The impacts of cyanobacteria on pulp-and-paper wastewater toxicity and biodegradation of wastewater contaminants

This study investigated the effects of cyanobacteria from pulp-and-paper waste-treatment systems on biological toxicity removal and biodegradation of certain wastewater contaminants. In field and batch studies, using the Microtox assay, cyanobacterial biomass and final wastewater toxicity were significantly correlated. In softwood-based wastewater, a decrease in toxicity was negatively correlated with cyanobacterial biomass, but the correlation was positive in hardwood-based wastewater. In the softwood-based wastewater, toxicity remained higher in the light than it was in the dark, whereas in hardwood-based wastewater, toxicity was lower in the light than it was in the dark. All of these results were light-dependent, suggesting that the photosynthetic growth of cyanobacteria is required to induce significant effects. When grown in mixed cultures with bacterial degraders, cyanobacteria from pulp-and-paper waste-treatment systems generally impeded the biodegradation of the wastewater contaminants phenol and dichloroacetate (DCA). However, there was one case where the cyanobacterium Phormidium insigne improved the bacterial degradation of DCA. Doubling inorganic nutrient concentrations did not improve phenol or DCA biodegradation in the majority of cases, indicating that nutrient competition is not a major factor. These data suggest that cyanobacteria play an important role during the biological treatment of contaminants, and, hence, toxicity removal in pulp-and-paper waste-treatment systems.     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

A novel cyanolytic bacterium,Pseudomonas fluorescens BG-E as a potential biological control agent for freshwater bloom-forming cyanobacteria Pseudanabaena spp.

The majority of bacterial antagonists identified to date are active against Microcystis. Therefore, this study aimed to isolate and characterize novel cyanolytic bacterial strains antagonistic against bloom-forming filamentous cyanobacteria. The bacterial strain BG-E isolated from the Bandagiriya Wewa in Sri Lanka was identified as Pseudomonas fluorescens (MZ007859) based on the 16S rRNA gene sequencing. BG-E showed 82% and 73% cyanolytic activity (CA) against Pseudanabaena sp. LW2 (MW288948) and Pseudanabaena lonchoides LW1 (MW288940), respectively, after 10 days of inoculation. The light microscopic images affirmed the complete disintegration in the filamentous structures of the tested Pseudanabaena species. The bacterial cell density of 15% v/v showed the CA with 95% and 89% cell lysis, respectively, in P. lonchoides and Pseudanabaena sp. LW2. Moreover, the results showed that >50% CA could be achieved by 0.100 and 1.00 (OD₇₃₀) cell densities for these same species. The highest CA of the cell-free supernatant of BG-E against P. lonchoides and bacterial culture against Pseudanabaena sp. LW2 illustrated the species-specific mode of action of BG-E. Although BG-E efficiently lysed the tested cyanobacterial species, the results of the MC-biodegradation assay confirmed its inability to degrade MC-LR cyanotoxin. Further, the BG-E strain lacks the mlrABCD gene cluster which is known to be responsible for the enzymatic degradation of MCs. The overall findings highlighted the applicability of P. fluorescens BG-E as a biological controlling agent to terminate blooms of freshwater filamentous cyanobacteria genus Pseudanabaena. The incorporation of cyanotoxin-degrading heterotrophic bacteria is recommended as a means of controlling toxic Pseudanabaena blooms.     

Diversity of cyanobacterial species and phylotypes in biofilms from the littoral zone of Lake Baikal

The majority of naturally occurring biofilms contain numerous microorganisms that have not yet been cultured. Additionally, there is little information available regarding the genetic structure and species diversity of these communities. Therefore, we characterised the species diversity, structure and metagenome of biofilms grown on stones and steel plates in the littoral zone of Lake Baikal (East Siberia, Russia) by applying three different approaches. First, light microscopy enabled identification of the species diversity of biofilm-forming cyanobacteria on different substrates with the dominance of Rivularia rufescens, Tolypothrix limbata, Chamaesiphon fuscus, Ch. subglobosus, and Heteroleibleinia pusilla. Additionally, scanning electron microscopy was used to show the spatial structure of biofilms. Finally, sequence analysis of 30,660 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to Cyanobacteria (8–46% sequences), Proteobacteria (14–43%), and Bacteroidetes (10–41%). Rivularia sp., Pseudanabaena sp., and Chamaesiphon spp. were the dominant cyanobacterial phylotypes.     

