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Plastocyanin and cytochrome c(6), abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c(6) spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(l). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR / SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem I accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the di-iron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5’ extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.

During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO4 2−) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivation-triggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO4 2− transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO4 2− transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.

There are numerous sources of bioenergy that are generated by photosynthetic processes, for example, lipids, alcohols, hydrogen, and polysaccharides. However, generally only a small fraction of solar energy absorbed by photosynthetic organisms is converted to a form of energy that can be readily exploited. To more efficiently use the solar energy harvested by photosynthetic organisms, we evaluated the feasibility of generating bioelectricity by directly extracting electrons from the photosynthetic electron transport chain before they are used to fix CO(2) into sugars and polysaccharides. From a living algal cell, Chlamydomonas reinhardtii, photosynthetic electrons (1.2 pA at 6000 mA/m(2)) were directly extracted without a mediator electron carrier by inserting a nanoelectrode into the algal chloroplast and applying an overvoltage. This result may represent an initial step in generating "high efficiency" bioelectricity by directly harvesting high energy photosynthetic electrons.

The Chlamydomonas reinhardtii PSR1 gene is required for proper acclimation of the cells to phosphorus (P) deficiency. P-starved psr1 mutants show signs of secondary sulfur (S) starvation, exemplified by the synthesis of extracellular arylsulfatase and the accumulation of transcripts encoding proteins involved in S scavenging and assimilation. Epistasis analysis reveals that induction of the S-starvation responses in P-limited psr1 cells requires the regulatory protein kinase SNRK2.1, but bypasses the membrane-targeted activator, SAC1. The inhibitory kinase SNRK2.2 is necessary for repression of S-starvation responses during both nutrient-replete growth and P limitation; arylsulfatase activity and S deficiency-responsive genes are partially induced in the P-deficient snrk2.2 mutants and become fully activated in the P-deficient psr1snrk2.2 double mutant. During P starvation, the sac1snrk2.2 double mutants or the psr1sac1snrk2.2 triple mutants exhibit reduced arylsulfatase activity compared to snrk2.2 or psr1snrk2.2, respectively, but the sac1 mutation has little effect on the abundance of S deficiency-responsive transcripts in these strains, suggesting a post-transcriptional role for SAC1 in elicitation of S-starvation responses. Interestingly, P-starved psr1snrk2.2 cells bleach and die more rapidly than wild-type or psr1 strains, suggesting that activation of S-starvation responses during P deprivation is deleterious to the cell. From these results we infer that (i) P-deficient growth causes some internal S limitation, but the S-deficiency responses are normally inhibited during acclimation to P deprivation; (ii) the S-deficiency responses are not completely suppressed in P-deficient psr1 cells and consequently these cells synthesize some arylsulfatase and exhibit elevated levels of transcripts for S-deprivation genes; and (iii) this increased expression is controlled by regulators that modulate transcription of S-responsive genes during S-deprivation conditions. Overall, the work strongly suggests integration of the different circuits that control nutrient-deprivation responses in Chlamydomonas.

Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu+ transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of “back-up” enzymes for plastocyanin in photosynthesis and the ferroxidase in iron assimilation plus activation of alternative oxidase to handle the electron “overflow” resulting from reduced cytochrome oxidase function. Oxygen-dependent enzymes in the tetrapyrrole pathway (coproporphyrinogen oxidase and aerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition and tetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC core sequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is ideal for future investigation of nutritional manganese deficiency and selenoenzyme function. It is also suited for studies of trace nutrient interactions, nutrition-dependent metabolic changes, the relationship between photo-oxidative stress and metal homeostasis, and the important questions of differential allocation of limiting metal nutrients (e.g., to respiration vs. photosynthesis).

