such rates are very low com-
trogen (N2 ) will serve as electron sink under such pared to uptake rates of glucose that are required to
conditions. sustain growth during fermentation in other micro-
organisms. As shown for Enterococcus faecalis
grown in glucose-limited chemostats, the glucose up-
7. Energetics of fermentation in cyanobacteria take rate increases with the speci¢c growth rate from
80 nmol min
1 3
(mg cell protein)
1
at 0.1 h
1 3
to 550
3
7.1. Maintenance requirements in cyanobacteria nmol min
1 3
(mg cell protein)
1
at 0.5 h
1 3
[107]. So it
3
appears likely that fermentation of glycogen in cya-
Compared to aerobic respiration the energy yield nobacteria primarily serves maintenance purposes
of fermentation is low. In the light, cyanobacteria because it does not aim to sustain growth [62].
accumulate energy storage material endogenously This view is in accordance with the low speci¢c ac-
which is subsequently utilized in the dark. That tivities of the key enzymes of the fermentation me-
this process does not serve solely maintenance pur- tabolism that are found in cell-free extracts
poses was demonstrated by Post et al. [7] who [22,37,60,61,63]. Most of the fermentation experi-
showed that the cyanobacterium O. agardhii when ments have been conducted with resting cell suspen-
grown in continuous culture under a light-dark cycle sion in bu¡ers which would not allow growth. How-
was capable of maintaining its growth rate at the ever, in those cases where cells were incubated in
expense of endogenous carbohydrate during the complete medium, indeed no growth was detected
dark period. These authors provided evidence that [44,62].
the energy yield of aerobic respiration was su¤cient From the degradation rates of glycogen and the
to sustain growth at the same rate as in the light. pathways likely to be involved, the ATP production
Apart from this work, remarkably little has been during fermentation is estimated to be in the range
published about the energetics of dark metabolism of 0.8^8.5 nmol min
1 3
(mg cell protein)
1
(Table 7).
3
in cyanobacteria. In general it is assumed that the It must be emphasized, however, that these numbers
198 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211
do not take into account that substrates other than During homolactic acid fermentation lactate is the
glycogen may be involved in fermentation as well. only fermentation product and also no CO2 is pro-
For instance, in O. limosa the osmoprotectant treha- duced. This means that the carbon recovery was only
lose is fermented as well [63] and the glucose part of 80%. For each molecule of glycogen-glucose that is
glucosyl-glycerol, the osmoprotectant of M. chthono- fermented to 2 molecules lactate 3 ATP are gener-
plastes is fermented when this organism contains low ated. Thus this fermentation would have resulted in
amounts of glycogen [61]. Moreover, Microcystis U
the formation of 2.4 mol of ATP (0.8 3) for each
PCC7806 [22], Nostoc strain Cc [62] and O. terebri- molecule of glycogen-glucose degraded. Assuming
formis [28] can also utilize exogenous glucose. that the carbon not recovered has been assimilated
Data on maintenance requirements of cyanobacte- in structural cell material (C-content is 50%) and
ria are scarce. Only for one organism, O. agardhii, that YATP equals 20 g biomass (mol ATP)31 , it

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

has this been examined thoroughly [106]. Whereas can be calculated that 1.44 mol of ATP are required
the speci¢c maintenance rate is independent of the to produce this cell material. Thus 0.96 mol of ATP
light intensity, the e¤ciency with which radiant en- would be available for maintenance purposes, which
ergy is converted into biochemical energy decreases is 40% of the ATP generated. In order to judge how
with increasing light intensity. At the lowest light much this would be in terms of biomass and rate the
intensity tested the speci¢c light energy uptake for qATP has to be known.
maintenance is estimated to equal a rate of ATP Oren and Shilo [44] calculated a rate of polyglu-
production of 4 nmol min31 (mg cell protein)31 cose utilization of about 5 Wmol glucose (mg chlo-
(see Appendix A). Although this value already agrees rophyll a)31 h31 in the presence of elemental sulfur.
reasonably well with the data obtained from fermen- They did not give a value for the degradation in the
tation experiments, the true speci¢c ATP production absence of elemental sulfur but this might have been
for maintenance may be even lower at lower light the same or lower. In order to obtain a protein-based
intensity. In the following sections the energetics of qATP the ratio protein:chlorophyll a has to be
fermentation in four cyanobacteria that have been known. O. limnetica contains about 2 Wmol glucose
studied in reasonable detail is considered. equivalents (mg cell protein)31 which is utilized in
about 20 h of dark anaerobic incubation. From these
7.2. Energetics of fermentation in Oscillatoria data it can be calculated that the ratio protein:chlo-
limnetica rophyll in O. limnetica must have been about 50.
This is about twice as high as for M. chthonoplastes
Oren and Shilo [44] were the ¢rst to report anae- [61] or O. limosa [108]. However, the relatively low
robic dark metabolism in a cyanobacterium. Their content of chlorophyll a in O. limnetica may have
choice to study O. limnetica, a strain isolated from been due to the anoxygenic conditions under which
Solar Lake (Sinai desert), was based on the fact that the organism was grown with high light intensity
this organism in its natural habitat thrives for pro- U
(2 1033 J cm32 s31 ) and sul¢de present. This gives
longed periods of time under anoxic conditions. O. a speci¢c rate of glucose utilization of 1.7 nmol
limnetica is also capable of anoxygenic photosynthe- min31 (mg cell protein)31 . The qATP is 4 nmol
sis, using sul¢de as electron donor, which is oxidized min31 (mg cell protein)31 (80% of the glucose uti-
to elemental sulfur and excreted from the cells [49]. lized is fermented). Since 40% of the ATP generated
Oren and Shilo [44] demonstrated that O. limnetica is available for maintenance, the
was capable of degrading of endogenous carbohy- qmATP
drate and excreting lactate. In the presence of ele-
mental sulfur, sul¢de was produced while the is estimated to be 1.6 nmol min31 (mg cell
amount of lactate produced decreased. Lactate was protein)31 . However, this number may be consider-
the only organic fermentation product produced by ably lower when the rate of glucose degradation is
O. limnetica. lower in the absence of elemental sulfur.
In the absence of elemental sulfur O. limnetica Another interesting observation made by Oren
produced 1.6 mol of lactate per glucose metabolized. and Shilo [44] was that in the presence of the inhib-
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 199

itor of protein synthesis, chloramphenicol, the culated for the other cyanobacteria. Whether the re-

amount of lactate produced per glucose metabolized duction of elemental sulfur is associated with energy

increased to 1.9 which was almost the amount that generation is still uncertain. The oxidation of glucose

would be expected when the glucose was completely through the OPP pathway does not yield any ATP

fermented to lactate. This also strongly indicated and therefore a role of sulfur solely as electron sink

that growth can occur during dark anaerobic incu- would represent a loss of energy.

bation. Oren and Shilo [44], who used a Y ATP of

10.5 g biomass (mol ATP)

31 but did not take into 7.3. Energetics of fermentation in Oscillatoria limosa
account a speci¢c rate of maintenance energy re-

quirement, also calculated that about 20% of poly- O. limosa is a non-heterocystous nitrogen-¢xing

glucose could have been assimilated into structural cyanobacterium. Heyer et al. [63] suggested that fer-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

cell material. This would have resulted in an increase mentation in O. limosa , in addition to meeting main-

of biomass of only 3.3%. It is not correct not to tenance requirements, might support other metabolic

include the rate of maintenance energy requirement processes such as growth and nitrogen ¢xation. Stal

in these calculations because it is a substantial part and Heyer [105] have demonstrated that this organ-

of the energy generated under dark anoxic condi- ism was capable of dark anaerobic acetylene reduc-

tions. On the other hand a Y ATP of 20 is probably tion (nitrogenase activity) for 12^24 h at a rate of

more realistic than 10.5 g biomass (mol ATP)

31 2 Wmol C2 H2 h31 (mg chlorophyll a)31 . The ratio pro-
[109]. tein :chlorophyll a in this organism is 23 [108] which

Oren and Shilo [44] argued that it would not make transforms this rate of acetylene reduction to 1.45

a big di¡erence if sul¢de respiration would occur. nmol min

31 (mg cell protein)
31 . Reduction of dini-

They assumed that 3.5 ATP could be generated per trogen by nitrogenase requires 4 ATP for each pair

glucose oxidized which is only 0.5 more than in the of electrons involved (16 ATP per N ) [110]. This 2
case of lactate fermentation. In the presence of ele- means that the reduction of one molecule C H 2 2 to

mental sulfur the carbon recovery of dark anaerobic C H 2 4 (ethylene) would require 4 ATP (assuming the

metabolism in O. limnetica was 92%. Even with ele- same mechanism as for N 2 reduction). To support

mental sulfur present some lactate was produced. Per the observed rate of dark anaerobic acetylene reduc-

molecule of glucose 0.8 mol lactate and 6.2 mol sul- tion 5.8 nmol ATP min
31 (mg cell protein)
31 are