Effects of barley straw (Hordeum vulgare) on freshwater and brackish phytoplankton and cyanobacteria

A short-term laboratory study was conductedto investigate the effect of barley strawin controlling several common phytoplanktonand cyanobacterial species. Following aone-month incubation of barley straw incoarsely filtered fresh Potomac River andbrackish Patuxent River waters, the growthof six autotrophic taxa was followed inculture. Barley straw slurry reduced theyield of three taxa (Ankistrodesmusfalcatus, Chlorella capsulata, Isochrysis sp.) in comparison withcultures not receiving the slurry. Although no significant changes in growthwere detected with three other taxa (Cyclotella sp., Prorocentrumminimum, freshwater Pseudanabaenasp.), some patterns indicated potentialimpacts of the barley straw. First, ahigher addition of straw to Cyclotella sp. resulted in a lower biomassaccumulation than in cultures receivinglower levels. Second, the bloom-formingdinoflagellate Prorcentrum minimumwas apparently stimulated at low barleystraw levels, perhaps suggesting conditionsassociated with the straw(metals-chelation, bacterial-producednutrients) might stimulate dinoflagellategrowth. Third, species shifts wereobserved in two of the cultures, withbarley straw favoring shifts from Isochrysis to a Cyclotella sp. –Thalassiosira sp. mixture and shiftsfrom Pseudanabaena to a Pseudanabaena – Scenedesmus mixture. These results provide new records for thesusceptibility of freshwater and brackishphytoplankton taxa to barley strawexposure, including species-specificresponses and shifts in species dominancein mixed assemblages.     

Effect of exogenous glucose on electron flow to photosystem I and respiration in cyanobacterial cells

The effects of exogenous glucose on the rates of alternative pathways of photosystem II (PSII)-independent electron flow to PSI and of dark respiration in Synechocystis sp. 6803 cells were studied. The presence of glucose was shown to accelerate the electron flow to P700⁺, the PSI primary electron donor oxidized with Far-red light (FRL), which excites specifically only PSI. An increase in the glucose concentration was accompanied by a further activation of electron flow to PSI, which was supported by the dark donation of reducing equivalents to the electron transport chain. An increase in the external glucose concentration resulted also in the disappearance of lag-phase in the kinetics of P700⁺ reduction, which was observed in the cells incubated without glucose after FRL switching off. A similarity of nonphotochemical processes of electron transfer to PSI in cyanobacteria and higher plants was supposed, basing on the earlier observed fact of the occurrence of such lagphase in higher plants and its dependence on the exhausting of stromal reductants in the light. Acceleration of dark electron flow to PSI in the presence of glucose, a major respiratory substrate, may indicate the coupling between nonphotochemical processes in the photosynthetic and respiratory chains of electron transport in cyanobacterial cells. A close correlation between photosynthesis and respiration in cyanobacterial cells is also confirmed by a sharp acceleration of respiration with an increase in the glucose concentration in medium.     

HETEROTROPHY OF FOUR MARINE PHYTOPLANKTERS AT LOW SUBSTRATE CONCENTRATIONS1

Three pelagic marine phytoplankters, Coccolithus huxleyi, Skeletonema costatum, and Thalassiosira ro-tula, and a facultative heterotroph, Cyclotella cryp-tica, have been exposed to three organic substrates, viz, glucose, acetate, and glutamate, at low concentrations (organic carbon 0.25 mg/liter). Experiments were performed in the dark and light and the net assimilation of substrate was measured by using radiocarbon. The dark uptake of carbon dioxide was also determined, together with photosynthesis at near optimum light intensity. The expected heterotrophy was detected with Cyclotella cryptica. Thalassiosira rotula was found to assimilate glutamate at an appreciable rate. In all cases, however, the short-term uptake of carbon dioxide in the dark was the greatest assimilation rate measured. Values are discussed in relation to their ecological significance and it is concluded that heterotrophic survival of these and probably most other algae in the open ocean xuould be impossible unless they were in contact with a high concentration of substrate in the form of particulate matter.     

