Sulfur-free Medium Research Articles

A novel sulfur supply strategy for maximizing lipid production in Tribonema minus (Xanthophyceae)

Tribonema minus, a promising filamentous oleaginous microalga, was cultured under different nutrient concentrations and different culture modes (fed-batch culture, two-step culture) to study the method of rapid regulation of its lipid metabolism. In contrast to many other oleaginous microalgae, T. minus did not show that nitrogen stress promoted lipid accumulation; however, sulfur deficiency promoted rapid lipid accumulation with a maximum lipid content of 54% of dry weight. Increasing the MgSO4 concentration significantly increased nitrogen uptake and biomass (10.09 g/L). Lipid productivity was significantly increased by the two-step culture using a medium with a high concentration of MgSO4 in the first step and a sulfur-free medium in the second step. In addition, it was found that the lipid content of T. minus was negatively correlated with the intracellular sulfur content when the intracellular sulfur content was below 0.6%. This study provides a new approach for industrial lipid production in T. minus.

Biodesulfurization of the mixture of dibenzothiophene and its alkylated derivatives by Sphingomonas subarctica T7b

Organosulfur compounds classified as dibenzothiophenes (DBTs) and their derivatives are contained in petroleum. When used as fuel, these substances release SOx emissions, thus contributing to air pollution, acid rain, and climate change. Therefore, it is necessary to reduce the content of these organic sulfur compounds in fuels and one way to achieve this is through bacterial desulfurization. This study reports the biodesulfurization process of a mixture of DBT, 4-hexyl DBT, 4,6-dibutyl DBT, and various organosulfur compounds in light gas oil (LGO). The experiment was conducted by treating 1 mL of aromatic organosulfur compounds with 100 mg/L in \textit{n}-tetradecane or 1 mL LGO with 5 mL mineral salts in sulfur-free medium, incubated at 27 °C for 5 days with shaking at 273 rpm. Gas chromatography analyses revealed that the growing Sphingomonas subarctica T7b cells desulfurized and converted 88.29% of DBT to 2-hydroxybiphenyl as a metabolite while a mixture of DBT and 4,6-dibutyl DBT was desulfurized at 86.40\% and 7.00%, respectively. Furthermore, the mixture of DBT, 4-hexyl DBT, and 4,6-dibutyl DBT had a desulfurization percentage of 84.40%, 41.00%, and 6.66%, respectively, after five days of incubation. The compounds were observed to desulfurize slightly better as single compounds compared to when mixed with other aromatic sulfur compounds.

Poly-β-Hydroxybutyrate Production by Rhodopseudomonas sp. Grown in Semi-Continuous Mode in a 4 L Photobioreactor

The synthesis of polyhydroxybutyrate (PHB) by photosynthetic non-sulfur bacteria is a potential approach for producing biodegradable plastics. In this work, acetate was used as a single carbon source to study the effect on PHB formation in Rhodopseudomonas sp. cultured in a cylindrical four-liter photobioreactor under semi-continuous mode. The cultivation process is divided into a symmetrical growth phase and a PHB accumulation phase separated temporally. The symmetrical growth phase (nutrient sufficient conditions) was followed by a sulfur-limited phase to promote PHB accumulation. The main novelty is the progressive lowering of the sulfur concentration into Rhodopseudomonas culture, which was obtained by two concomitant conditions: (1) sulfur consumption during the bacterial growth and (2) semi-continuous growth strategy. This caused a progressive lowering of the sulfur concentration into Rhodopseudomonas culturedue to the sulfur-free medium used to replace 2 L of culture (50% of the total) that was withdrawn from the photobioreactor at each dilution. The PHB content ranged from 9.26% to 15.24% of cell dry weight. At the steady state phase, the average cumulative PHB was >210 mg/L. Sulfur deficiency proved to be one of the most suitable conditions to obtain high cumulative PHB in Rhodopseudomonas culture.