¢de are produced. In order to produce 6.2 mol sul- required. Fermentation of glycogen in nitrogen-¢x-

¢de 0.52 mol glucose must be oxidized. Add the 0.4 ing O. limosa yields 2.2 nmol ATP min
31 (mg cell

mol glucose that was fermented to lactate, only 0.08 protein)

31 (Table 7). However, this organism also

mol of the glucose could have been assimilated into ferments its osmoprotectant trehalose via the homo-

structural cell material. With 50% carbon content acetic pathway [63]. The homoacetic fermentation of

this would give an increase in structural cell material glucose results in a net yield of 4 ATP (Fig. 2). The

of 11.52 g and with a Y ATP of 20 g biomass (mol net yield of ATP produced during the formation of

ATP)
31 , this would cost 0.58 ATP. This could easily acetate from CO 2 is zero (Fig. 2). However, energy

be produced by lactate fermentation. The 0.4 mol from this reaction may be conserved electrochemi-

glucose fermented to lactate would have yielded 1.2 cally, e.g. as a Na

‡ gradient [72], which would pre-

ATP. The speci¢c rate of glucose utilization is 1.7 sumably add another equivalent of ATP. Although

nmol min
31 (mg cell protein)
31 , of which 40% is only 8 Wmol (mg chlorophyll a)31 of the disaccharide

diverted to lactate fermentation. Assuming ATP gen- trehalose are degraded in 24 h, the high energy yield

eration exclusively through lactate fermentation the of homoacetate fermentation more than doubles the

qATP = 2 nmol min

31 (mg cell protein)31 . Half of this qATP to 4.6 nmol min
31 (mg cell protein)
31 . This is

ATP production is required for the assimilation of obviously not su¤cient to explain the observed rate

carbon into structural cell material. This leaves a of acetylene reduction. The possibility that qATP is

qmATP = 1 nmol min

31 (mg cell protein)
31 . These spe- underestimated should be considered. For instance,

ci¢c rates of maintenance energy requirements seem the transport of acetic and lactic acid over the cyto-

very reasonable when compared with what was cal- plasmic membrane may generate metabolic energy
200 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211

Table 8
[111]. If this possibility is considered we estimate a
Stoichiometry of glycogen degradation and product formation
qATP of 6.1. This would be su¤cient to support the
during fermentation in Microcystis PCC7806

observed rate of acetylene reduction but leaves

L cells L-D cells
hardly any ATP for other metabolic processes (e.g.
Substrate glucose (glycogen) 8.9 3.5
maintenance). The carbon and redox balances of fer-
Products ethanol 5.3 4.9
mentation in O. limosa were good, which indicated
acetate 4.9 1.1

that no carbon was used for biosynthesis. H 2 2.5 1.8

CO 2 10.2 6.0

7.4. Energetics of fermentation in Microcystis L-lactate 0.3 nd

aeruginosa C recovery 59% 86%

O/R balance 1.56 1.03

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Washed cells (10 ml, 2.0 mg protein ml
31 ) were incubated in a 30
Moezelaar and Stal [37] found that the glycogen
ml serum bottle under an argon atmosphere for 8 h. The cells were
content of Microcystis PCC7806 ( M. aeruginosa) de-
grown in batch culture under continuous light (L) or under an
pended on the light regime under which it was culti- alternating light-dark (16-8 h) cycle (L-D) and harvested at

vated. When the organism was grown under an al- OD 750 0.8^1.0. Amounts of substrate and products are expressed

ternating light-dark (16-8 h) cycle the maximum in Wmol. C balance is the amount of carbon atoms ( Wmol) in the

substrate(s) which is (are) metabolized, divided by the amount of

amount of glycogen (at the end of the light phase)

was 1.5 Wmol glucose (mg cell protein)

31 . Under
carbon atoms recovered in the products, times 100%. The C bal-

ance should be 100% and a lower value indicates that products

continuous light this organism contained twice as
31 ).
may be missing. The O/R balance is the sum of all oxidized sub-

much glycogen (3 Wmol glucose (mg cell protein) strates and products, divided by the sum of all reduced substrates

and products. Each compound receives a redox number which

The fermentation patterns of both cultures showed
indicates the number of H atoms in the compound deviating
marked di¡erences. Whereas fermentation in the
from water (which therefore has the redox number 0). Excess of
light-dark grown culture had a reasonable carbon
H atoms gives a negative redox number, a shortage is indicated by
balance (86%) and a good oxidation/reduction (O/ a positive sign. The redox numbers are multiplied by the molar

R) balance (1.03) [22], this was not the case in the amount of the substrate used or product formed. The O/R balance

should be 1. A greater value indicates a lack of reduced com-

culture grown in continuous light (carbon recovery
pounds. Data from [22,37].
59%, O/R balance 1.56) [37,112] (Table 8). The car-

bon balance is the amount of carbon atoms ( Wmol)

in the substrate(s) which is (are) metabolized, divided tion, the culture grown under continuous light also

by the amount of carbon atoms recovered in the produced some lactate which was not the case in the

products, times 100%. The carbon balance should light-dark grown cells. These di¡erences could not be

be 100% and a lower value indicates that products attributed to di¡erences in speci¢c activities of en-

may be missing. The O/R balance is the sum of all zymes involved in fermentation since these were

oxidized substrates and products, divided by the sum identical in both cultures and su¤cient to explain

of all reduced substrates and products. Each com- the highest rates of product formation. The rates

pound receives a redox number which indicates the of glycogen degradation in the light-dark and the

number of H atoms in the compound deviating from continuous light grown cultures were 0.4 and 0.9

water (which therefore has the redox number 0). Ex- nmol glucose min
31 (mg cell protein)
31 , respectively.
cess of H atoms gives a negative redox number, a In the culture of Microcystis PCC7806 grown

shortage is indicated by a positive sign. The redox under a light-dark regime the carbon recovery was

numbers are multiplied by the molar amount of the 86% and the O/R balance 1.03 [22] (Table 8). Assum-

substrate used or product formed. The O/R balance ing that the missing carbon had been converted into

should be 1. A greater value indicates a lack of re- cell material which of course would result in 100%

duced compounds. Furthermore the light-dark carbon recovery, the O/R balance becomes 0.99. Re-

grown culture produced much more ethanol relative assimilation of carbon from glycogen could proceed

to acetate as compared to the culture grown in con- via acetyl-CoA [1] which might explain the relative

tinuous light. The latter produced approximately low amount of acetate produced by this culture. Cell

equimolar amounts of ethanol and acetate. In addi- material is slightly reduced and a redox number of
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 201

30.37 (mol C)31 is calculated on the basis of atomic phosphoenolpyruvate ‡ CO2 ‡ H2 O !

ratios of phytoplankton given by Atkinson and
Smith [113]. The amount of ATP produced during oxaloacetate ‡ Pi
fermentation in this culture can be calculated taking
into account the amount of glucose converted into PEP carboxylase, the enzyme that catalyzes this re-
fermentation products (3 Wmol, Table 8). Three ATP action, is a very important enzyme for CO2 metab-
are produced per glycogen-glucose fermented and 1 olism in cyanobacteria. The activity of this enzyme
for each acetate produced. This gives a total amount results in the synthesis of C4 products. It has been
of ATP of 10.2 Wmol and a qATP of 1 nmol min31 estimated that in cyanobacteria up to 20% of carbon
(mg cell protein)31 , which is slightly lower than in- assimilation can be attributed to PEP carboxylase
dicated in Table 7 where it was based on the decrease [114].