HETEROTROPHY OF FOUR MARINE PHYTOPLANKTERS AT LOW SUBSTRATE CONCENTRATIONS1

Three pelagic marine phytoplankters, Coccolithus huxleyi, Skeletonema costatum, and Thalassiosira rotula, and a facultative heterotroph, Cyclotella cryptica, have been exposed to three organic substrates, viz, glucose, acetate, and glutamate, at low concentrations (organic carbon 0.25 mg/liter). Experiments were performed in the dark and light and the net assimilation of substrate was measured by using radiocarbon. The dark uptake of carbon dioxide was also determined, together with photosynthesis at near optimum light intensity. The expected heterotrophy was detected with Cyclotella cryptica. Thalassiosira rotula was found to assimilate glutamate at an appreciable rate. In all cases, however, the short-term uptake of carbon dioxide in the dark was the greatest assimilation rate measured. Values are discussed in relation to their ecological significance and it is concluded that heterotrophic survival of these and probably most other algae in the open ocean would be impossible unless they were in contact with a high concentration of substrate in the form of particulate matter.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

Identification of two cyanobacterial strains isolated from the Kotel’nikovskii hot spring of the Baikal rift

Two cyanobacterial strains, Pseudanabaena sp. 0411 and Synechococcus sp. 0431, were isolated from a sample collected in the Kotel’nikovskii hot spring of the Baikal rift. According to the results of light and transmission electron microscopy, as well as of the phylogenetic analysis of the 16S rRNA gene, these cyanobacteria were classified as Pseudanabaena sp. nov. and Synechococcus bigranulatus Skuja. The constructed phylogenetic tree shows that the studied strains are positioned in the clades of cyanobacteria isolated from hydrothermal vents of Asia and New Zealand, separately from marine and freshwater members of these genera, including those isolated from Lake Baikal.     

Physiological characterization and electron microscopic investigation of cyanobacteria associated with wheat rhizosphere

Physiological attributes of a set of cyanobacterial strains, isolated from the rhizosphere of wheat (var. HD 2687), identified as belonging to the genera Calothrix (n = 3), Westiellopsis (1), Hapalosiphon (2) and Nostoc (2), were axenized and evaluated. The concentrated culture filtrates of three cyanobacterial strains — C. ghosei, H. intricatus and Nostoc sp. were able to enhance germination percentage, radicle and coleoptile length in inhibition experiments with wheat seeds. Indole-3-acetic acid (IAA) production was recorded in light and dark (+0.5 % glucose) incubated cultures. Incubation in the presence of tryptophan significantly enhanced IAA production. Acetylene-reducing activity was higher in light incubated cultures of Nostoc sp. followed by C. ghosei, while in the dark, C. ghosei recorded highest values. TLC of the filtrates revealed the presence of several amino acids such as histidine, and auxin-like compounds. Co-culturing with selected strains recorded significant enhancement in plant chlorophyll. Root sections of wheat seedlings co-cultured with C. ghosei revealed the presence of short filaments inside the root hairs and cortical region. Such strains can be promising candidates for developing plant growth promoting associations for wheat crop, besides serving as model systems for understanding the metabolic interactions of cyanobacteria with host plant, such as wheat.     

Use of glucose biosensors to measure extracellular glucose exudation by intertidal microphytobenthos in southern Tasmania

Micro glucose biosensors were used to measure net extracellular glucose produced by natural microphytobenthos and three diatom cultures (Amphora coffeaeformis, Navicula menisculus, Nitzschia longissima) from southern Tasmania, Australia. They were exposed to a light gradient in either nutrient‐replete or nutrient‐limiting conditions. Glucose exudation in the natural communities increased with increased light but the response in the cultures was variable. Similarly, nutrient‐replete conditions elicited lower rates of glucose exudation in the natural communities but produced variable species‐specific responses in the cultures. Increased glucose exudation mostly correlated with a reduction in maximum quantum yield (F_v/F_m). The same trend was observed in the natural communities for relative maximum electron transfer rates (rETR_max) but responses in the cultures were again variable and species‐specific. Responses of the three species to increased light and nutrient deficiency were variable, although glucose exudation, F_v/F_m and rETR_max was mostly lower in the nutrient‐limited media. In a second set of experiments species/communities were treated with/without antibiotics. In the dark, glucose concentrations in treatments with antibiotics remained unchanged, while in those with bacteria, it fell rapidly. In the sediment communities, glucose consumption in the dark was ~25% the rate of exudation at the highest light level. In culture, exudation rates were up to 100% greater than those with active bacteria. Rates of glucose consumption in the dark in the antibiotic–treated samples were negligible and up to 10⁴ times lower than those with active bacteria. These results demonstrate the important role extracellular glucose exudation has on maintaining an active microbial loop.     