The Chlamydomonas reinhardtii transcription factor PSR1 is required for the control of activities involved in scavenging phosphate from the environment during periods of phosphorus limitation. Increased scavenging activity reflects the development of high-affinity phosphate transport and the expression of extracellular phosphatases that can cleave phosphate from organic compounds in the environment. A comparison of gene expression patterns using microarray analyses and quantitative PCRs with wild-type and psr1 mutant cells deprived of phosphorus has revealed that PSR1 also controls genes encoding proteins with potential “electron valve” functions—these proteins can serve as alternative electron acceptors that help prevent photodamage caused by overexcitation of the photosynthetic electron transport system. In accordance with this finding, phosphorus-starved psr1 mutants die when subjected to elevated light intensities; at these intensities, the wild-type cells still exhibit rapid growth. Acclimation to phosphorus deprivation also involves a reduction in the levels of transcripts encoding proteins involved in photosynthesis and both cytoplasmic and chloroplast translation as well as an increase in the levels of transcripts encoding stress-associated chaperones and proteases. Surprisingly, phosphorus-deficient psr1 cells (but not wild-type cells) also display expression patterns associated with specific responses to sulfur deprivation, suggesting a hitherto unsuspected link between the signal transduction pathways involved in controlling phosphorus and sulfur starvation responses. Together, these results demonstrate that PSR1 is critical for the survival of cells under conditions of suboptimal phosphorus availability and that it plays a key role in controlling both scavenging responses and the ability of the cell to manage excess absorbed excitation energy. While phosphorus (P) is an abundant element in the Earth’s crust, its availability can limit the growth of organisms present in both aquatic and terrestrial environments. P is essential for many fundamental processes that sustain life, including nucleic acid synthesis, membrane synthesis, energy metabolism, signaling, redox reactions, and modification of protein activities. The major form of P readily assimilated and utilized by most or

The unicellular green alga Chlamydomonas reinhardtii is a valuable model for studying metal metabolism in a photosynthetic background. A search of the Chlamydomonas expressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally for Saccharomyces cerevisiae . They include a multicopper ferroxidase (encoded by Fox1 ), an iron permease (encoded by Ftr1 ), a copper chaperone (encoded by Atx1 ), and a copper-transporting ATPase. A cDNA, Fer1 , encoding ferritin for iron storage also was identified. Expression analysis demonstrated that Fox1 and Ftr1 were coordinately induced by iron deficiency, as were Atx1 and Fer1 , although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 of C. reinhardtii is also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression in Chlamydomonas indicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth of C. reinhardtii under copper- and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficient Chlamydomonas cells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.

Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.

Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c(6) and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper- conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper-deficient conditions, the crd strains fail to accumulate photosystem I and light-harvesting complex I, and they contain reduced amounts of light-harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate-bridged di-iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper- or oxygen-deficient cells, and Crd1 function is required for adaptation to these conditions.

Publisher Summary In this chapter, some basic metabolic processes that are highly sensitive to cellular P levels, explore P acquisition and assimilation in Chlamydomonas are enumerated. Our current understanding of how limitation for available P alters metabolic and regulatory processes in this alga is summarized. It integrates biochemical, physiological, genetic, molecular, and genomic information to define the interactions of the key regulator PSR1 with the promoters of target genes and potential partner proteins with P deprivation. The sustainability of agricultural yields and the quality of aquatic ecosystems will benefit from more efficient use of Pi by crop plants, which would also decrease the dependence of farmers on rock Pi reserves, the mining of which can have serious economic and ecological consequences Genetic and molecular characteristics coupled with genome-wide analyses have made Chlamydomonas an excellent model for elucidating the ways in which photosynthetic eukaryotes sense and respond to their nutrient environment. It is also critical that we define the full range of genes controlled by PSR1 and the functions of the Polypeptides that they encode if we are to understand the ways in which photosynthetic organisms, whether microbe or plant, survive conditions that severely limit their growth potential and enhance the potentially toxic effects that may be triggered by the absorption of excess excitation energy and the concomitant generation of reactive oxygen species.

Photosynthetic organisms have developed elaborate mechanisms to acquire macronutrients and to adjust to conditions in which those nutrients become limiting to growth. Some of the responses of photosynthetic organisms to macronutrient limitation may be specific for a particular nutrient and involve the development of various mechanisms to scavenge the limiting nutrient from the external milieu, which may require elevated synthesis of high affinity transport systems, the redistribution of internal nutrient stores and the synthesis of hydrolytic enzymes that release the nutrient from organic substrates in the soil. Other responses may be of a more general nature, occurring during a number of different nutrient limitation conditions, and involve modifying the biosynthetic machinery of the cell, including the photosynthetic apparatus. In this review we focus on the acquisition of the macronutrients nitrogen, sulfur and phosphorus from the environment, and the ways in which the unicellular green alga Chlamydomonas reinhardtii acclimates to changes in its nutrient environment.

