International Journal of Hydrogen Energy 27 (2002) 1421–1430www.elsevier.com/locate/ijhydene

Accumulation of O2-tolerant phenotypes in H2-producingstrains of Chlamydomonas reinhardtii by sequentialapplications of chemical mutagenesis and selection

Timothy Flynn1, Maria Lucia Ghirardi, Michael Seibert ∗

National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401, USA

Abstract

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidlyinactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H2-evolving activities of up to10 times the O2-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consistingof random chemical mutagenesis, enrichment for H2-metabolism clones following exposure to increasing amounts of O2, andscreening using a chemochromic sensor. The selected strains were characterized by two types of assays and classi4ed as thosethat (a) can evolve H2 following exposure to O2 concentrations that inactive the WT strain and (b) in addition, are able toquickly reactivate H2-production activity once O2 is removed. These results suggest that O2-tolerance can be increased bysuccessive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by geneticmeans. Other results show that the hydrogenase is less sensitive to O2 when it is actively catalyzing H2 evolution.Published by Elsevier Science Ltd on behalf of the International Association for Hydrogen Energy.

Keywords: Green algae; Chlamydomonas reinhardtii; H2 photoproduction; Hydrogenase; O2 tolerance

1. Introduction

Both the nitrogenase and hydrogenase enzyme systems,present in various photosynthetic bacteria, cyanobacteria,and green algae, can catalyze the photobiological productionof molecular H2 [1–7]. Nitrogenase-catalyzed reactions arecomparatively energy intensive, requiring adenosine triphos-phate (ATP) input and a 4xed carbon intermediate to driveeither nitrogen 4xation or H2 photoproduction [1,5]. On theother hand, green algae can photoevolve H2 directly fromwater [8,9] using the hydrogenase system, and hence havethe theoretical potential to produce H2 at 2–3 times the lightconversion e?ciency compared to a nitrogenase system. Be-sides light-induced H2 production, hydrogenases catalyze

∗ Corresponding author. Tel.: +1-303-384-6279; fax: +1-303-384-6150.

E-mail address: mike seibert@nrel.gov (M. Seibert).1 Current address: Primordial Solutions, 463 Gunnison Ave.,

Grand Junction, CO 81501, USA.

dark fermentative H2 production, but at much lower rates[5,8].In algae, electrons produced from the oxidation of water

by photosystem II are transferred to photosystem I in thechloroplast thylakoid membrane, where they reduce ferre-doxin, a low oxidation–reduction potential carrier [10]. Nor-mally, reduced ferredoxin (via NADPH) is the source ofelectrons for the Benson–Calvin cycle that 4xes CO2. How-ever, during dark, anaerobic treatment, algal cells induce thede novo synthesis of the reversible hydrogenase [8,11], achloroplast stromal enzyme [12] that catalyzes both H2 pro-duction and H2 uptake. This nuclear-encoded protein [12]accepts electrons from ferredoxin [11], binds protons, andreleases molecular H2.The major challenge that currently precludes the use of

algae in applied H2-producing systems is the sensitivity ofthe hydrogenase to inactivation by O2 co-produced by thephotosynthetic water-splitting process [9,13,14]. To start toaddress this challenge, our laboratory has examined twoapproaches to select for mutant H2-producing strains of

0360-3199/02/$ 22.00 Published by Elsevier Science Ltd on behalf of the International Association for Hydrogen Energy.PII: S 0360 -3199(02)00117 -9

1422 T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430

Nomenclature

ATP adenosine triphosphate,BS basal salts,5BU 5-bromouracil,Chl chlorophyll,DBMIB dibromothymoquinone,DCMU 3-(3,4-dichlorophenyl-)-1,1-dimethyl urea,EMS ethyl methanesulfonate,Light-CE Light=Clark electrode assay,MNZ metronidazole,

