Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Heterologous Lactate Synthesis in Synechocystis sp. Strain PCC 6803 Causes a Growth Condition-Dependent Carbon Sink Effect.

Cyanobacteria are considered promising hosts for product synthesis directly from CO2 via photosynthetic carbon assimilation. The introduction of heterologous carbon sinks in terms of product synthesis has been reported to induce the so-called "carbon sink effect," described as the release of unused photosynthetic capacity by the introduction of additional carbon. This effect is thought to arise from a limitation of carbon metabolism that represents a bottleneck in carbon and electron flow, thus enforcing a downregulation of photosynthetic efficiency. It is not known so far how the cellular source/sink balance under different growth conditions influences the extent of the carbon sink effect and in turn product formation from CO2, constituting a heterologous carbon sink. We compared the Synechocystis sp. strain PCC 6803 wild type (WT) with an engineered lactate-producing strain (SAA023) in defined metabolic states. Unexpectedly, high-light conditions combined with carbon limitation enabled additional carbon assimilation for lactate production without affecting biomass formation. Thus, a strong carbon sink effect only was observed under carbon and thus sink limitation, but not under high-sink conditions. We show that the carbon sink effect was accompanied by an increased rate of alternative electron flow (AEF). Thus, AEF plays a crucial role in the equilibration of source/sink imbalances, presumably via ATP/NADPH balancing. This study emphasizes that the evaluation of the biotechnological potential of cyanobacteria profits from cultivation approaches enabling the establishment of defined metabolic states and respective quantitative analytics. Factors stimulating photosynthesis and carbon fixation are discussed. IMPORTANCE Previous studies reported various and differing effects of the heterologous production of carbon-based molecules on photosynthetic and growth efficiency of cyanobacteria. The typically applied cultivation in batch mode, with continuously changing growth conditions, however, precludes a clear differentiation between the impact of cultivation conditions on cell physiology and effects related to the specific nature of the product and its synthesis pathway. In this study, we employed a continuous cultivation system to maintain defined source/sink conditions and thus metabolic states. This allowed a systematic and quantitative analysis of the effect of NADPH-consuming lactate production on photosynthetic and growth efficiency. This approach enables a realistic evaluation of the biotechnological potential of engineered cyanobacterial strains. For example, the quantum requirement for carbon production was found to constitute an excellent indicator of the source/sink balance and thus a key parameter for photobioprocess optimization. Such knowledge is fundamental for rational and efficient strain and process development.

The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins.

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC50  = 4.1 µM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.

Effects of temperature and salinity on respiratory losses and the ratio of photosynthesis to respiration in representative Antarctic phytoplankton species.

The Southern Ocean (SO) is a net sink for atmospheric CO2 whereby the photosynthetic activity of phytoplankton and sequestration of organic carbon (biological pump) plays an important role. Global climate change will tremendously influence the dynamics of environmental conditions for the phytoplankton community, and the phytoplankton will have to acclimate to a combination of changes of e.g. water temperature, salinity, pH, and nutrient supply. The efficiency of the biological pump is not only determined by the photosynthetic activity but also by the extent of respiratory carbon losses of phytoplankton cells. Thus, the present study investigated the effect of different temperature and salinity combinations on the ratio of gross photosynthesis to respiration (rGP/R) in two representative phytoplankton species of the SO. In the comparison of phytoplankton grown at 1 and 4°C the rGP/R decreased from 11.5 to 7.7 in Chaetoceros sp., from 9.1 to 3.2 in Phaeocystis antarctica strain 109, and from 12.4 to 7.0 in P. antarctica strain 764, respectively. The decrease of rGP/R was primarily dependent on temperature whereas salinity was only of minor importance. Moreover, the different rGP/R at 1 and 4°C were caused by changes of temperature-dependent respiration rates but were independent of changes of photosynthetic rates. For further interpretation, net primary production (NPP) was calculated for different seasonal conditions in the SO with specific combinations of irradiance, temperature, and salinity. Whereas, maximum photosynthetic rates significantly correlated with calculated NPP under experimental 'Spring', 'Summer', and 'Autumn' conditions, there was no correlation between rGP/R and the respective values of NPP. The study revealed species-specific differences in the acclimation to temperature and salinity changes that could be linked to their different original habitats.