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

of glycogen rather than on the formation of fermen- If only 1.5 of the 6.5 Wmol CO2 were re-¢xed dur-
tation products. The carbon that was not recovered ing fermentation both the C and O/R balances are
(3 Wmol) could give rise to 72 Wg cell material (as- satis¢ed (Table 9). The ¢xation of this amount of
suming 50% of cell material is carbon). Its synthesis CO2 via the carboxylation of phosphoenolpyruvate
would cost 3.6 Wmol ATP, assuming a YATP of 20 g would cost 1.5 Wmol ATP. When taking into account
biomass (mol ATP)31 , which is considered as realis- the qm 31 31
ATP of 0.7 nmol min (mg cell protein) and a
tic value in this case [109]. It is assumed that the 3 1
YATP of 20 g biomass (mol ATP) , su¤cient energy
3
remaining 6.6 Wmol ATP (10.2 3.6) covers the re- is available for the synthesis of 180 Wg structural cell
quirements for maintenance. It equals 0.7 nmol ATP material (assuming 50% (w/w) of cell matter is car-
min31 (mg cell protein)31 . This rate seems low but it bon). This ¢ts the 7.2 Wmol C (equals 172 Wg cell
is in the range of the theoretical value calculated for material) that must have been assimilated (Table 9).
Escherichia coli (0.5 nmol ATP min31 (mg Some of the assumptions used above were rather
protein)31 ) [109]. Measured rates of maintenance en- conservative. For instance, YATP normally includes
ergy in E. coli are 10^100 times this theoretical rate energy for maintenance purposes. Furthermore, no
[109] but cyanobacteria are known for their low energy for the transport of substrate is necessary
maintenance requirements [106]. The qm ATP of 0.7 since the glucose is already inside the cell. Moreover,
nmol min31 (mg cell protein)31 is still more than many cyanobacteria contain the polypeptide cyano-
5 times lower than calculated for O. agardhii (see phycin (multi-L-arginyl poly-L-aspartate) [69] which
Appendix A). However, the qm ATP of 4 for this organ- can provide the cell with ready to use amino acids
ism was calculated for growth in the light and it is for biosynthesis. The excretion of acids such as ace-
known that the qm ATP increases with light intensity. tate and lactate may also yield energy [111]. We con-
The qm ATP of 0.7 we have derived seems therefore a clude that even though the qATP seems rather low,
good estimate for maintenance energy in cyanobac- fermentation of endogenous carbohydrate storage
teria thriving under anaerobic conditions in the dark. may support a limited amount of growth in cyano-
It is therefore reasonable to apply this value also for bacteria. However, due to the limited amount of
the culture of Microcystis PCC 7806 grown under storage carbohydrate this would not result in a meas-
continuous light. If we assume the missing carbon urable increase of biomass. This conclusion sheds
from fermentation in this organism also to be con- some light on the fermentation in M. chthonoplastes.
verted in cell material in order to make up the car-
bon balance to 100% it makes the O/R balance only 7.5. Energetics of fermentation in Microcoleus
slightly better (1.35). This high O/R balance is most chthonoplastes
probably caused by an erroneous value for CO2 . On
the basis of the fermentation pathway [22] one CO2 The glycogen content in M. chthonoplastes may
is produced for each molecule ethanol and acetate vary with culture conditions as in Microcystis. Cells
produced. Moezelaar and Stal [37] hypothesized from the exponential growth phase contained rela-
that some re-¢xation of CO2 via the carboxylation tively low amounts of glycogen (0.3 Wmol glucose
of phosphoenolpyruvate had occurred: (mg cell protein)31 ) whereas cells from the stationary
202 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211
Table 9
Stoichiometry of fermentation of endogenous glucose (glycogen) in a culture of the cyanobacterium Microcystis aeruginosa PCC7806
grown under continuous light
Compound Wmol Wmol C Redox number Redox value
Glucose 4.3 325.8 0 0
Ethanol 3.5 +7.0 34 314
Acetate 3.0 +6.0 0 0
H2 1.6 0 32 33.2
CO2 5.0 +5.0 +4 +20
Cell carbon 7.2 +7.2 3 0.37a 32.7
Lactate 0.2 +0.6 0 0
3
+25.8/ 25.8 3
+20/ 19.9

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Balance 100% 1.01
The numbers in italics are calculated (see text), the other amounts were measured [37]. Incubation 6 h, total biomass 15 mg protein.
a
Per Wmol C.

growth phase contained signi¢cantly larger amounts ture also showed a larger amount of acetate formed
(2 Wmol glucose (mg cell protein)31 ) [61]. This huge than expected on the basis of the fermentation path-
di¡erence in glycogen content had only a moderately way. Moezelaar et al. [61] supposed that a homo-
e¡ect on the speci¢c rate of glucose fermentation. acetic fermentation pathway existed in M. chthono-
This rate was 0.40 nmol glucose min31 (mg cell plastes in addition to the mixed acid fermentation.
protein)31 in the stationary phase cells and 0.33 However, attempts to detect the key enzymes of the
nmol glucose min31 (mg cell protein)31 in the expo- homoacetic pathway failed [61]. Moreover, the as-
nentially growing cells. This was partly caused by the sumption of the presence of homoacetic fermentation
fact that the low glycogen containing cells also de- improved the O/R balance not su¤ciently (the O/R
graded the osmoprotectant glucosyl-glycerol. Only balance decreased from 1.51 to 1.22). In order to
the glucose of this compound was utilized and glyc- explain these high O/R balances of fermentation in
erol was excreted into the medium. The degradation M. chthonoplastes Moezelaar et al. [61] assumed that
of glucosyl-glycerol contributed 0.12 nmol glucose ferric iron could have served as electron acceptor.
min31 (mg cell protein)31 to the rate of glucose fer- They conceived that part of the formic acid is oxi-
mentation, leaving 0.21 nmol glucose min31 (mg cell dized to CO2 by ferric iron according to the follow-
protein)31 for the degradation of glycogen. This is ing equation [115]:
about half the rate of glycogen degradation of the
stationary phase cultures. The latter cultures did not HCOO3 ‡ 2Fe3‡ !CO2 ‡ H‡ ‡ 2Fe2‡ …2†
degrade the osmoprotectant glucosyl-glycerol. In
fact, the glycogen content of stationary phase cul- M. chthonoplastes was grown with an elevated
tures and the rate with which it is decomposed would amount of ferric-citrate in the medium because it
allow the organism to continue for 3.5 days. We resulted in homogeneous growth of this organism
have indeed observed that M. chthonoplastes
sur- [61]. Similarly, the reduction of ferric iron could
vived 4^5 days of incubation under dark anoxic con- also (in part) explain the high O/R balance of 1.30
ditions before it started to lyse. Due to rather similar in the case of fermentation in the presence of ele-
q q
glucose in both cultures the ATP were also quite com- mental sulfur [61]. With elemental sulfur present a
parable in both cultures: 1.65 and 1.32 nmol min31 reduction to sul¢de will take place. However, sul¢de
(mg cell protein)31 in the stationary and exponential will be oxidized back to elemental sulfur by ferric
phase cultures, respectively. iron [115] (see equation on p. 23). Thus, the amount
The fermentation patterns showed good carbon of sul¢de formed will be underestimated.
recoveries but rather poor O/R balances of 1.55 Recently, we have investigated the possibility of
and 1.22 in the exponential and stationary phase ferric iron reduction by cultures of M. chthono-
cultures, respectively [61]. The stationary phase cul- plastes. It was shown that Eq. 2 was indeed carried
 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211 203

out by this cyanobacterium [76]. However, the rates culture because of the much higher production of
at which it occurred were far from su¤cient to serve formate in that culture. Iron reduction in M. chtho-
as an important electron acceptor in fermentation noplastes has a rather low a¤nity for formate.
and taking iron reduction into account would have The ATP yield of fermentation in M. chthono-
only a minor in£uence on the O/R balance of fer- plastes can be calculated as follows. For every glu-
mentation in M. chthonoplastes. cose degraded 3 ATP are formed and 1 additional
In the light of what has been calculated for Micro- for each acetate produced. We calculated the amount
cystis PCC7806 it may be hypothesized that also in of glucose degraded as half of the sum of the
M. chthonoplastes some re-assimilation of CO2 could amounts of ethanol, acetate and lactate formed.
have take place. According to the proven fermenta- This gives 28.8 and 84.8 Wmol ATP for the low
tion pathway in this organism [61] the amount of and high glycogen containing cultures, respectively.