MV-GC dark methyl viologen=gas chromatographyassay,

NMR nuclear magnetic resonance,O2 I50 the concentration of added O2 that de-

creases V0 by one-half,PAR photosynthetically active radiation,V0 maximum rate of H2 evolution by algae that

have not been pre-inactivated by O2,WT wild-type

the green alga, Chlamydomonas reinhardtii, with improvedO2-tolerance. The two selective pressures depend on main-taining the reversible activities of the hydrogenase enzyme(i.e., the H2-producing and H2-uptake reactions) underconditions of O2 stress. The hydrogen-production selectivepressure is based on the toxic eKects of the drug metro-nidazole (MNZ) on algal photosynthesis in the absence ofCO2. When reduced, MNZ reacts with O2, releasing su-peroxide radicals and hydrogen peroxide that kill the cells[15]. However, hydrogenase can compete with MNZ forelectrons available from light-reduced ferredoxin. If the hy-drogenase is active, some of the electrons from ferredoxinwill be used for H2 production, instead of MNZ reduc-tion, and decrease the toxic eKects of the drug [14]. Theselective conditions that we developed require addition ofO2 to the system during MNZ treatment in order to elimi-nate wild-type (WT) hydrogenase activity. This selectionis not very speci4c, and survivors should have one of thefollowing three phenotypes: (a) lower rates of photosyn-thetic electron transport to ferredoxin due to mutations inthe antenna complexes or the electron transport chain [15],(b) an over-expressed O2-scavenging system that removesphotosynthetically evolved O2 more quickly than in theWT or (c) a more O2-tolerant hydrogenase that can divertelectrons away from MNZ before active O2 species can begenerated [14].The H2-uptake selective pressure was 4rst conceived by

McBride et al. [16], who subjected a mutagenized popu-lation of WT algal cells to photoreductive conditions inthe presence of controlled concentrations of O2. The sur-viving cells grew by 4xing CO2 with electrons obtainedfrom the oxidation of H2 (hydrogenase H2-uptake activity)and ATP generated by cyclic electron transfer around pho-tosystem I [13]. Photosynthetic water oxidation, and con-sequently O2 evolution, was blocked by the presence ofthe photosystem II inhibitor, DCMU, following a periodof starvation to deplete the cells of internal sources of re-ductant. This selective pressure is much more speci4c forO2-tolerance than H2-production selection, but can possiblygenerate herbicide-resistant mutants [17]. Moreover, cells

that are not completely pre-starved can potentially survivethe selective pressure.We have applied both types of selective pressures to pop-

ulations of C. reinhardtii in order to 4nd better strategies forobtaining H2-producing strains with improved O2-tolerance.This paper describes the application of two rounds of bothtypes of selective pressures to mutagenized populations ofWT C. reinhardtii cells. Since neither selective pressureis completely stringent and the standard H2-production as-says used to con4rm the selection of positive mutants areextremely time consuming, we have included an additionalstep to facilitate the rapid identi4cation of desirable strains[18,19]. This involves the use of a chemochromic sensor4lm that is normally transparent, but turns blue speci4callyin the presence of H2 gas [19–21]. The sensor can detectas little as 4 nmol of H2 emitted in a 2–3 min period froma 2 to 3 mm diameter colony growing on an agar Petri dish[18]. When the sensor is used as a screening tool to iden-tify desirable isolates, colonies from cells that survive theselective pressures can be examined rapidly on agar platesfor their ability to produce H2 after exposure to O2 dosesthat eliminate H2 production in the WT [22]. In this study,we demonstrate that the combination of classical randommutagenesis, selection, and screening techniques can actu-ally yield H2-producing Chlamydomonas strains with im-proved levels of O2-tolerance compared to the parental WTstrain.

2. Materials and methods

2.1. Cultivation of algae

Wild-type (WT) Chlamydomonas reinhardtii Dangeard137c (+) was grown photoheterotrophically as seed cul-tures in liquid basal salts (BS) medium [22] supplementedwith 10-mM sodium acetate. The cells were grown at 24

◦C

with shaking under illumination (70 �E m−2 s−1 PAR, coolwhite Lorescent lamps) to late-log phase, at a 4nal celldensity equivalent of 20–30 �g ml−1 chlorophyll (Chl).

T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430 1423

The experimental cultures used for mutagenesis, selection,and assays for H2-production were grown photoautotrophi-cally, and Lasks containing 600 ml of BS were inoculated ata Chl concentration of 2:5 �g ml−1, 24 h prior to use. Thecultures were stirred, exposed to cool white Lorescent light(70–80 �E m−2 s−1 PAR), and sparged with 2% CO2 in airat a rate of 50–100 ml min−1. The cells were harvested bycentrifugation at 2000 x g for 10 min. Chlorophyll concen-tration was determined spectrophotometrically in 95% v=vethanol [23].