Photocalorespirometry (Photo-CR): A Novel Method for Access to Photosynthetic Energy Conversion Efficiency.

One key parameter for assessing the CO2 fixation in aquatic ecosystems but also for the productivity of photobioreactors is the energy conversion efficiency (PE) by the photosynthetic apparatus. PE strictly depends on a range of different fluctuating environmental conditions and is therefore highly variable. PE is the result of complex metabolic control. At the moment PE can only be determined indirectly. Furthermore, the currently available techniques either capture only short time processes, thus reflecting only parts of the photosynthetic engine, or quantify the total process but only with limited time resolution. To close this gap, we suggest for the first time the direct measurement of the fixed energy combined with respirometry, called photocalorespirometry (Photo-CR). The proof of the principle of Photo-CR was established with the microalga Chlamydomonas reinhardtii. The simultaneous measurement of oxygen production and energy fixation provides an calorespirometric ratio of -(437.9 ± 0.7) kJ mol-1 under low light conditions. The elevated calorespirometric ratio under high light conditions provides an indication of photo-protective mechanisms. The Photo-CR delivers the PE in real time, depending on the light intensity. Energetic differences less than 0.14% at radiation densities of up to 800 µE m-2 s-1 can be quantified. Other photosynthetic growth parameters (e.g. the specific growth rate of 0.071 h-1, the cell specific energy conservation of 30.9 ± 1.3 pW cell-1 at 150 µE m-2 s-1 and the number of photons (86.8) required to fix one molecule of CO2) can easily be derived from the Photo-CR data.

Electron balancing under different sink conditions reveals positive effects on photon efficiency and metabolic activity of Synechocystis sp. PCC 6803.

BACKGROUND: Cyanobacteria are ideal model organisms to exploit photosynthetically derived electrons or fixed carbon for the biotechnological synthesis of high value compounds and energy carriers. Much effort is spent on the rational design of heterologous pathways to produce value-added chemicals. Much less focus is drawn on the basic physiological responses and potentials of phototrophs to deal with natural or artificial electron and carbon sinks. However, an understanding of how electron sinks influence or regulate cellular physiology is essential for the efficient application of phototrophic organisms in an industrial setting, i.e., to achieve high productivities and product yields. RESULTS: The physiological responses of the cyanobacterium Synechocystis sp. PCC 6803 to electron sink variation were investigated in a systematic and quantitative manner. A variation in electron demand was achieved by providing two N sources with different degrees of reduction. By additionally varying light and CO2 availabilities, steady state conditions with strongly differing source-sink ratios were established. Balancing absorbed photons and electrons used for different metabolic processes revealed physiological responses to sink/source ratio variation. Surprisingly, an additional electron sink under light and thus energy limitation was found not to hamper growth, but was compensated by improved photosynthetic efficiency and activity. In the absence of carbon and light limitation, an increase in electron demand even stimulated carbon assimilation and growth. CONCLUSION: The metabolism of Synechocystis sp. PCC 6803 is highly flexible regarding the compensation of additional electron demands. Under light limitation, photosynthesis obviously does not necessarily run at its maximal capacity, possibly for the sake of robustness. Increased electron demands can even boost photosynthetic activity and growth.

Glycolate from microalgae: an efficient carbon source for biotechnological applications.

Glycolate is produced in autotrophic cells under high temperatures and Ci -limitation via oxygenation of ribulose-1,5-bisphosphate. In unicellular algae, glycolate is lost via excretion or metabolized via the C2 cycle by consuming reductants, ATP and CO2 emission (photorespiration). Therefore, photorespiration is an inhibitory process for biomass production. However, cells can be manipulated in a way that they become glycolate-producing 'cell factories', when the ratio carboxylation/oxygenation is 2. If under these conditions the C2 cycle is blocked, glycolate excretion becomes the only pathway of photosynthetic carbon flow. The study aims to proof the biotechnological applicability of algal-based glycolate excretion as a new biotechnological platform. It is shown that cells of Chlamydomonas can be cultivated under specific conditions to establish a constant and long-term stable glycolate excretion during the light phase. The cultures achieved a high efficiency of 82% of assimilated carbon transferred into glycolate biosynthesis without losses of function in cell vitality. Moreover, the glycolate accumulation in the medium is high enough to be directly used for microbial fermentation but does not show toxic effects to the glycolate-producing cells.