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

CO2 produced must equal the sum of the amounts Assuming CO2 assimilation by carboxylation of
of ethanol and acetate, minus the amount of for- phosphoenolpyruvate (see above) (which would
mate. Moreover, the amount of CO2 should equal cost 1 ATP (CO2 )31 ), YATP of 20 g biomass (mol
the amount of H2 . From Table 10 it is clear that ATP)31 , and a carbon content of 50% of cell dry
this was not the case. It is assumed that the missing weight it is calculated that 1.39 and 0.88 nmol
H2 had been used for the synthesis of structural cell ATP min31 (mg cell protein)31 are available for
material. This amount can be calculated as to equal maintenance purposes in the stationary phase and
the sum of the amounts of ethanol and acetate minus exponentially growing culture, respectively. These
the amounts of formate and H2 . The amount of re- numbers are well above what was calculated for Mi-
assimilated CO2 can than be calculated as half of the crocystis. Thus, from an energetic point of view the
molar amount of the missing H2 (assuming CH2 O as assumed re-assimilation of CO2 would be possible. It
the formula for structural cell material). From the would result in an increase of cell protein of 79 and
calculated amounts of CO2 reassimilated and cell 53 Wg (assuming 50% of cell material is protein) in
material produced, reasonable carbon recoveries the stationary phase and exponential culture, respec-
and O/R balances are obtained for both the expo- tively. This increase is very small on a total protein
nential (low glycogen) and stationary (high glycogen) content of respectively 35 and 15 mg.
cultures (Tables 10 and 11). Notwithstanding the fact that the stationary phase
The deviations from the ideal O/R balance of 1 culture of M. chthonoplastes contained almost seven
may be found in a possibly too high value for the times as much glycogen as the exponentially growing
reduced state of structural cell material and because culture, this resulted hardly in a higher rate of fer-
the reduction of iron was not included in these cal- mentation and supposed increase in biomass. In part
culations. Formate-mediated iron reduction may this was due to the fact that the exponentially grow-
have been more important in the stationary phase ing culture also utilized its osmoticum glucosyl-glyc-

Table 10
Stoichiometry of fermentation of endogenous glucose (glycogen and glucosyl-glycerol) in an exponentially growing (low glycogen) culture
of the cyanobacterium Microcoleus chthonoplastes
Compound Wmol Wmol C Redox number Redox value
Glucose 7.1 42.6 0 0
Ethanol 7.4 14.8 34 329.6
Acetate 7.1 14.2 0 0
Formate 5.1 5.1 +2 +10.2
H2 0.6 0 32 31.2
CO2 5 5 +4 +20
Cell carbon 4.4 4.4 30.37 31.6
43.5/42.6 +30.2/332.4
Balance 102% 0.93
The numbers in italics are calculated (see text), the other amounts were measured [61]. Incubation 24 h, total biomass 15 mg protein.
204 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211
Table 11
Stoichiometry of fermentation of endogenous glucose (glycogen) in a stationary phase (high glycogen) culture of the cyanobacterium Mi-
crocoleus chthonoplastes
Compound Wmol Wmol C Redox number Redox value

Glucose 20 120 0 0
Ethanol 17.6 35.2 34 370.4
Acetate 22.9 45.8 0 0
Formate 25.9 25.9 +2 +51.8
H2 1.4 0 32 32.8
CO2 8 8 +4 +32
Cell carbon 6.6 6.6 30.37 32.4
Lactate 0.8 2.4 0 0
3

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

123.9/120 +83.8/ 75.6
Balance 103% 1.11

The numbers in italics are calculated (see text), the other amounts were measured [61]. Incubation 24 h, total biomass 35 mg protein.

erol. Apparently, a faster growth was not possible. The limited rate of glycogen degradation in M.
Although the fermentation experiments were carried chthonoplastes may serve an important ecological
out in a nutrient-free bu¡er, it can be assumed that goal. It has been shown that this organism can sur-
su¤cient nutrients for growth must have been vive 4^5 days under anoxic conditions in the dark. In
present in the cells or as contaminants in the bu¡er. microbial mats, the environment in which M. chtho-
Growth of cyanobacteria under anaerobic conditions noplastes occurs anoxic dark conditions may persist
is not trivial. For instance, notwithstanding the fact for prolonged periods of time, particularly during
that M. chthonoplastes can perform sul¢de-depend- periods of increased rates of deposition. The impor-
ent anoxygenic photosynthesis and fermentation it is tance of a low rate of glycogen degradation can be
not capable of growth in the complete absence of exempli¢ed by the case of O. terebriformis
. Under
oxygen. Oxygen appears to be an essential nutrient aerobic conditions in the dark this organism depletes
for this organism [116]. On the other hand the activ- its energy storage quickly after which it dies. How-
ity of certain enzymes may have limited faster deg- ever, under anoxic conditions glycogen is degraded
radation of glycogen, although the measured enzyme much slower, allowing the organism to survive the
activities of the mixed acid fermentation in M. chtho- night period [28]. In fact, in order to prevent aerobic
noplastes were su¤cient to explain the rates of prod- (and fast) degradation of glycogen this organism
uct formation. The formation of lactate in the high moves into the anoxic part of the sediment during
glycogen containing culture hinted to an over£ow the dark [117].
metabolism caused by high intracellular concentra-
tions of fructose-1,6-bisphosphate and/or pyruvate
[75]. Relative to acetate the low production of etha- 8. Concluding remarks
nol in this culture may be explained by the low spe-
ci¢c activity of alcohol dehydrogenase relative to Most of the research on cyanobacteria concen-
acetate kinase [61]. Whatever caused the limited trates on their photoautotrophic mode of life.
rate of glycogen degradation it is likely to be respon- This, however, does not give credit to the fact
sible for the higher rest (maintenance) rate of ATP that these organisms are frequently faced with sit-
production of 1.39 nmol min31 (mg cell protein)31 . uations in which light is not available. This is not
The rest (maintenance) rate of the low glycogen con- only the case during the night but also during the
taining culture is with 0.88 slightly higher than the daytime cyanobacteria may be deprived of light and
one derived for Microcystis
. The fact that this cul- some symbiotic species live permanently in the
ture degrades part of its osmoticum glucosyl-glycerol dark. In order to survive short periods of darkness
may cost the organism some additional energy in cyanobacteria use endogenous carbohydrate (glyco-
order to maintain osmotic equilibrium [67]. gen) which is synthesized and stored in the light.
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 205
Glycogen is mobilized via the OPP pathway and thesis does not prevent fermentation. The constitu-
under aerobic conditions respiration may yield suf- tive property of fermentation has the advantage for
¢cient energy to allow growth. A few species are the organism that it can react immediately when an-
even capable of taking up a limited number of or- oxic conditions are established, which may occur
ganic compounds (mainly glucose, fructose and su- within minutes in some environments. On the other
crose) and grow chemoorganotrophically in the hand it can be asked how fermentation is regulated.
dark. Very little work has been done on the chemo- In O. limnetica the inhibition of aerobic respiration
organotrophic metabolism of cyanobacteria under by cyanide was su¤cient to start fermentation and in
anoxic conditions. Cyanobacteria exposed in their symbiotic Nostoc sp. fermentation did not even re-
natural environment to anoxic dark conditions pos- quire completely anoxic conditions and started at
sess the capacity to ferment endogenous storage low levels of oxygen. Thus neither light nor oxygen

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

carbohydrate and some species can even take up has a negative regulatory e¡ect on fermentation in
exogenous carbohydrate. The marine mat-forming these cyanobacteria. From the results obtained thus
cyanobacteria O. limosa and M. chthonoplastes far it is clear that in none of the cyanobacteria
also partly degraded their organic solutes that serve studied fermentation is regulated at the level of ex-
as osmoprotectants in these organisms. In M. pression of genes. It is possible that the fermentation
chthonoplastes the degradation of osmoprotectant pathways in these cyanobacteria are regulated (acti-
is particularly important when the amount of gly- vated or inhibited) by a particular metabolite. This
cogen is low. It is not clear how osmotic equilib- was for instance the case with lactate dehydrogenase
rium of the cell is maintained when the organic in Microcystis PCC 7806 (see Section 4). This type of
solute is degraded, but it is assumed that inorganic regulation should also be present when (part of) the
ions (probably K‡ ) are temporarily taking over this pentose phosphate pathway is involved as is the case
function. Although the maintenance of osmotic in heterolactic fermentation in O. limosa. Metabolic
equilibrium by potassium ions would take energy, control must ensure that the reductive pentose phos-
the energy content of the organic osmoprotectant is phate cycle operates only in the light and the oxida-
apparently of such importance for the organism tive process in the dark. However, in the majority of
that its mobilization is essential for dark anaerobic cyanobacterial fermentations the EMP pathway is
energy generation and weighs more than its func- involved and therefore the possibility that fermenta-
tion as maintaining osmotic equilibrium. The con- tion in these cyanobacteria is not subject to regula-
sequences of the catabolic degradation of the osmo- tion and is itself constitutive cannot be excluded.
protectant in cyanobacteria deserves further study, This would mean that in this case a small part of
both with regard of the precise mechanism of the the carbon ¢xed during the light is lost by fermenta-
achievement of osmotic equilibrium under anoxic tion. Another observation that supports this is the
dark conditions and its energetics. fact that M. chthonoplastes reduces ferric iron in the
The cyanobacteria capable of fermentation show a light as well as in the dark, both under aerobic and
variety of di¡erent pathways. These include homo- anoxic conditions at the same rate. The reduction of
and heterolactic acid fermentation, homoacetic acid ferric iron was shown to be enzyme catalyzed and
fermentation and mixed acid fermentations. In a few coupled to the oxidation of the fermentation product
species the pathways have been established by the formate [76,102]. Apparently, the advantage of being
identi¢cation of the enzymes. In all species investi- capable of reacting instantaneously to changing en-
gated the fermentation pathways appeared to be con- vironmental conditions is more important for the
stitutive. All enzymes were present in photoauto- organism than saving energy by inducing fermenta-
trophically grown cells. When cell suspensions were tion when it is needed. On the other hand the excre-
transferred to dark anoxic conditions fermentation tion of low-molecular organic compounds is of great
commenced without a lag. Pre-incubation in the importance for structure and functioning of the eco-
dark or under anoxic conditions did not increase system since it will provide substrates for growth of
enzyme activities or changed the rate of fermenta- other microorganisms (e.g. sulfate-reducing bacteria
tion. Also the addition of inhibitors of protein syn- in marine microbial mats [118]). It is evident that the
206 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211
subject of regulation of fermentation in cyanobacte- occurred. The advantage of sulfur reduction was
ria deserves more attention. mainly the possibility of a higher production of ace-
There is no doubt that the energy yield of fermen- tate which would yield additional ATP. An excep-
tation is low compared to phototrophic or respira- tion was probably O. limnetica but calculations
tory metabolism and therefore it was generally as- showed that the energy yield of sulfur respiration
sumed that fermentation in cyanobacteria would was only slightly higher as compared to homolactic
probably only be su¤cient to cover maintenance re- acid fermentation.
quirements. However, a number of observations are The property of fermentation is essential for those
not in agreement with this assumption. Species M. cyanobacteria that in their natural environment are
aeruginosa and M. chthonoplastes showed di¡erent exposed to anoxic conditions in the dark. Species
rates of fermentation depending on the amount of that did not possess this capacity died and lysed