2.2. Mutagenesis

Replicate suspensions containing 7 × 107 cells were ad-justed to a 4nal concentration of 46 mM ethyl methane-sulfonate (EMS). The number of cells per sample corre-sponded to the estimatedC. reinhardtii genome size of about3 × 107 bp [24] and was chosen to ensure statistically thateach surviving cell would contain a unique mutation, andthat all base pairs in the genome would have the probabil-ity of being aKected by the mutagen. The cell suspensionswere then incubated in the dark, harvested, washed, and sus-pended in BS medium at the end of 5, 10, 15 and 20 minEMS treatments, respectively.Alternatively, cell cultures of 1 �g=ml Chl were grown

photoautotrophically overnight to establish logarithmicgrowth. The following day, 5-bromouracil (5BU) wasadded to a 4nal concentration of 1 mM. This culture wasgrown under the same conditions for an additional 48 h,harvested, and then re-suspended in BS lacking 5BU.Survivors from both mutagenesis treatments were grown

photoautotrophically for at least another 7 days in order toeliminate auxotrophic mutants.

2.3. H2-production selective pressure

This procedure includes the following modi4cation to themethod described previously [14]. A suspension of 5:25 ×107 mutagenized algal cells (1:05 × 107 cells ml−1) wasanaerobically induced in the dark for 4 h at room temper-ature and stored at 4

◦C overnight in the dark. Anaerobic

MNZ and sodium azide solution was added to the cells, to 4-nal concentrations of 40 mMMNZ, 1 mM sodium azide and2:1× 106 cells ml−1. The azide was included to inhibit theendogenous catalase activity. While maintaining darkness,O2 was added to the desired concentration in the gas phase,and the suspensions were vigorously mixed for 4–5 min andthen exposed to incandescent light (320 �E m−2 h−1 PAR4ltered through a solution of 1% CuSO4) for 6 min. Earlierexperiments had shown that longer exposure to light resultedin little further decrease in cell survival. After the 6-min il-lumination, the cells were washed, re-suspended in BS, andgrown photoautotrophically to eliminate antenna and pho-tosynthetic electron transport mutants.

2.4. H2-uptake selective pressure

A total of 7 × 108 mutagenized cells were suspended inBS to a cell density 2:8 × 106 cells ml−1, treated with amixture of DCMU and atrazine, and placed in the dark withstirring for 2 days to allow the cells to use up internal stor-age reserves. The two herbicides in combination at 15 �Meach will not allow C. reinhardtii cells to survive under nor-mal photoautotrophic conditions. Following this treatment,Lasks of starved cells were transferred to anaerobic jars(Oxoid Anaerobic System, Unipath Limited, Hampshire,UK), 4lled with N2, and then placed in darkness for 4 hto induce the algal hydrogenase activity. The N2 was thenreplaced with 15% H2, 2% CO2, 20–30% O2, and Ar(balance), and the cultures were then incubated at roomtemperature with stirring and illuminated with cool whiteLuorescent light (70 �E m−2 s−1 PAR). The gas in the jarswas replaced daily to compensate for the consumption ofO2 by any remaining respiration for 7–10 days until the cul-tures became chlorotic. At this point, gas replacement wasreduced to three times per week. When the cultures werevisibly devoid of chlorophyll and appeared as a turbid whitesuspension (ca. 10 days after placement in the anaerobicjars), the cells were harvested, washed with BS medium,and then revived in liquid BS medium plus 10 mM sodiumacetate.

2.5. Chemochromic screening for H2-producing colonies

Individual colonies surviving the selection pressureswere plated on 1:8% w=v agar containing BS mediumsupplemented with 10 mM sodium acetate and 0:5 g l−1

yeast extract, and transferred to square Petri dishes to form8 × 8 colony matrices. Following a growth period of 7–14days, the agar plates were exposed to anaerobic condi-tions overnight to induce algal hydrogenase activity in thecolonies. When screening for O2-tolerance, the plates were4rst pre-exposed to O2 (21–100%) for diKerent periods oftime in the dark to inactivate any remaining cells with WTphenotype and then transferred immediately to an anaerobicglove box. A H2-sensor was applied and the plates wereilluminated for 3 min to photoevolve H2 [21]. Followingillumination, the sensors were analyzed for the locationof visible blue dots corresponding to algal colonies thatevolved H2 following the O2 pre-treatment. The best clonesidenti4ed in this manner were transferred from the originalplate to liquid BS containing 10 mM acetate, and the cellswere cultivated for further characterization.