GROWTH,PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM,CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N₂ fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O₂-sensitive N₂ fixation from photosynthetic O₂ evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.     

Molecular specificity and detection for Pseudanabaena (cyanobacteria) species based on rbcLX sequences

In decades, Pseudanabaena species have been frequently reported as cyanobacterial bloom components. However, these species are always overlooked because they cannot be observed easily owing to their small sizes. This study aimed to characterize Pseudanabaena species and detect them in field waters by developing an effective DNA marker. A region with approximately 600 bp in the rbcLX sequences was specific to Pseudanabaena species alone compared with other cyanobacterial genera. Therefore, new primers specific for Pseudanabaena were generated. By using this molecular tool, seasonal and spatial dynamics of Pseudanabaena populations of six sites in Lushui Reservoir, China were measured from July 2010 to August 2011. Pseudanabaena species existed in the reservoir throughout the year, and cell abundance ranged between 10⁴ and 10⁶ equivalent cells/L, with the peak at 8.07 × 10⁶ equivalent cells/L. The seasonal dynamics pattern of Pseudanabaena was similar to that of Microcystis but showed a slight lag at the transitional points of water temperature in the reservoir.     

Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso Sea bacterioplankton

Bacterioplankton are the primary trophic conduit for dissolved organic carbon (DOC) and linking community structure with DOC utilization is central to understanding global carbon cycling. We coupled stable isotope probing (SIP) with 16S rRNA pyrosequencing in dark seawater culture experiments on euphotic and mesopelagic communities from the Sargasso Sea. Parallel cultures were amended with equimolar quantities of four DO¹³C substrates to simultaneously evaluate community utilization and population‐specific incorporation. Of the substrates tested – two cyanobacterial products (exudates or lysates from a culture of Synechococcus) and two defined monosaccharides (glucose or gluconic acid) – the cyanobacterial exudates were incorporated by the greatest diversity of oligotrophic bacterioplankton populations in surface waters, including taxa from > 10 major subclades within the Flavobacteria, Actinobacteria, Verrucomicrobia and Proteobacteria (including SAR11). In contrast, the monosaccharide glucose was not incorporated by any taxa belonging to extant oligotrophic oceanic clades. Conversely, proteobacterial copiotrophs, which were rare in the ambient water (< 0.1% of sequences), grew rapidly on all DOC amendments at both depths, but with different substrate preferences among lineages. We present a new analytical framework for using SIP to detect DOC incorporation across diverse oligotrophic bacterioplankton and discuss implications for the ecology of bacterial–DOC interactions among populations of diverging trophic strategies.     

Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803

The plasmid hik31 operon (P3, slr6039‐slr6041) is located on the pSYSX plasmid in Synechocystis sp. PCC 6803. A P3 mutant (ΔP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed larger cells with division defects, glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light‐dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, energy metabolism genes; and an upregulation of cell envelope and transport and binding genes in the mutant. ΔP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism and cell division in the dark.     

More than just a pair of blue genes: how cyanobacteria adapt to changes in their light environment

Cyanobacteria require light to perform photosynthesis, but not all colors of light are equally useable for them. In particular, blue light-grown cyanobacterial strains, including the well-studied model organism Synechocystis sp. PCC 6803 (Synechocystis), have been observed to exhibit slower growth rates than white or red light-grown cells. In this issue of Physiologia Plantarum, Luimstra et al. (2020) have attempted to understand why cyanobacterial cells suffer under blue light. They measured the molecular and genetic responses of Synechocystis cells to being shifted from white light to blue light. They found that blue light-grown cells make changes that lead to a redistribution of energy flow between the two photosystems that power photosynthesis. These findings could help researchers identify avenues for optimizing photosynthesis in cyanobacterial species, a group of organisms which show great promise as potential solar-powered factories for the production of biofuels and other high-value products.     

Einfluss verschiedener Lichtwellenlängen auf die Zusammensetzung von Chlorella in Glucosekultur bei gehemmter Photosynthese

Summary Increasing blue light intensity inhibits the growth of Chlorella pyrenoidosa in glucose culture in which photosynthesis is blocked by DCMU, whereas red light supports growth which is the same as or better than that in dark controls.The action spectrum of light induced protein synthesis from exogenous glucose (photosynthesis inhibited, blue light addition resulting in growth >90% of the dark control) shows only one broad maximum at 450–490 nm which resembles the absorption spectrum of carotenoids.     