Bioremediation of copper polluted wastewater-like nutrient media and simultaneous synthesis of stable copper nanoparticles by a viable green alga

Environmentally benign, algae-mediated biosynthesis of valuable copper-based materials in wastewater, combined with cupric ion (Cu2+) bioremediation, has the potential to reduce the cost of wastewater treatment and generate high quality biosolids. This study investigated the ability of a wild-type strain of Chlamydomonas reinhardtii to bioremediate free Cu2+ in wastewater-like nutrient media during the biosynthesis of copper nanoparticles (Cu NPs). The color of supernatants from treated Cu-polluted media provided a first indication of Cu-based NPs formation in the aqueous phase. Analysis by fluorescein diacetate hydrolysis, observations of cell morphology, and algal regrowth experiments after treatment showed that algal viability was crucial for efficient Cu2+ reduction to Cu NPs. UV–vis absorption spectroscopy demonstrated that sulfur-free medium, which enables sustained hydrogen photoproduction in alga, was not the most efficient in NPs formation. Dark-field scanning transmission electron microscopy (STEM) images overlapped with Cu signal map from energy dispersive X-ray spectrometry (EDS) and high-resolution transmission electron microscopy confirmed the presence of polydisperse, spherical, and well-dispersed sub-10 nm Cu NPs crystallites exclusively in algae-treated heavily Cu-polluted media (10 mg L−1). STEM and EDS also demonstrated the affinity of Cu NPs for carbon-rich (organic) objects. Overall, this study demonstrates the feasibility of Cu2+ bioremediation from the wastewater-like complex nutrient media and the simultaneous biosynthesis of Cu NPs by viable green microalga.

Штамм Gordonia alkanivorans 135 - перспективный деструктор дибензотиофена

Genes involved in the dibenzothiophene degradation have been identified in the genome of Gordonia alkanivorans 135. The efficiency of the degradation was evaluated by high-performance liquid chromatography after the strain cultivation in mineral sulfur-free medium with glucose (hexadecane) as a carbon source at a temperature of 28 °C. The results obtained in this work allow us to consider the Gordonia alkanivorans 135 strain as promising for development of bio technological method for microbial oil desulfurization. Gordonia, dibenzothiophene, biodegradation. This work was financially supported by the Russian Science Foundation (Grant no. 19-74-00097).

Identification of enzyme(s) capable of degrading endosulfan and endosulfan sulfate using in silico techniques

Endosulfan is one of the most widely used organochlorine cyclodiene insecticides. Microbial oxidation of endosulfan forms endosulfan sulfate, which is more or less toxic and persistent as endosulfan. Due to lack of specificity and efficiency of microbial bioremediation technique in the field conditions, enzymatic bioremediation is receiving huge attention to clean-up the environment. In the present study, X-ray crystal structures of enzymes from Brookhaven Protein Data Bank were screened for their potential to degrade endosulfan and endosulfan sulfate using molecular docking and molecular dynamics simulation techniques. A phenol hydroxylase, 1PN0 from Trichosporon cutaneum was found to have the potential to degrade both α-endosulfan and endosulfan sulfate while a bacterial CotA laccase, 3ZDW from Bacillus subtilis has the potential to degrade α-endosulfan. The in silico result correlate with in vitro degradation study using two different strains of Trichosporon cutaneum. In vitro degradation study found that the fungal strain was capable of degrading 60.36% α-endosulfan, 70.73% β-endosulfan, and 52.08% endosulfan sulfate. The presence of phenol hydroxylase inhibitor in the sulfur-free medium with endosulfan and endosulfan sulfate as sole sulfur source inhibits the growth of both the fungal strains. Such in silico techniques can provide an easy and reliable way to speed up the development of bioremediation processes through rapid identification of potential enzymes and microbes to counter the ever-increasing number of toxic compounds in the environment.

Recovery of Elemental Tellurium Nanoparticles by the Reduction of Tellurium Oxyanions in a Methanogenic Microbial Consortium.

This research focuses on the microbial recovery of elemental tellurium (Te(0)) from aqueous streams containing soluble tellurium oxyanions, tellurate (Te(VI)), and tellurite (Te(IV)). An anaerobic mixed microbial culture occurring in methanogenic granular sludge was able to biocatalyze the reduction of both Te oxyanions to produce Te(0) nanoparticles (NPs) in sulfur-free medium. Te(IV) reduction was seven times faster than that of Te(VI), such that Te(IV) did not accumulate to a great extent during Te(VI) reduction. Endogenous substrates in the granular sludge provided the electron equivalents required to reduce Te oxyanions; however, the reduction rates were modestly increased with an exogenous electron donor such as H2. The effect of four redox mediators (anthraquinone-2,6-disulfonate, hydroxocobalamin, riboflavin, and lawsone) was also tested. Riboflavin increased the rate of Te(IV) reduction eleven-fold and also enhanced the fraction Te recovered as extracellular Te(0) NPs from 21% to 64%. Lawsone increased the rate of Te(VI) reduction five-fold, and the fraction of Te recovered as extracellular material increased from 49% to 83%. The redox mediators and electron donors also impacted the morphologies and localization of Te(0) NPs, suggesting that NP production can be tailored for a particular application.