2.6. H2-evolution assays

2.6.1. Anaerobic inductionMid-log phase algal cells were harvested and resuspended

at 200 �g ml−1 Chl in a potassium phosphate buKer [14]supplemented with 15 mM glucose and 0:5% v=v ethanol.Concurrently, an enzymatic oxygen scrubbing system [25]

1424 T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430

that consisted of 1 mg ml−1 glucose oxidase (C100, Sigma,St. Louis, MO) and 27 720 units ml−1 catalase (Sigma, St.Louis, MO) was prepared, and 2 ml of the enzymatic mix-ture was dispensed into dialysis tubing (6–8 kDMWcutoK).In an anaerobic glove-box, the dialysis bags were added tovials containing 20 ml of cell suspensions. The vials werethen sealed, covered with aluminum foil to ensure darkness,and incubated at room temperature for 4 h. Following thisinduction treatment, the cell suspensions were kept at 4

◦C

overnight and assayed for H2-evolution activity using eithera Clark-electrode or a methyl viologen=gas chromatographyassay.

2.6.2. Light=Clark electrode (light-CE) assayAnaerobically induced cells were treated with diKerent

amounts of O2 to inactivate hydrogenase activity, and thenilluminated to initiate H2 production. Hydrogen evolutionwas monitored with a Clark electrode, as described previ-ously [14]. The O2 concentration in the chamber was setby bubbling with Ar until the desired initial value wasreached (0–5% depending on the experiment), and thenthe anaerobically induced cell suspension was injected intothe buKer. The cells were exposed to the pre-set O2 con-centration for 2 min in the dark. Since some respiratoryconsumption of O2 occurred in the dark (between 15 and30 �mol O2 mg Chl−1 h−1), the O2 concentration was notabsolutely constant during the dark deactivation treatment.Following O2 inactivation, the cells suspension was illu-minated (320 �E m−2 s−1 PAR incandescent light 4lteredthrough 9 cm of 1% CuSO4) for 3 min. The initial rates ofH2 production were plotted as a function of the initial O2concentration.

2.6.3. Dark methyl viologen=gas chromatography(MV-GC) assayThis assay measures H2 evolution in the dark, using

methyl viologen, pre-reduced by sodium dithionite, as theelectron donor. Anaerobically induced cells were treatedwith O2 (100% was injected into capped serum vials to givevarious 4nal O2 concentrations in the gas phase), and thevials were then incubated with stirring in the dark for 2 min.Following the O2 pre-treatment to inactivate WT hydroge-nase activity, the vials were Lushed with Ar gas and thentreated with 5 mM methyl viologen and 0:1% w=v TritonX-100. After reestablishing anaerobiosis, sodium dithionitein 30 mM NaOH was added to a 4nal concentration of5 mM. The samples were then incubated in a water bath(30

◦C) with shaking for 15 min. Hydrogen evolution was

stopped by injecting 100 �l of 20% (w=v) trichloroaceticacid into the vials. The amount of H2 evolved during the15-min incubation was measured by gas chromatography(Model 3700, Varian, Sugar Lane, TX) equipped with a 5 PAmolecular sieve column and a thermoconductivity detector.Argon was used as the carrier gas.