Irradiance Regulation of Photosynthesis and Respiration in Modern Marine Microbialites Built by Benthic Cyanobacteria in a Tropical Lagoon (New Caledonia)

Microbialites are organosedimentary deposits that have built up as a result of the growth and binding of detrital sediment by a benthic microbial community. This study focuses on microbialites built by monospecific populations of cyanobacteria in the south-west lagoon of New Caledonia, where they have been observed down to 20–25 m depth. The aim was to study their photosynthetic and respiratory responses to various light intensities. The Phormidium sp. TK1 microbialite was collected at 19 m depth and the P. crosbyanum (Tilden) microbialite was collected at 0.5 and 13 m depth. Phormidium sp. TK1 showed all the characteristic features of a low-light adapted species. The initial slope of the Photosynthesis versus Irradiance curve for this microbialite was close to the maximum quantum yield indicating an efficient light absorption and utilization at low light. The photosynthesis maximum was located 0.2–0.4 mm below the surface and did not shift with changing light intensity. Respiration rates were low and not enhanced by light; photoinhibition was observed at higher light intensities. In Phormidium crosbyanum (Tilden) microbialites, the photosynthesis maximum shifted downward to lower depths with increasing light, probably as a result of phototactic migration of cyanobacterial filaments, and light-enhanced respiration was observed at light intensities above light saturation. The photosynthetic para- meters measured in P. crosbyanum indicate that P. crosbyanum is capable of photo-acclimation at high light intensities. The gross productivity of the different microbialites was comparable to values measured in cyanobacterial stromatolites observed in other shallow environments. However, the microbialites studied here were characterized by a lower respiration / production ratio which indicates a higher growth efficiency.     

Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts

The photosynthetic properties of a range of lichens containing both green algal (11 species) and cyanobacterial (6 species) photobionts were examined with the aim of determining if there was clear evidence for the operation of a CO₂-concentrating mechanism (CCM) within the photobionts. Using a CO₂-gas-exchange system, which allowed resolution of fast transients, evidence was obtained for the existence of an inorganic carbon pool which accumulated in the light and was released in the dark. The pool was large (500–1000 nmol · mg Chl) in cyanobacterial lichens and about tenfold smaller in green algal lichens. In Hypogymnia physodes (L.) Nyl., which contains the green alga Trebouxia jamesii, a small inorganic carbon pool was rapidly formed in the light. Carbon dioxide was released from this pool into the gas phase upon darkening within about 20 s when photosynthesis was inhibited by the carbon-reduction-cycle inhibitor glycolaldehyde. In the absence of this inhibitor, release appeared to be obscured by carboxylation of ribulose bisphosphate. The kinetics of CO₂ uptake and release were monophasic. The operation of an active CCM could be distinguished from passive accumulation and release accompanying the reversible light-dependent alkalization of the stroma by the presence of saturation characteristics with respect to external CO₂. In Peltigera canina (L.) Willd., which contains the cyanobacterium Nostoc sp., a larger CO₂ pool was taken up over a longer period in the light and the release of this pool in the dark was slow, lasting 3–5 min. This pool also accumulated in the presence of glycolaldehyde, and under these conditions the CO₂ release was biphasic. In both species, photosynthesis at low CO₂ was inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide (EZ). Inhibition could be reversed fully or to a considerable extent by high CO₂. In Peltigera, EZ decreased both the accumulation of the CO₂ pool by the CCM and the rate of photosynthesis. Free-living cultures of Nostoc sp. showed a similar effect of EZ on photosynthesis, although it was more dramatic than that seen with the lichen thalli. In contrast, in Hypogymnia, EZ actually increased the size of the CO₂ pool, although it inhibited photosynthesis. This effect was also seen when glycolaldehyde was present together with EZ. Surprisingly, EZ did not alter the kinetics of either CO₂ uptake or release. Taken together, the evidence indicates the operation in cyanobacterial lichens of a CCM which is capable of considerable elevation of internal CO₂ and is similar to that reported for free-living cyanobacteria. The CCM of green algal lichens accumulates much less CO₂ and is probably less effective than that which operates in cyanobacterial lichens.