Importance and Determinants of Induction of Cold-Induced DEAD RNA Helicase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes.

Hydrogen production using an algae photoelectrochemical cell

In this paper we present a photoelectrocatalytic-enzymatic hybrid system for simultaneous hydrogen production and organic pollutants’ reduction. The destruction of the antibiotic Chloramphenicol, which acts as a model pollutant, will take place in the anodic compartment, while simultaneously hydrogen is produced in the cathode compartment of an algae photoelectrochemical cell. The photogenerated electrons in a TiO2 anode are transferred to the cathode, where the hydrogenase enzymes derived from the algae Chlamydomonas Reinhardtii strain CC-124, catalyze the reduction of the H+ species to H2. Parameters like the growing medium, detergent, electron relay and algae concentration have been optimized. Fifty percent reduction in the organic carbon content and almost complete destruction of the Chloramphenicol molecule is possible at the anode under photoelectrocatalytic conditions. Simultaneously in the cathode compartment and in the presence of the algae culture C. Reinhardtii strain CC-124, 216μM H2 is produced over a reaction time of 450min, using 0.4mM Triton X 100 for the cell membranes rapture and 0.05mM Methyl Viologen (MV+2), which acts as an electron relay, in a Tris-acetate-phosphate (TAP) sulphur free medium. The expression of the hydrogenase enzymes under photoheterotrophic and sulphur deprived conditions has been confirmed by a reverse transcription-polymerase chain reaction (RT-PCR).

Thermophilic desulfurization of dibenzothiophene and different petroleum oils by Klebsiella sp. 13T

Biodesulfurization (BDS) has the potential to desulfurize dibenzothiophene (DBT) and its alkylated derivatives, the compounds that are otherwise refractory to hydrodesulfurization (HDS). Thermophilic microorganisms are more appropriate to be used for BDS applications following HDS. The aim of the present study was to isolate a thermophilic microorganism and to explore its commercial relevance for BDS process. The desulfurizing thermophilic strain was isolated and enriched from various soil and water samples using sulfur free medium (SFM) supplemented with DBT. Microbiological and genomic approach was used to characterize the strain. Desulfurization reactions were carried out using DBT and petroleum oils at 45°C followed by different analytical procedures. We report the isolation of a thermophilic bacterium Klebsiella sp. 13T from contaminated soils collected from petroleum refinery. HPLC analysis revealed that Klebsiella sp. 13T could desulfurize DBT to 2-hydroxybiphenyl (2-HBP) at 45°C through 4S pathway. In addition, adapted cells of Klebsiella sp. 13T were found to remove 22-53% of sulfur from different petroleum oils with highest sulfur removal from light crude oil. Klebsiella sp. 13T is a potential candidate for BDS because of its thermophilic nature and capability to desulfurize petroleum oils.

Biodegradation of endosulfan and endosulfan sulfate by Achromobacter xylosoxidans strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO(2) from the culture media with endosulfan as sulfur source as compared to MgSO(4) suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.

Sulphur depletion altered somatic embryogenesis in Theobroma cacao L. Biochemical difference related to sulphur metabolism between embryogenic and non embryogenic calli

Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO 4 2 - ion deficiency in culture media on somatic embryogenesis was studied through sequential replacement of MgSO 4 and K 2 SO 4 by MgCl 2 and KCl, respectively, at different steps of somatic embryogenesis. It appears that explants gradually lost their embryogenic competence as the period of exposition to sulphur free medium increases. These results suggest that, sulphur availability and the duration to sulphur exposition might modulate the expression of genes involved in somatic embryo differentiation in T. cacao . Cysteine, glutathione, reducing sugars, cysteine synthase and cysteine desulfurase activities were analysed in different morphogenetic structures obtained in vitro . Cysteine and reducing sugars contents appeared to be higher in embryogenic calli than their nonembryogenic homologues, whereas glutathione content appears to be lower in embryogenic calli. Cysteine synthase activities also discriminate the embryogenic calli from non embryogenic calli. In the embryogenic calli, the ratio cysteine synthase/cysteine desulfurase activities were above unit. The assimilation of exogenous sulphur (sulphate) for the synthesis of cysteine might hence be crucial for somatic embryogenesis in T. cacao . This explains the reduction and the absence of somatic embryo response observed during sulphur depletion in culture media. Sulphur nutrition is therefore critical in cacao somatic embryogenesis. Keyswords: Cacao, embryo, sulphate, cysteine synthase, glutathione, deficiency African Journal of Biotechnology Vol. 9(35), pp. 5665-5675, 30 August, 2010

Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii

The coding region of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of Chlamydomonas reinhardtii. As a result, transgenic C. reinhardtii cultures more rapidly consumed O 2 and increased H 2 output compared with controls in both sulfur-free and sulfur-containing medium. H 2 production of the transgenic algal cultures in sulfur-free medium was 4-fold greater than that of control cultures, ∼3.3 ml bottle −1. Maximum expression of the hemH-lba fusion protein on day 5 coincided with the lowest levels of O 2 content and the highest H 2 evolution rate detected over 7 days of anaerobic induction in sulfur-free medium. When the concentration of sulfate in the growth medium was restored to 12.5 or 50 μM, O 2 consumption and H 2 yield decreased more slowly in the transgenic algal cultures than in the control cultures. These results demonstrate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii improved their H 2 yield by decreasing O 2 content in the medium, thereby representing the potential for H 2 production in green algae to be improved.

Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O 2 consumption, lower O 2 content and higher H 2 output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O 2 contents and maximal H 2 evolution occurred. The highest H 2 production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H 2 production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H 2 yield by decreasing O 2 content in the medium. This protein had potential to be used in improvement of H 2 production in green algae.

Simultaneous biodegradation of chloro- and methylthio-s-triazines using charcoal enriched with a newly developed bacterial consortium

A special type of charcoal, Charcoal A100, was enriched with a newly developed bacterial consortium using a perfusion method. The bacterial consortium consisted of a methylthio-s-triazine-degrading bacterium (Rhodococcus sp. FJ1117YT) and the chloro-s-triazine-degrading bacterial consortium CD7 (containing Bradyrhizobium japonicam CSB1, Arthrobacter sp. CD7w and β-Proteobacteria CDB21). Enriched charcoal was capable of degrading chloro-s-triazines (simazine and atrazine) and methylthio-s-triazines (simetryn and dimethametryn) simultaneously in sulfur-free medium. Almost complete degradation was observed after 4-day cultivation of chloro-s-triazines and 9-day cultivation of methylthio-s-triazines. These triazines were mineralized via their 2-hydroxy analogues.

Nutrient regulation of oligopeptide transport in Saccharomyces cerevisiae

Small peptides (2-5 amino acid residues) are transported into Saccharomyces cerevisiae via two transport systems: PTR (Peptide TRansport) for di-/tripeptides and OPT (OligoPeptide Transport) for oligopeptides of 4-5 amino acids in length. Although regulation of the PTR system has been studied in some detail, neither the regulation of the OPT family nor the environmental conditions under which family members are normally expressed have been well studied in S. cerevisiae. Using a lacZ reporter gene construct fused to 1 kb DNA from upstream of the genes OPT1 and OPT2, which encode the two S. cerevisiae oligopeptide transporters, the relative expression levels of these genes were measured in a variety of environmental conditions. Uptake assays were also conducted to measure functional protein levels at the plasma membrane. It was found that OPT1 was up-regulated in sulfur-free medium, and that Ptr3p and Ssy1p, proteins involved in regulating the di-/tripeptide transporter encoding gene PTR2 via amino acid sensing, were required for OPT1 expression in a sulfur-free environment. In contrast, as measured by response to toxic tetrapeptide and by real-time PCR, OPT1 was not regulated through Cup9p, which is a repressor for PTR2 expression, although Cup9p did repress OPT2 expression. In addition, all of the 20 naturally occurring amino acids, except the sulfur-containing amino acids methionine and cysteine, up-regulated OPT1, with the greatest change in expression observed when cells were grown in sulfur-free medium. These data demonstrate that regulation of the OPT system has both similarities and differences to regulation of the PTR system, allowing the yeast cell to adapt its utilization of small peptides to various environmental conditions.