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

storage carbohydrate in the cell. A higher rate of within 2^3 h after exposure to dark anoxic condi-
fermentation allows a higher rate of ATP produc- tions (L.J. Stal, unpublished results). Dark anaerobic
tion. Since it is not likely that maintenance require- metabolism expands the metabolic versatility of cya-
ments are di¡erent in cultures with low or high gly- nobacteria and also makes possible their ecological
cogen content it is evident that the additional ATP success. Moreover, the excretion of fermentation
production can be used for non-maintenance pur- products is essential for the structure and function-
poses. Moreover, O. limosa was capable of maintain- ing of ecosystems such as microbial mats in which
ing the high-energy-requiring process of nitrogen ¢x- photosynthesis by cyanobacteria is the driving force
ation under anoxic conditions in the dark. Carbon [121,122].
and redox balances indicated that in high glycogen
cultures some carbon must have been re-¢xed, ap-
parently at the expense of the ATP produced in ad- Acknowledgments
dition of maintenance requirement. Maintenance
requirements in cyanobacteria appeared to be ex- The comments of two anonymous reviewers on an
tremely low but were in the same order of magnitude earlier version of the paper are gratefully acknowl-
as the theoretical value which was calculated for edged.
E. coli .
Nothing is known about the intracellular levels of
the adenylate and pyridine nucleotide pools during Appendix
dark anoxic incubations of cyanobacteria capable of
fermentation. In Synechococcus sp. an abrupt change Estimation of the ATP production in Oscillatoria
of concentrations of ATP and NADPH occurs when agardhii required for maintenance
the culture is transferred from the light to the dark
under aerobic conditions. The ATP concentration According to Gons and Mur [123] the light-limited
returns to the light level within 15^20 min in the growth of phototrophic microorganisms is described
dark, whereas this was not the case with NADPH by :
[119]. This was taken as evidence for an e¤cient dark
Sy- dE
x dt c ˆ W ‡ Wm …A1†
1
energy generation in this organism. However, in
nechococcus
W W
g
sp. this energy generation was shown to
be dependent on oxygen [120]. It would be very in- where
3
x is the biomass (J), dE dt
/ the light uptake rate
teresting to carry out comparable studies with cya- (J h 1 ), Wg the speci¢c growth rate (h31 ), Wm the
nobacteria capable of fermentation. speci¢c maintenance rate (h31 ), and c the e¤ciency
Sulfur appeared to be the only electron acceptor of the conversion of light energy into biomass. Eq.
that is used during dark metabolism in many of the A1 can be arranged to :
cyanobacteria tested. In most cases it must be con-
cluded that it was unlikely that sulfur respiration
Wg ˆ q c3W
EW m …A2†
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 207

in which qE is the biomass-speci¢c light energy up- ATP being formed for every 3 H‡ . Thus, 1 mol of
take (h
31 ) : ATP is formed per 3 mol of light quanta absorbed:
dE
xATP ˆ 13.
qE ˆx1
W
dt
…A3† Substituting the above values in Eq. A5 gives:

For the lower speci¢c growth rates a plot of Wg ver- qmATP ˆ 2 01900410225 160
: W :
W
1
3
ˆ
sus qE results in a straight line with slope c. The
: U W

intercept with the abscissa corresponds to the speci¢c 2U1039 mol ATP min31 …mg dry weight†31
light energy uptake required for maintenance, qm E,
and extrapolation to the ordinate provides an esti- Since the protein content of biomass is 55% [6], this
3
mate for Wm . The relation between qm E and the cor- value corresponds to a qm ATP of approximately 4

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

responding rate of ATP production qm ATP is given by: nmol ATP min31 (mg protein)31 during growth at
a light intensity of 0.5 W m32 .
qmATP ˆ qmE xATP
O W …A4†
[124] in which qm is expressed in mol ATP per
ATP
hour per joule biomass, O is the energy per mol of References
light quanta (J mol31 ), and xATP the photochemical
[1] Smith, A.J. (1982) Modes of cyanobacterial carbon metabo-
e¤ciency of ATP formed per light quanta absorbed. lism. In: The Biology of Cyanobacteria (Carr, N.G. and
In order to express qm
ATP in mol ATP per min per mg Whitton, B.A., Eds.), pp. 47^85. Blackwell Scienti¢c, Oxford.
biomass, the value obtained with Eq. A4 has to be [2] Bergman, B., Gallon, J.R., Rai, A.N. and Stal, L.J. (1997) N2
multiplied by the heat of combustion of biomass Q ¢xation by non-heterocystous cyanobacteria. FEMS Micro-
(J mg31 ) and divided by 60: biol. Rev. 19, 139^185.
[3] Bergman, B., Johansson, C. and Soëderbaëck, E. (1992) The
xATP
qmATP ˆ qE QO 60
m
W W
…A5† Nostoc-Gunnera symbiosis. New Phytol. 122, 379^400.
W
[4] Matthijs, H.C.P. and Lubberding, H.J. (1988) Dark respira-
tion in cyanobacteria. In: Biochemistry of the Algae and Cy-
The speci¢c maintenance light energy uptake qm E is
anobacteria (Rogers, L.J. and Gallon, J.R., Eds), pp. 131^145.
not constant but increases with incident light inten- Clarendon Press, Oxford.

sity [125]. For the cyanobacterium O. agardhii qm

[5] Van Liere, L., Mur, L.R., Gibson, C.E. and Herdman, M.
E (1979) Growth and physiology of Oscillatoria agardhii Go-
values ranged from 0.004 h31 at 0.5 W m32 to mont cultivated in continuous culture with a light-dark cycle.
0.02 h31 at 40 W m32 [126]. Assuming that the Arch. Microbiol. 123, 315^318.
data obtained with the lowest light intensity result [6] Post, A.F., Loogman, J.G. and Mur, L.R. (1985) Regulation
in the most accurate estimation of qm of growth and photosynthesis by Oscillatoria agardhii grown
E , we have
used the qm value of 0.004 h31 to calculate qm . with a light/dark cycle. FEMS Microbiol. Ecol. 31, 97^102.
E ATP [7] Post, A.F., Loogman, J.G. and Mur, L.R. (1986) Photosyn-
The energy of the photosynthetically active radiation thesis, carbon £ows and growth of Oscillatoria agardhii Go-
(400^700 nm) of the lamps used to grow O. agardhii mont in environments with a periodic supply of light. J. Gen.
U
was 2.19 105 J (mol of quanta)31 [124]. The heat of Microbiol. 132, 2129^2136.
combustion of O. agardhii cells grown under light- [8] Tandeau de Marsac, N. and Houmard, J. (1994) Adaptation

limiting continuous culture was 22.1 J mg31 [5].