We have developed a single cell/organelle platform to provide experimental access and control over the electrochemical reactions involved in metabolic pathways of biological microorganisms. As sensing elements we have been using unique needle shaped Atomic Force Microscope (AFM) probes with integrated single or dual electrochemical (EC) nano electrodes. The probe system is designed for local probing of the cytosolic cell environment and cell organelles using amperometric, potentiometric and impedance spectroscopic methods. The sharpness of the probe tip is less than 30nm and allows for a reliable penetration of cell membranes. Reliable immobilization of living single cells and full control of the chemical micro environment close to the single cell has been achieved by introducing a open micro channel systems. Furthermore, this probing set up significantly reduces parasitic electrical capacitances and allows for current detection in sub pico-ampere regime at high signal band widths. Fabrication strategies of the AFM/EC nano probe system as well as open channel micro fluidic systems for single cell/organelle probing will be introduced. Further, the results of using this platform to explore in situ energy conversion reactions in single algae cells and chloroplast and their potential for bio energy sources will be presented and discussed.

A genetic screen for Chlamydomonas reinhardtii mutants with copper-dependent growth or nonphotosynthetic phenotypes revealed three loci, COPPER RESPONSE REGULATOR 1 (CRR1), COPPER RESPONSE DEFECT 1 (CRD1), and COPPER RESPONSE DEFECT 2 (CRD2), distinguished as regulatory or target genes on the basis of phenotype. CRR1 was shown previously to be required for transcriptional activation of target genes like CYC6, CPX1, and CRD1, encoding, respectively, cytochrome c6 (which is a heme-containing substitute for copper-containing plastocyanin), coproporphyrinogen III oxidase, and Mg-protoporphyrin IX monomethylester cyclase. We show here that CRR1 is required also for normal accumulation of copper proteins like plastocyanin and ferroxidase in copper-replete medium and for apoplastocyanin degradation in copper-deficient medium, indicating that a single pathway controls nutritional copper homeostasis at multiple levels. CRR1 is linked to the SUPPRESSOR OF PCY1-AC208 13 (SOP13) locus, which corresponds to a gain-of-function mutation resulting in copper-independent expression of CYC6. CRR1 is required also for hypoxic growth, pointing to a physiologically meaningful regulatory connection between copper deficiency and hypoxia. The growth phenotype of crr1 strains results primarily from secondary iron deficiency owing to reduced ferroxidase abundance, suggesting a role for CRR1 in copper distribution to a multicopper ferroxidase involved in iron assimilation. Mutations at the CRD2 locus also result in copper-conditional iron deficiency, which is consistent with a function for CRD2 in a pathway for copper delivery to the ferroxidase. Taken together, the observations argue for a specialized copper-deficiency adaptation for iron uptake in Chlamydomonas.

The molecular mechanisms underlying the onset of Fe-deficiency chlorosis and the maintenance of photosynthetic function in chlorotic chloroplasts are relevant to global photosynthetic productivity. We describe a series of graded responses of the photosynthetic apparatus to Fe-deficiency, including a novel response that occurs prior to the onset of chlorosis, namely the disconnection of the LHCI antenna from photosystem I (PSI). We propose that disconnection is mediated by a change in the physical properties of PSI-K in PSI in response to a change in plastid Fe content, which is sensed through the occupancy, and hence activity, of the Fe-containing active site in Crd1. We show further that progression of the response involves remodeling of the antenna complexes—specific degradation of existing proteins coupled to the synthesis of new ones, and establishment of a new steady state with decreased stoichiometry of electron transfer complexes. We suggest that these responses are typical of a dynamic photosynthetic apparatus where photosynthetic function is optimized and photooxidative damage is minimized in graduated responses to a combination of nutrients, light quantity and quality.