Towards an understanding of the molecular regulation of carbon allocation in diatoms: the interaction of energy and carbon allocation.

In microalgae, the photosynthesis-driven CO2 assimilation delivers cell building blocks that are used in different biosynthetic pathways. Little is known about how the cell regulates the subsequent carbon allocation to, for example, cell growth or for storage. However, knowledge about these regulatory mechanisms is of high biotechnological and ecological importance. In diatoms, the situation becomes even more complex because, as a consequence of their secondary endosymbiotic origin, the compartmentation of the pathways for the primary metabolic routes is different from green algae. Therefore, the mechanisms to manipulate the carbon allocation pattern cannot be adopted from the green lineage. This review describes the general pathways of cellular energy distribution from light absorption towards the final allocation of carbon into macromolecules and summarizes the current knowledge of diatom-specific allocation patterns. We further describe the (limited) knowledge of regulatory mechanisms of carbon partitioning between lipids, carbohydrates and proteins in diatoms. We present solutions to overcome the problems that hinder the identification of regulatory elements of carbon metabolism.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

PtAUREO1a and PtAUREO1b knockout mutants of the diatom Phaeodactylum tricornutum are blocked in photoacclimation to blue light.

Aureochromes are blue light receptors specifically found in photosynthetic Stramenopiles (algae). Four different Aureochromes have been identified in the marine diatom Phaeodactylum tricornutum (PtAUREO 1a, 1b, 1c, and 2). Since blue light is necessary for high light acclimation in diatoms, it has been hypothesized that Aureochromes might play an important role in the light acclimation capacity of diatoms. This hypothesis was supported by an RNAi knockdown line of PtAUREO1a, which showed a phenotype different from wild type cells when grown in either blue or red light. Here, we show for the first time the phenotype and the photoacclimation reaction of TALEN-mediated knockout mutants of PtAUREO1a and PtAUREO1b, clearly proving the necessity of Aureochromes for light acclimation under blue light. However, both mutants do also show specific differences in their respective phenotypes. Hence, PtAUREO1a and 1b are not functionally redundant in photoacclimation to blue light, and their specific contribution needs to be clarified further.

Assessing in situ dominance pattern of phytoplankton classes by dominance analysis as a proxy for realized niches.

This study looks at two facets of dominant phytoplankton classes during phytoplankton succession. A detailed assessment of this issue is of special interest with regard to realized niches from a theoretical point of view but also for lake management as practical application. A realized niche mirrors the functional adaptability of an organism in a lake-specific constellation of environmental parameters. Therefore, the characterization of realized niches could be a key factor for management of problematic waters. Different strategies exist to control eutrophication and the risk of blooms by harmful algae. During the last decades, many restoration measures were initiated to manage eutrophicated inland lakes. In the past, it has become evident several times that restoration strategies do not necessarily lead to a reduction of biomass of undesirable cyanobacteria but can even promote their development. Due to this uncertainty of success and the high costs for remediation strategies, new prediction tools are required - ideally, based on routine monitoring data. Therefore, we developed a new method to extract potential optimal growth conditions (POGC) as indicators of realized niches for different phytoplankton taxa from existing data to improve existing strategies used in lake remediation and restoration. The analysis presented in this work is based on dominance pattern of different phytoplankton groups relative to environmental variables. Interpretation of these dominance patterns as indicators of POGC showed distinct pattern for several phytoplankton classes for all investigated objects. We identified low nitrogen and phosphate concentrations as favorable condition for cyanobacteria in Lake Auensee and Lake Feldberger Haussee. The reservoir Bleilochtalsperre showed a high N/P-concentration and cyanobacteria dominance was generally very low.

Photosystem II cycle activity and alternative electron transport in the diatom Phaeodactylum tricornutum under dynamic light conditions and nitrogen limitation.