of cyanobacteria to environmental stimuli: new steps towards
molecular mechanisms. FEMS Microbiol. Rev. 104, 119^190.
[9] Saku, J. and Adhikary, S.P. (1982) Heterotrophic growth and
In oxygenic photosynthesis, eight quanta are min- pigment composition of four ¢lamentous blue-green algae.
imally required to release one molecule of O2 from Arch. Hydrobiol. (Suppl.) 63, 189^200.
water and to transport four electrons over the thyla- [10] De Philippis, R., Margheri, M.C. and Vincenzini, M. (1996)
koid membrane to ferredoxin. As a result of water Fermentation in symbiotic and free-living cyanobacteria.
Arch. Hydrobiol. Algol. Stud. 83, 459^468.
splitting and electron transport, eight protons accu-
[11] Wolk, C.P. and Sha¡er, P.W. (1976) Heterotrophic micro-
mulate inside the thylakoid lumen forming a proton and macro-cultures of a nitrogen-¢xing cyanobacterium.
motive force. ATP is generated by H‡ e¥ux from Arch. Microbiol. 102, 123^127.
the thylakoid lumen through ATP synthetase, one [12] Watanabe, A. and Yamamoto, Y. (1967) Heterotrophic nitro-
208 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211
gen ¢xation by the blue-green alga Anabaenopsis circularis. photoheterotrophic growth of a heterotrophic blue-green bac-
Nature 214, 738. terium. J. Bacteriol. 114, 695^700.
[13] Rippka, R. (1972) Photoheterotrophy and chemoheterotrophy [30] Smoker, J.A. and Barnum, S.R. (1990) Nitrogen ¢xation ac-
among unicellular blue-green algae. Arch. Mikrobiol. 87, 93^ tivity of a ¢lamentous, nonheterocystous cyanobacterium in
98. the presence and absence of exogenous, organic substrates.
[14] Khoja, T.M. and Whitton, B.A. (1971) Heterotrophic growth Arch. Microbiol. 153, 417^421.
of blue-green algae. Arch. Mikrobiol. 79, 280^282. [31] Raboy, B., Padan, E. and Shilo, M. (1976) Heterotrophic
[15] Khoja, T.H. and Whitton, B.A. (1975) Heterotrophic growth capacities of Plectonema boryanum. Arch. Microbiol. 110,
of ¢lamentous blue-green algae. Br. Phycol. J. 10, 139^148. 77^85.
[16] Fay, P. (1965) Heterotrophy and nitrogen ¢xation in Chloro- [32] White, A.W. and Shilo, M. (1975) Heterotrophic growth of
gloea fritschii. J. Gen. Microbiol. 39, 11^20. the ¢lamentous blue-green alga Plectonema boryanum. Arch.
[17] Miller, J.S. and Allen, M.M. (1972) Carbon utilization pat- Microbiol. 102, 123^127.
terns in the heterotrophic blue-green alga Chlorogloea fritschii. [33] Marquez, F.J., Sasaki, K., Kakizono, T., Nishio, N. and Na-

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

Arch. Microbiol. 86, 1^12. gai, S. (1993) Growth characteristics of Spirulina platensis in
[18] Hutson, K.G., Rogers, L.J., Haslett, B.G. and Boulter, D. mixotrophic and heterotrophic conditions. J. Ferment. Bio-
(1980) Ferredoxins from two cyanobacteria capable of heter- eng. 76, 408^410.
otrophic growth in the dark. FEMS Microbiol. Lett. 7, 279^ [34] Marquez, F.J., Nishio, N. and Nagai, S. (1995) Enhancement
284. of biomass and pigment production during growth of Spiru-
[19] Smith, A.J. and Hoare, D.S. (1977) Specialist phototrophs, lina platensis in mixotrophic culture. J. Chem. Tech. Biotech-
lithotrophs, and methylotrophs: a unity among a diversity nol. 62, 159^164.
of procaryotes? Bacteriol. Rev. 41, 419^448. [35] Anderson, S.L. and Mcintosh, L. (1991) Light-activated het-
[20] Joset-Espardellier, F., Astier, C., Evans, E.H. and Carr, N.G. erotrophic growth of the cyanobacterium Synechocystis sp.
(1978) Cyanobacteria grown under photoautotrophic, photo- strain PCC-6803 ^ A blue-light-requiring process. J. Bacteriol.
heterotrophic and heterotrophic regimes: sugar metabolism 173, 2761^2767.
and carbon dioxide ¢xation. FEMS Microbiol. Lett. 4, 261^ [36] Kiyohara, T.Y., Fujita, Y., Hattori, A. and Watanabe, A.
264. (1960) Heterotrophic culture of a blue-green alga, Tolypothrix
[21] Diako¡, S. and Scheibe, J. (1975) Cultivation in the dark of tenuis I. J. Gen. Appl. Microbiol. 6, 176^182.
the blue-green alga Fremyella diplosiphon : a photoreversible [37] Moezelaar, R. and Stal, L.J. (1997) A comparison of fermen-
e¡ect of green and red light on growth rate. Physiol. Plant. tation in the cyanobacterium Microcystis PCC7806 grown
34, 125^128. under a light-dark cycle and continuous light. Eur. J. Phycol.
[22] Moezelaar, R. and Stal, L.J. (1994) Fermentation in the uni- (in press).
cellular cyanobacterium Microcystis PCC7806. Arch. Micro- [38] Gorrell, T.E. and U¡en, R.L. (1977) Fermentative metabolism
biol. 162, 63^69. of pyruvate by Rhodospirillum rubrum after anaerobic growth
[23] Harder, R. (1917) Ernahrungsphysiologische Untersuchungen in darkness. J. Bacteriol. 131, 533^543.
an Cyanophyceen, hauptsaëchlich dem endophytischen Nostoc [39] Schoën, G. and Voelskow, H. (1976) Pyruvate fermentation in
punctiforme. Z. Bot. 9, 145. Rhodospirillum rubrum and after transfer from aerobic to
[24] Hoare, D.S., Ingram, L.O., Thurston, E.L. and Walkup, R. anaerobic conditions in the dark. Arch. Microbiol. 107, 87^92.
(1971) Dark heterotrophic growth of an endophytic blue- [40] Yen, H.C. and Marrs, B. (1977) Growth of Rhodopseudomo-
green alga. Arch. Mikrobiol. 78, 310^321. nas capsulata under anaerobic dark conditions with dimethyl
[25] Bottomley, P.J. and Van Baalen, C. (1978) Characteristics of sulfoxide. Arch. Biochem. Biophys. 181, 411^418.
heterotrophic growth in the blue-green alga Nostoc sp. strain [41] Madigan, M.T. and Gest, H. (1978) Growth of a photosyn-
Mac. J. Gen. Microbiol. 107, 309^318. thetic bacterium anaerobically in darkness, supported by `ox-
[26] Bottomley, P.J. and Van Baalen, C. (1978) Dark hexose me- idant-dependent' sugar fermentation. Arch. Microbiol. 117,
tabolism by photoautotrophically and heterotrophically 119^122.
grown cells of the blue-green alga (cyanobacterium) Nostoc [42] Van Gemerden, H. (1968) On the ATP generation by Chro-
sp. strain Mac. J. Bacteriol. 135, 888^894. matium in darkness. Arch. Mikrobiol. 64, 118^124.
[27] Beauclerk, A.D. and Smith, A.J. (1978) Transport of D-glu- [43] Cohen, Y., JÖrgensen, B.B., Revsbech, N.P. and Poplawski,
cose and 3-O-methyl-D-glucose in the cyanobacteria Aphano- R. (1986) Adaptation to hydrogen sul¢de of oxygenic and
capsa 6714 and Nostoc strain Mac. Eur. J. Biochem. 82, 187^ anoxygenic photosynthesis among cyanobacteria. Appl. Envi-
197. ron. Microbiol. 51, 398^407.
[28] Richardson, L.L. and Castenholz, R.W. (1987) Enhanced sur- [44] Oren, A. and Shilo, M. (1979) Anaerobic heterotrophic dark
vival of the cyanobacterium Oscillatoria terebriformis in dark- metabolism in the cyanobacterium Oscillatoria limnetica : sul-
ness under anaerobic conditions. Appl. Environ. Microbiol. fur respiration and lactate fermentation. Arch. Microbiol. 122,
53, 2151^2158. 77^84.
[29] Ingram, L.O., Calder, J.A., Van Baalen, C., Plucker, F.E. and [45] Gfeller, R.P. and Gibbs, M. (1984) Fermentative metabolism
Parker, P.L. (1973) Role of reduced exogenous organic com- of Chlamydomonas reinhardii. I. Photoassimilation of acetate.
pounds in the physiology of the blue-green bacteria (algae): Plant Physiol. 82, 160^166.
 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211 209

[46] Kreuzberg, K. and Martin, W. (1984) Oscillatory starch deg- terium Oscillatoria limosa incubated anaerobically in the dark.

radation and fermentation in the green alga Chlamydomonas Arch. Microbiol. 151, 558^564.

reinhardii . Biochim. Biophys. Acta 799, 291^297. [64] Heyer, H. and Krumbein, W.E. (1991) Excretion of fermenta-

[47] Cohen, Y., Krumbein, W.E., Goldberg, M. and Shilo, M. tion products in dark and anaerobically incubated cyanobac-

(1977) Solar Lake (Sinai). 1. Physical and chemical limnology. teria. Arch. Microbiol. 155, 284^287.