3. Results

The general approach to isolate H2-producing mutantswith higher levels of tolerance to O2 is summarized in Fig. 1.It consisted of a combination of random chemical mutagene-sis, using either ethylmethane sulfonate (EMS on the left) or5-bromouracil (5BU on the right), followed by applicationof one of two types of selective pressures (H2-productionon the left or H2-uptake on the right). The colonies that sur-vived the selective pressures were then chemochromicallyscreened for their ability to produce H2 on plates, followinginactivation by exposure to O2. The strains that passed thescreen were further characterized for their H2-production tol-erance to O2, and the best strains were resubmitted to a sec-ond round of mutagenesis=selection=screening under morestringent O2 conditions.The generation of random mutants of C. reinhardtii was

done by treating suspensions of algae with either EMS or5BU. These mutagens induce A-T ↔ G-C transition mu-tations, some of which will be lethal to the organism [26].Although it is currently impossible to predict the theoret-ical relationship between lethality and mutation rate for agiven mutagen, we know empirically [27] that the rate ofmultiple mutations within a given genome will decreasealong with the rate of lethal mutations. Therefore, lethal doserates of around 50% were chosen for our experiments in an

H2-Production lineage

H2-Uptake lineage

EMS mutagenesis: 15min, 56% survival

H2-production selection: 5% O2,4 min, 0.12% survival

Strain 76D4

EMS mutagenesis: 20min, 61% survival

H2-production selection: 40%O2, 5 min, 0.31% survival

Strain 141F2

Chemochromic screening

5BU mutagenesis: 2 d

Chemochromicscreening

H2-uptake selection: 20% O2,14 d

Strain 104G5

5BU mutagenesis: 3 d

H2-uptake selection: 30% O2,17 d

Chemochromic screening

Strain 155G6

Chemochromicscreening

WT

Fig. 1. Sequence of events leading to the isolation of the twogenerations of H2-production and H2-uptake selection mutants. Seetext for details.

T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430 1425

2520151050

100

10

Exposure to EMS (min)

Sur

viva

l (%

)

Fig. 2. Killing curves for two diKerent EMS treatments. The per-centage of surviving cells is plotted as a function of the time ofexposure to 46 mM EMS. The parental strains were either the WTstrain (open circles) or a 4rst generation mutant (76D4 strain,closed circles) produced by EMS mutagenesis of the WT.

attempt to average one point mutation per genome. Typicalkill curves for EMS were shown previously [22], but it isinteresting to note that the rate of lethality as a function ofEMS exposure decreases with subsequent rounds of muta-genesis. Fig. 2 shows the killing eKect of 46 mM EMS onthe WT strain compared to its eKect on a 4rst-generationmutant obtained by EMS mutagenesis of the WT (76D4).It is clear that the 76D4 mutant accumulated lethal muta-tions at a lower rate than the WT upon exposure to the sameconcentration of EMS, suggesting lower sensitivity to themutagen. To compensate for this decreased rate of mutage-nesis, the incubation time for this mutant strain in EMS wasincreased from 15 to 20 min (see Fig. 1). Curiously, whenthe second-generation mutant strain (141F2) was exposed tothe same 20 min of EMS treatment, almost no lethal eKectwas observed (Z. Huang, data not shown). The procedureused for 5BU mutagenesis does not allow for a similar typeof analysis, given that the mutagen is incorporated into thealgal genome during cell replication that occurs during the48 h incubation.Following mutagenesis, surviving cells were submitted to

one of two selective pressures. One lineage underwent twocycles of EMS mutagenesis and H2-production selection (onthe left in Fig. 1), resulting in strains 76D4 and 141F2, whilethe other underwent two cycles of 5BU mutagenesis andH2-uptake selection (on the right in Fig. 1), resulting in thestrains 104G5 and 155G6.The 4rst generation strain 76D4 was obtained by

application of H2-production selective pressure onEMS-mutagenized WT cultures. This selective procedureused a 4-min pre-exposure to 5% O2 that resulted in 0.12%survival. Three replicates of this selective procedure weredone, yielding approximately the same percentage ofsurvivors [22]. Strain 76D4 was identi4ed among theearly survivors by chemochromic screening and biochemical

Fig. 3. Chemochromic screening of plate #141. Agar-grown singlecolonies were pre-exposed to 100% O2 for 5 min. The plate wasthen transferred to an anaerobic chamber, where the sensor was ap-plied and the system was illuminated for 3 min. The 4gure showsthe resulting sensor, photographed on a white background. Manypositive clones were identi4ed and tested. Mutant 141F2 was ob-tained from position F2 on the sensor. The WT and the 76D4 con-trol strains were located at positions H8 and H7, respectively, anddid not evolve signi4cant amounts of H2 under these conditions.The original photograph shows a substantial glare on the bottomleft side which shows up as an apparent bleaching of the spotson the sensor in that area. This, however, did not interfere withthe actual analysis described in the text. Note that this plate in-corporated the best identi4ed mutants, but many other plates wereassayed (see text).