Alternative electron sinks are an important regulatory mechanism to dissipate excessively absorbed light energy particularly under fast changing dynamic light conditions. In diatoms, the cyclic electron transport (CET) around Photosystem II (PS II) is an alternative electron transport pathway (AET) that contributes to avoidance of overexcitation under high light illumination. The combination of nitrogen limitation and high-intensity irradiance regularly occurs under natural conditions and is expected to force the imbalance between light absorption and the metabolic use of light energy. The present study demonstrates that under N limitation, the amount of AET and the activity of CETPSII in the diatom Phaeodactylum tricornutum were increased. Thereby, the activity of CETPSII was linearly correlated with the amount of AET rates. It is concluded that CETPSII significantly contributes to AET in P. tricornutum. Surprisingly, CETPSII was found to be activated already at the end of the dark period under N-limited conditions. This coincided with a significantly increased degree of reduction of the plastoquinone (PQ) pool. The analysis of the macromolecular composition of cells of P. tricornutum under N-limited conditions revealed a carbon allocation in favor of carbohydrates during the light period and their degradation during the dark phase. A possible linkage between the activity of CETPSII and degree of reduction of the PQ pool on the one side and the macromolecular changes on the other is discussed.

The acclimation of Phaeodactylum tricornutum to blue and red light does not influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern.

Diatoms are major contributors to the aquatic primary productivity and show an efficient acclimation ability to changing light intensities. Here, we investigated the acclimation of Phaeodactylum tricornutum to different light quality with respect to growth rate, photosynthesis rate, macromolecular composition and the metabolic profile by shifting the light quality from red light (RL) to blue light (BL) and vice versa. Our results show that cultures pre-acclimated to BL and RL exhibited similar growth performance, photosynthesis rates and metabolite profiles. However, light shift experiments revealed rapid and severe changes in the metabolite profile within 15 min as the initial reaction of light acclimation. Thus, during the shift from RL to BL, increased concentrations of amino acids and TCA cycle intermediates were observed whereas during the BL to RL shift the levels of amino acids were decreased and intermediates of glycolysis accumulated. Accordingly, on the time scale of hours the RL to BL shift led to a redirection of carbon into the synthesis of proteins, whereas during the BL to RL shift an accumulation of carbohydrates occurred. Thus, a vast metabolic reorganization of the cells was observed as the initial reaction to changes in light quality. The results are discussed with respect to a putative direct regulation of cellular enzymes by light quality and by transcriptional regulation. Interestingly, the short-term changes in the metabolome were accompanied by changes in the degree of reduction of the plastoquinone pool. Surprisingly, the RL to BL shift led to a severe inhibition of growth within the first 48 h which was not observed during the BL to RL shift. Furthermore, during the phase of growth arrest the photosynthetic performance did not change. We propose arguments that the growth arrest could have been caused by the reorganization of intracellular carbon partitioning.

Unusual features of the high light acclimation of Chromera velia.

In the present study, the high light (HL) acclimation of Chromera velia (Chromerida) was studied. HL-grown cells exhibited an increased cell volume and dry weight compared to cells grown at medium light (ML). The chlorophyll (Chl) a-specific absorption spectra ([Formula: see text]) of the HL cells showed an increased absorption efficiency over a wavelength range from 400 to 750 nm, possibly due to differences in the packaging of Chl a molecules. In HL cells, the size of the violaxanthin (V) cycle pigment pool was strongly increased. Despite a higher concentration of de-epoxidized V cycle pigments, non-photochemical quenching (NPQ) of the HL cells was slightly reduced compared to ML cells. The analysis of NPQ recovery during low light (LL) after a short illumination with excess light showed a fast NPQ relaxation and zeaxanthin epoxidation. Purification of the pigment-protein complexes demonstrated that the HL-synthesized V was associated with the chromera light-harvesting complex (CLH). However, the difference absorption spectrum of HL minus ML CLH, together with the 77 K fluorescence excitation spectra, suggested that the additional V was not protein bound but localized in a lipid phase associated with the CLH. The polypeptide analysis of the pigment-protein complexes showed that one out of three known LHCr proteins was associated in higher concentration with photosystem I in the HL cells, whereas in ML cells, it was enriched in the CLH fraction. In conclusion, the acclimation of C. velia to HL illumination shows features that are comparable to those of diatoms, while other characteristics more closely resemble those of higher plants and green algae.