Limnol. Oceanogr. 22, 597^608. [65] Aoyama, K., Uemura, I., Miyake, J. and Asada, Y. (1997)

[48] Cohen, Y., Krumbein, W.E. and Shilo, M. (1977) Solar Lake Fermentative metabolism to produce hydrogen gas and organ-

(Sinai). 2. Distribution of photosynthetic microorganisms and ic compounds in a cyanobacterium, Spirulina platensis . J. Fer-

primary production. Limnol. Oceanogr. 22, 609^620. ment. Bioeng. 83, 17^20.

[49] Cohen, Y., Padan, E. and Shilo, M. (1975) Facultative anoxy- [66] Stal, L.J. and Reed, R.H. (1987) Low-molecular mass carbo-

genic photosynthesis in the cyanobacterium Oscillatoria limne- hydrate accumulation in cyanobacteria from a marine micro-

tica . J. Bacteriol. 123, 855^861. bial mat in response to salt. FEMS Microbiol. Ecol. 45, 305^

[50] Stal, L.J. (1995) Physiological ecology of cyanobacteria in 312.

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

microbial mats and other communities. New Phytol. 131, 1^ [67] Reed, R.H. and Stewart, W.D.P. (1988) The responses of

32. cyanobacteria to salt stress. In : Biochemistry of Algae and

[51] Revsbech, N.P., JÖrgensen, B.B., Blackburn, T.H. and Cohen, Cyanobacteria (Rogers, L.J. and Gallon, J.R., Eds.), pp.

Y. (1983) Microelectrode studies of the photosynthesis and 217^231. Clarendon Press, Oxford.

O2 , H2 S and pH pro¢les of a microbial mat. Limnol. Ocea- [68] Stal, L.J. (1992) Poly(hydroxyalkanoate) in cyanobacteria : an

nogr. 28, 1062^1074. overview. FEMS Microbiol. Rev. 103, 169^180.

[52] Reynolds, C.S. (1987) Cyanobacterial waterblooms. Adv. Bot. [69] Simon, R.D. (1987) Inclusion bodies in the cyanobacteria :

Res. 13, 67^143. cyanophycin, polyphosphate, polyhedral bodies. In : The Cy-

[53] Fallon, R.D. and Brock, T.D. (1981) Overwintering of Micro- anobacteria (Fay, P. and Van Baalen, C., Eds.), pp. 199^225.

cystis in Lake Mendota. Freshwater Biol. 11, 217^226. Elsevier, Amsterdam.

[54] Reynolds, C.S., Jaworski, G.H.M., Cmiech, H.A. and Lee- [70] Stal, L.J., Heyer, H., Bekker, B., Villbrandt, M. and Krum-

dale, G.F. (1981) On the annual cycle of the blue-green alga bein, W.E. (1989) Aerobic-anaerobic metabolism in the cya-

Microcystis aeruginosa Ku
ë tz Emend Elenkin. Phil. Trans. R. nobacterium Oscillatoria limosa . In : Microbial Mats. Physio-

Soc. Lond. Biol. 293, 419^477. logical Ecology of Benthic Microbial Communities (Cohen, Y.

[55] Reynolds, C.S. and Walsby, A.E. (1975) Waterblooms. Biol. and Rosenberg, E., Eds.), pp. 255^276. ASM, Washington,

Rev. 50, 437^481. DC.

[56] Ibelings, B.W. and Mur, L.R. (1992) Micropro¢les of photo- [71] Van der Oost, J. (1989) The Hydrogen Metabolism of the

synthesis and oxygen concentration in Microcystis scums. Unicellular Cyanobacterium Cyanothece PCC7822. PhD The-

FEMS Microbiol. Ecol. 86, 195^203. sis, Vrije Universiteit, Amsterdam.

[57] Stewart, W.D.P., Rowell, P. and Rai, A.N. (1983) Cyanobac- [72] Heise, R., Muller, V. and Gottschalk, G. (1989) Sodium de-

teria-eukaryotic plant symbioses. Ann. Microbiol. (Paris) 134 pendence of acetate formation by the acetogenic bacterium

B, 205^228. Acetobacterium woodii . J. Bacteriol. 171, 5473^5478.

[58] Tredici, M.R., Margheri, M.C., Giovannetti, L., De Philippis, [73] Heyer, H. (1989) Der anaerobe Dunkelmetabolismus von Os-
R. and Vincenzini, M. (1988) Heterotrophic metabolism and cillatoria limosa . PhD Thesis, Universita
ë t Oldenburg, Olden-

diazotrophic growth of Nostoc sp. from Cycas circinalis . Plant burg.

Soil 110, 199^206. [74] Stal, L.J., Bekker, S. and Meier, F. (1988) Hydrogenase activ-

[59] Stal, L.J. and Krumbein, W.E. (1986) Metabolism of cyano- ities in the marine nitrogen-¢xing cyanobacterium Oscillatoria
éme colloque
bacteria in anaerobic marine sediments. Deuxie limosa . Abstr. VI Int. Symp. Phot. Prok. p. 21.

international de è riologie
bacte marine, Actes de Colloques [75] Moezelaar, R., Teixeira de Mattos, M.J. and Stal, L.J. (1995)

Vol. 3, pp. 301^309. Gerbam, Ifremer, Brest. Lactate dehydrogenase in the cyanobacterium Microcystis
[60] Van der Oost, J., Bulthuis, B.A., Feitz, S., Krab, K. and PCC7806. FEMS Microbiol. Lett. 127, 47^50.

Kraayenhof, R. (1989) Fermentation metabolism of the uni- [76] Schaub, B.E.M. and Stal, L.J. (1996) Anaerobic reduction of

cellular cyanobacterium Cyanothece PCC 7822. Arch. Micro- iron and sulfur by the marine benthic cyanobacterium Micro-
biol. 152, 415^419. coleus chthonoplastes . Abstr. 5th Eur. Mar. Microbiol. Symp.

[61] Moezelaar, R., Bijvank, S.M. and Stal, L.J. (1996) Fermenta- p. 46.

tion and sulfur reduction in the mat-building cyanobacterium [77] Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy

Microcoleus chthonoplastes . Appl. Environ. Microbiol. 62, conservation in chemotrophic anaerobic bacteria. Bacteriol.

1752^1758. Rev. 41, 100^180.

[62] Margheri, M.C. and Allotta, G. (1993) Homoacetic fermenta- [78] Leach, C.K. and Carr, N.G. (1971) Pyruvate : ferredoxin ox-

tion in the cyanobacterium Nostoc sp. strain Cc from Cycas idoreductase and its activation by ATP in the blue-green alga

circinalis . FEMS Microbiol. Lett. 111, 213^218. Anabaena variabilis . Biochim. Biophys. Acta 245, 165^174.

[63] Heyer H., Stal, L.J. and Krumbein, W.E. (1989) Simultaneous [79] Bothe, H., Falkenberg, B. and Nolteernsting, U. (1974) Prop-

heterolactic and acetate fermentation in the marine cyanobac- erties and function of the pyruvate :ferredoxin oxidoreductase
210 L.J. Stal, R. Moezelaar / FEMS Microbiology Reviews 21 (1997) 179^211
from the blue-green alga Anabaena cylindrica. Arch. Mikro- [98] Sheridan, R.P. (1973) Hydrogen sul¢de production by Syne-
biol. 96, 291^304. chococcus lividus. J. Phycol. 9, 437^445.
[80] Neuer, G. and Bothe, H. (1982) The pyruvate :ferredoxin ox- [99] Pfennig, N. and Biebl, H. (1976) Desulfuromonas acetoxidans

idoreductase in heterocysts of the cyanobacterium Anabaena gen.nov. and sp. nov., a new anaerobic, sulfur-reducing ace-

cylindrica. Biochim. Biophys. Acta 716, 358^365. tate-oxidizing bacterium. Arch. Microbiol. 110, 3^12.

[81] Hallenbeck, P.C., Kochian, L.V. and Benemann, J.R.. (1981) [100] Stal, L.J. (1991) The sulfur metabolism of mat-building cya-

Hydrogen evolution catalyzed by hydrogenase in cultures of nobacteria in anoxic marine sediments. Kieler Meeresforsch.

cyanobacteria. Z. Naturforsch. 36C, 87^92. 8, 152^157.

[82] Houchins, J.P. and Burris, R.H.. (1981) Comparative charac- [101] Mishra, D. and Schmidt, A. (1992) Regulation and partly

terization of two distinct hydrogenases from Anabaena sp. puri¢cation of the ATP-sulfurylase from the cyanobacterium

strain 7120. J. Bacteriol. 146, 215^221. Synechococcus-6301. Z. Naturforsch. C47, 95^101.