Biodesulfurization of alkylated forms of dibenzothiophene and benzothiophene by Sphingomonas subarctica T7b

Sphingomonas subarctica T7b was isolated from soil in Toyotomi, Hokkaido, Japan as an organism capable of desulfurizing aromatic hydrocarbons in light gas oil (LGO) through enrichment culture. S. subarctica T7b could grow on mineral salt sulfur-free (MSSF) medium with the n-tetradecane oil phase containing dibenzothiophene (DBT), alkyl dibenzothiophenes (alkyl DBTs) or alkyl benzothiophenes (alkyl BTs) as the sole sulfur source and desulfurize these compounds, but could not utilize the tetradecane as a carbon source. This is the first report of a gram-negative bacterium which can desulfurize 4,6-dibutyl DBT and 4,6-dipentyl DBT. The desulfurized product of DBT produced by this strain was 2-hydroxybiphenyl, as in the case of other DBT-desulfurizing bacteria. S. subarctica T7b could desulfurize LGO and the sulfur content was decreased to 41% within 36 h.

Dilution methods to deprive Chlamydomonas reinhardtii cultures of sulfur for subsequent hydrogen photoproduction

Sulfur deprivation of Chlamydomonas reinhardtii cultures gradually inactivates photosynthetic O 2 evolution and leads to the establishment of anaerobiosis in the medium. Sulfur-deprived algal cultures kept under anaerobic conditions will then produce H 2 gas for 3–5 days under continuous illumination. Currently, sulfur deprivation is achieved by mechanical centrifugation of cultures grown in sulfur-replete medium, followed by extensive and costly washing. The cells are finally resuspended in sulfur-free medium. The current study investigates two procedures to deprive algal cultures of sulfur that eliminate the centrifugation step. These procedures involve sulfur deprivation by dilution of sulfur-replete cultures into either sulfur-limited medium or sulfur-free medium. We demonstrate that efficient H 2 photoproduction can be achieved on a timely basis using either procedure. However, the dilution of sulfate-replete algal cultures 1 :10 v/ v into sulfur-free medium is the most appropriate procedure. These observations serve as the basis for developing an algal H 2-production system that is cheaper, less time-consuming, and less amenable to contamination with other microorganisms than systems employing centrifugation for sulfur deprivation.

Reduction of selenium oxyanions by unicellular, polymorphic and filamentous fungi: Cellular location of reduced selenium and implications for tolerance

The ability of several filamentous, polymorphic and unicellular fungi to reduce selenite to elemental selenium on solid medium was examined.Fusarium sp. andTrichoderma reeii were the only filamentous fungi, of those tested, which reduced selenite to elemental selenium on Czapek-Dox agar resulting in a red colouration of colonies. Other organisms (Aspergillus niger, Coriolus versicolor, Mucor SK, andRhizopus arrhizus) were able to reduce selenite only on malt extract agar. Several fungi were able to grow in the presence of sodium selenite but were apparently unable to reduce selenite to elemental selenium, indicating that other mechanisms of selenite tolerance were employed, such as reduced uptake and/or biomethylation to less toxic, volatile derivatives. Sodium selenate was more toxic toFusarium sp. than selenite, and the toxicity of both oxyanions was increased in sulphur-free medium, with this effect being more marked for selenate. Scanning electron microscopy ofAspergillus funiculosus andFusarium sp. incubated with sodium selenite showed the presence of needle-like crystals of elemental selenium on the surfaces of hyphae and conidia, while transmission electron microscopy ofA. funiculosus revealed the deposition of electron-dense granules in vacuoles of selenite-treated fungi. Several yeasts were able to grow on MYGP agar containing sodium selenate or sodium selenite at millimolar concentrations. Sone, notablyRhodotorula rubra andCandida lipolytica, and the polymorphic fungusAureobasidium pullulans were also effective at reducing selenite to elemental selenium, resulting in red-coloured colonies.Schizosaccharomyces pombe was able to grow at selenite concentrations up to 5 mmol L−1 without any evidence of reduction, again indicating the operation of other tolerance mechanisms.

Characterization of Sulfate Transport in Chlamydomonas reinhardtii during Sulfur-Limited and Sulfur-Sufficient Growth

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.