Light acclimation in diatoms: from phenomenology to mechanisms.

This review summarizes the current knowledge about light acclimation processes in diatoms. Against the background of the phenomenological description of the process in the 70s-80s, the recent progress in diatom genetics has generated new information about the underlying mechanisms. Although the general responses of diatoms to changes in the light climate are comparable to the green algal lineage, many differences in the underlying mechanisms have been observed in the last ten years, yielding clear evidence that the regulatory network in diatoms has unique traits that might explain their ecological success.

Correction: Aureochrome 1a Is Involved in the Photoacclimation of the Diatom Phaeodactylum tricornutum.

[This corrects the article on p. e74451 in vol. 8.].

Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum.

Aureochromes constitute a family of blue light (BL) receptors which are found exclusively in heterokont algae such as diatoms (Bacillariophyceae) and yellow-green algae (Xanthophyceae). Previous studies on the diatom Phaeodactylum tricornutum indicate that the formation of a high light acclimated phenotype is mediated by the absorption of BL and that aureochromes might play an important role in this process. P. tricornutum possesses four genes encoding aureochromes. In this study we confirm the nuclear localisation of the PtAUREO1a, 1b and 2 proteins. Furthermore we studied the physiology of light quality acclimation in genetically transformed P. tricornutum cell lines with reduced expression of the aureochrome 1a gene. The results demonstrate that the AUREO1a protein has a distinct function in light acclimation. However, rather unexpectedly AUREO1a seems to repress high light acclimation which resulted in a state of 'hyper' high light acclimation in aureo1a silenced strains. This was indicated by characteristic changes of several photosynthetic parameters, including increased maximum photosynthesis rates, decreased chlorophyll a contents per cell and increased values of non-photochemical quenching in AUREO1a silenced strains compared to wild type cultures. Strikingly, AUREO1a silenced strains exhibited phenotypic differences compared to wild type cells during cultivation under BL as well as under red light (RL) conditions. Therefore, AUREO1a might influence the RL signalling process, suggesting an interaction of AUREO1a with RL perception pathways.

Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum.

The objective of the present study was to test the hypothesis that the acclimation to different light intensities in the diatom Phaeodactylum tricornutum is controlled by light quality perception mechanisms. Therefore, semi-continuous cultures of P. tricornutum were illuminated with equal amounts of photosynthetically absorbed radiation of blue (BL), white (WL), and red light (RL) and in combination of two intensities of irradiance, low (LL) and medium light (ML). Under LL conditions, growth rates and photosynthesis rates were similar for all cultures. However, BL cultures were found to be in an acclimation state with an increased photoprotective potential. This was deduced from an increased capacity of non-photochemical quenching, a larger pool of xanthophyll cycle pigments, and a higher de-epoxidation state of xanthophyll cycle pigments compared to WL and RL cultures. Furthermore, in the chloroplast membrane proteome of BL cells, an upregulation of proteins involved in photoprotection, e.g. the Lhcx1 protein and zeaxanthin epoxidase, was evident. ML conditions induced increased photosynthesis rates and a further enhanced photoprotective potential for algae grown under BL and WL. In contrast, RL cultures exhibited no signs of acclimation towards increased irradiance. The data implicate that in diatoms the photoacclimation to high light intensities requires the perception of blue light.

Methane production from glycolate excreting algae as a new concept in the production of biofuels.

It is the aim of the present work to introduce a new concept for methane production by the interaction of a glycolate-excreting alga (Chlamydomonas reinhardtii) and methanogenic microbes operating in separate compartments within one photobioreactor. This approach requires a minimum number of metabolic steps to convert light energy to methane thereby reducing the energetic and financial costs of biomass formation, harvest and refinement. In this feasibility study it is shown that the physiological limitations for sustained glycolate production can be circumvented by the use of C. reinhardtii mutants whose carbon concentrating mechanisms or glycolate dehydrogenase are suppressed. The results also demonstrate that methanogenic microbes are able to thrive on glycolate as single carbon source for a long time period, delivering biogas composed of CO(2)/methane with only very minor contamination.