Crd1 (Copper response defect 1), which is required for the maintenance of photosystem I and its associated light-harvesting complexes in copper-deficient (-Cu) and oxygen-deficient (-O(2)) Chlamydomonas reinhardtii cells, is localized to the thylakoid membrane. A related protein, Cth1 (Copper target homolog 1), is shown to have a similar but not identical function by genetic suppressor analysis of gain-of-function sct1 (suppressor of copper target 1) strains that are transposon-containing alleles at CTH1. The pattern of Crd1 versus Cth1 accumulation is reciprocal; Crd1 abundance is increased in -Cu or -O(2) cells, whereas Cth1 accumulates in copper-sufficient (+Cu), oxygenated cells. This expression pattern is determined by a single trans-acting regulatory locus, CRR1 (COPPER RESPONSE REGULATOR 1), which activates transcription in -Cu cells. In +Cu cells, a 2.1-kb Cth1 mRNA is produced and translated, whereas Crd1 is transcribed only at basal levels, leading to Cth1 accumulation in +Cu cells. In -Cu cells, CRR1 function determines the activation of Crd1 expression and the production of an alternative 3.1-kb Cth1 mRNA that is extended at the 5' end relative to the 2.1-kb mRNA. Synthesis of the 3.1-kb mRNA, which encodes six small upstream open reading frames that possibly result in poor translation, blocks the downstream promoter through transcriptional occlusion. Fluorescence analysis of wild-type, crd1, and sct1 strains indicates that copper-responsive adjustment of the Cth1:Crd1 ratio results in modification of the interactions between photosystem I and associated light-harvesting complexes. The tightly coordinated CRR1-dependent regulation of isoenzymes Cth1 and Crd1 reinforces the notion that copper plays a specific role in the maintenance of chlorophyll proteins.

Chlamydomonas reinhardtii activatesCpx1, Cyc6, and Crd1, encoding, respectively, coproporphyrinogen oxidase, cytochromec 6, and a novel di-iron enzyme when transferred to oxygen-deficient growth conditions. This response is physiologically relevant because C. reinhardtiiexperiences these growth conditions routinely, and furthermore, one of the target genes, Crd1, is functionally required for normal growth under oxygen-depleted conditions. The same genes are activated also in response to copper-deficiency through copper-response elements that function as target sites for a transcriptional activator. The core of the copper-response element, GTAC, is required also for the hypoxic response, as is a trans-acting locus, CRR1. Mercuric ions, which antagonize the copper-deficiency response, also antagonize the oxygen-deficiency response of these target genes. Taken together, these observations suggest that the oxygen- and copper-deficiency responses share signal transduction components. Nevertheless, whereas the copper-response element is sufficient for the nutritional copper response, the oxygen-deficiency response requires, in addition, a second cis-element, indicating that the response to oxygen depletion is not identical to the nutritional copper response. The distinction between the two responses is also supported by comparative analysis of the response of the target genes,Cyc6, Cpx1, and Crd1, to copper versus oxygen deficiency. A Crr1-independent pathway forHyd1 expression in oxygen-depleted C. reinhardtii demonstrates the existence of multiple oxygen/redox-responsive circuits in this model organism.

The nuclear genome of Chlamydomonas reinhardtii contains genetic information for the functionally equivalent photosynthetic electron carriers plastocyanin and cytochrome c6 (cyt c6). Plastocyanin is the major copper sink in Chlamydomonas, binding a single copper ion at its active site which is required for the redox activity of the protein. Expression of plastocyanin and cyt c6 is controlled by the availability of copper ions, with approximately 9 × 106 copper ions per cell required to maintain a full complement of plastocyanin molecules (1). If the cellular copper ion concentration decreases below this amount, cyt c6 is synthesized to restore the photosynthetic carrier capacity. Chlamydomonas also responds to copper deficiency by coordinate induction of the tetrapyrrole biosynthetic enzyme coproporphyrinogen (coprogen) oxidase (2), presumably to accommodate the increased demand for heme. High affinity copper transport and copper reductase activities are also induced, and the production of cyt c6 enables diversion of copper from plastocyanin to essential copper proteins such as mitochondrial cytochrome oxidase. Copper-responsive elements have been identified by site-directed mutational analysis of the promoters of the Cyc6 and Cpxl genes. We are using insertional mutagenesis to isolate genes involved in the regulation of copper-responsive gene expression as well as genes encoding additional targets of the pathway. In addition, we describe the phenotypes of mutants arising from serendipitous insertions of the Arg7 marker and promoter/reporter constructs into novel targets of copper-dependent gene expression. In these copper response target (crt) mutants the chlorophyll content of copper-deficient cells is reduced and photosynthetic electron transfer is inhibited. Further analysis of the crtl mutant revealed that in this strain PSI and LHC polypeptides are less abundant in copper-deficient cells, whereas in wild-type cells these proteins accumulate independently of copper.

Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.