[83] Sanchez, J.J., Palleroni, N.J. and Doudoro¡, M. (1975) Lac- [102] Stal, L.J. (1994) Microbial mats in coastal environments. In :

tate dehydrogenases in cyanobacteria. Arch. Microbiol. 104, Microbial Mats. Structure, Development and Environmental

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

57^65. Signi¢cance (Stal, L.J. and Caumette, P., Eds.), NATO ASI

[84] Garvie, E.I. (1980) Bacterial lactate dehydrogenases. Micro- Series, Vol. G35, pp. 21^32. Springer Verlag, Heidelberg.

biol. Rev. 44, 106^139. [103] Van Bergeijk, S.A. and Stal, L.J. (1996) The role of oxygenic

[85] Pearce, J. and Carr, N.G. (1967) The metabolism of acetate by phototrophic microorganisms in production and conversion

the blue-green algae Anabaena variabilis and Anacystis nidu- of dimethylsulfoniopropionate and dimethylsul¢de in micro-

lans. J. Gen. Microbiol. 49, 301^313. bial mats. In : Biological and Environmental Chemistry of

[86] Pelroy, R.A., Rippka, R. and Stanier, R.Y. (1972) Metabo- DMSP and Related Sulfonium Compounds (Kiene, R.P.,

lism of glucose by unicellular blue-green algae. Arch. Mikro- Visscher, P.T., Keller, M.D. and Kirst, G.O., Eds.), pp.

biol. 87, 303^322. 369^379. Plenum, New York.

[87] Fewson, C.A., Al-Ha¢dh, M. and Gibbs, M. (1962) Role of [104] Stewart, W.D.P., Fitzgerald, G.P. and Burris, R.H. (1968)

aldolase in photosynthesis. I. Enzyme studies with photosyn- Acetylene reduction by nitrogen-¢xing blue-green algae.

thetic organisms with special reference to blue-green algae. Arch. Mikrobiol. 62, 336^348.

Plant Physiol. 37, 402^406. [105] Stal, L.J. and Heyer, H. (1987) Dark anaerobic nitrogen

[88] Pearce, J. and Carr, N.G. (1969) The incorporation and me- ¢xation (acetylene reduction) in the cyanobacterium Oscilla-
tabolism of glucose by Anabaena variabilis. J. Gen. Microbiol. toria sp. FEMS Microbiol. Ecol. 45, 227^232.
54, 451^462. [106] Van Liere, L. and Mur, L.R.. (1979) Growth kinetics of

[89] Bishop, R.H. (1979) Regulatory characteristics of a fructose Oscillatoria agardhii Gomont in continuous culture, limited

bisphosphatase from the blue-green bacterium Anacystis nidu- in its growth by the light energy supply. J. Gen. Microbiol.

lans. Arch. Biochem. Biophys. 196, 295^300. 115, 153^160.

[90] Houchins, J.P. (1984) The physiology and biochemistry of [107] Snoep, J.L. (1992) Regulation of Pyruvate Catabolism in

hydrogen metabolism in cyanobacteria. Biochim. Biophys. Enterococcus faecalis. A Molecular Approach to Physiology.
Acta 768, 227^255. PhD Thesis, Universiteit van Amsterdam, Amsterdam.

[91] Howarth, D.C. and Codd, G.A. (1985) The uptake and pro- [108] Stal, L.J. and Krumbein, W.E. (1985) Oxygen protection of

duction of molecular hydrogen by unicellular cyanobacteria. nitrogenase in the aerobically nitrogen ¢xing nonheterocys-

J. Gen. Microbiol. 131, 1561^1569. tous cyanobacterium Oscillatoria sp. Arch. Microbiol. 143,

[92] Asada, Y. and Kawamura, S. (1984) Hydrogen evolution by 72^76.

Microcystis aeruginosa in darkness. Agric. Biol. Chem. 48, [109] Russell, J.B. and Cook, G.M. (1995) Energetics of bacterial

2595^2596. growth : Balance of anabolic and catabolic reactions. Micro-

[93] Asada, Y. and Kawamura, S. (1986) Screening for cyanobac- biol. Rev. 59, 48^62.

teria that evolve molecular hydrogen under dark and anaero- [110] Ljones, T. (1979) Nitrogen ¢xation and bioenergetics : the

bic conditions. J. Ferment. Technol. 64, 553^556. role of ATP in nitrogenase catalysis. FEBS Lett. 98, 1^8.

[94] Asada, Y., Kawamura, S. and Ho, K.-K. (1987) Hydrogenase [111] Konings, W.N., Lolkema, J.S. and Poolman, B. (1995) The

from the unicellular cyanobacterium Microcystis aeruginosa. generation of metabolic energy by solute transport. Arch.

Phytochemistry 26, 637^640. Microbiol. 164, 235^242.

[95] Almon, H. and Bo

ë ger, P. (1988) Hydrogen metabolism of the [112] Moezelaar, R. (1995) Fermentation in the Cyanobacteria Mi-
unicellular cyanobacterium Chroococcidiopsis thermalis crocystis aeruginosa and Microcoleus chthonoplastes. PhD

ATCC29380. FEMS Microbiol. Lett. 49, 445^449. Thesis, University of Amsterdam, Amsterdam.

[96] Serebriakova, L., Zorin, N.A. and Lindblad, P. (1994) Rever- [113] Atkinson, M.J. and Smith, S.V. (1983) C :N :P ratios of

sible hydrogenase in Anabaena variabilis ATCC29413. Pres- benthic marine plants. Limnol. Oceanogr. 28, 568^574.

ence and localization in non-N2 -¢xing cells. Arch. Microbiol. [114] Coleman, J.R. and Colman, B. (1981) Inorganic carbon ac-

161, 140^144. cumulation and photosynthesis in a blue-green alga as a

[97] Sheridan, R.P. and Castenholz, R.W. (1968) Production of function of external pH. Plant Physiol. 67, 917^921.

hydrogen sulphide by a thermophilic blue-green alga. Nature [115] Ehrlich, H.L. (1996) Geomicrobiology. Marcel Dekker, New

217, 1063^1064. York.

 L.J. Stal, R. Moezelaar/FEMS Microbiology Reviews 21 (1997) 179^211 211

Microcoleus chthono-
[116] De Wit, R., van Boekel, W.H.M. and van Gemerden, H. ing and fermentation in hot spring microbial mat commun-

plastes
(1988) Growth of the cyanobacterium ities. Appl. Environ. Microbiol. 62, 4598^4607.

on sul¢de. FEMS Microbiol. Ecol. 53, 203^209. [123] Gons, H.J. and Mur, L.R. (1975) An energy balance for algal

Oscillatoria terebriformis
[117] Richardson, L.L. and Castenholz, R.W. (1987) Diel vertical populations in light-limiting conditions. Verh. Int. Verein.

Scenedes-
movements of the cyanobacterium Limnol. 19, 2729^2733.

mus protuberans
in a sul¢de-rich hot spring microbial mat. Appl. Environ. [124] Gons, H.J. (1977) On the Light-limited Growth of

Microbiol. 53, 2142^2150. Fritsch. Ecological and Physiological As-

[118] Van Gemerden, H. (1993) Microbial mats : A joint venture. pects of the Energy Requirements for Growth and Mainte-

Mar. Geol. 113, 3^25. nance of a Planktonic Green Alga. PhD Thesis, University of

Anacystis nidulans Synechococcus

[119] Ihlenfeldt, M.J.A. and Gibson, J. (1975) CO2 ¢xation and its Amsterdam, Amsterdam.

Scenedes-
regulation in ( ). Arch. Mi- [125] Gons, H.J. and Mur, L.R. (1980) Growth rate and light

mus protuberans
crobiol. 102, 13^21. uptake rate in light-limited continuous cultures of

Oscillatoria agardhii
[120] Biggins, J. (1969) Respiration in blue-green algae. J. Bacter- Fritsch. Arch. Microbiol. 125, 9^17.

Downloaded from https://academic.oup.com/femsre/article/21/2/179/594506 by guest on 18 August 2020

iol. 99, 570^575. [126] Van Liere, L. (1979) On Gomont, Ex-

[121] Anderson, K.L., Tayne, T.A. and Ward, D.M. (1987) For- perimental Ecology and Physiology of a Nuisance Bloom-

mation and fate of fermentation products in hot spring cya- forming Cyanobacterium. PhD Thesis, University of Amster-

nobacterial mats. Appl. Environ. Microbiol. 53, 2343^2352. dam, Amsterdam.

[122] Nold, S.C. and Ward, D.M. (1996) Photosynthate partition-