assays (see below) as having higher tolerance to O2 thanthe WT. This strain was remutagenized, and the new mixedpopulation of mutants, after regrowth, was divided intothree equivalent cultures. Each culture was then subjectedto a H2-production selection that used a 5-min, pre-exposureto either 20%, 30% or 40% O2 in the dark. All three treat-ments yielded essentially a 0.3% survival rate. Strain 141F2was obtained from the 40% O2 treatment and identi4ed byapplication of chemochromic screening and biochemicalassays as the most tolerant to O2.The two generations of the H2-uptake selection lineage

were isolated in the presence of 20% and 30% O2, respec-tively, during the application of the selective pressure. Themutants with highest tolerance to O2 were similarly identi-4ed by chemochromic screening and biochemical analyses.Isolates from both selection lineages that survived the ap-

plication of the respective selective pressures were plated onagar as single colonies and screened. The cultures that hadbeen submitted to the H2 production selective pressure weresubsequently grown photoautotrophically to eliminate unin-teresting isolates, as mentioned before. We screened about12 600 colonies from both lineages. Fig. 3 shows the origi-nal chemochromic sensor used to screen the plate containingthe second-generation strain 141F2 (plate 141, grid position

1426 T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430

Table 1H2-production parameters of some 4rst-generation strains that sur-vived the application of the H2-production selective pressure

Replicate H2-production Strain V0b (�mol H2 % of V0c

selective pressurea mg Chl−1 h−1)

WT 39 0.261 72C1 81 142 76D4 78 182 76H3 72 352 74A4 64 152 75G8 50 262 75G1 82 172 75D4 88 182 75H3 67 272 75G3 73 293 78C8 64 9

aReplicate (1–3) H2-production selective pressure experimentswere done under the same experimental conditions to identifyO2-tolerant mutants from the same EMS-mutagenized culture (seetext).bMaximum rate of H2 evolution measured with a Clark electrode

without any exposure to O2.cRate of H2 evolution following deactivation by pre-exposure

of the cells to 2% O2 for 2 min (expressed as % of V0).

F2). This particular Petri dish, which happened to contain avery high number of positive clones, was exposed to 100%O2 for 5 min prior to a 3-min illumination under anaero-bic conditions. Control strains were located at positions H8(WT), and H7 (76D4). The master plates were routinelyreplicated several times so that several O2-inactivation treat-ments could be evaluated on the same day. The replicateplates allowed us to “titrate” the amount of O2 exposure re-quired to see diKerences between spots produced by the WTand the mutant colonies and to compensate for variationsin sensor quality, cell vigor, and level of anaerobic induc-tion. Often, several colonies (5–40) on each plate producedH2 following the inactivation treatment. The multiple pos-itive signals most likely represented replicates of the sameclone, since the mutagenized cultures were allowed to repli-cate both before and after the application of the selectivepressures and prior to the screening.In order to con4rm the e?cacy of the screening method,

many of the strains that were initially identi4ed as havinghigher O2 tolerance were further analyzed using a quick as-say, as shown in Table 1 (see also [22]). The assay consistedof measuring the rate of H2 evolution using a Clark electrode(see Materials and Methods) following a 2-min inactivationby exposure to 2% O2. That rate was compared to the max-imum rate measured in the absence of O2 inactivation (V0,see below), yielding a residual % rate that depends on thetolerance of a particular strain to O2 inactivation. Table 1compares the O2-tolerance of survivors from the 4rst roundof EMS mutagenesis followed by H2-producing selectivepressure and chemochromic screening. These steps resulted

543210

200

160

120

80

40

0

Oxygen concentration, %

WT

104G5

155G6

543210

200

160

120

80

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0

Oxygen concentration, %

Rat

e of

H2

evol

utio

n, µ

mol

es x

(mg

Chl

)-1 x

h-1

WT

76D4

141F2

Rat

e of

H2

evol

utio

n, µ

mol

es x

(mg

Chl

)-1 x

h-1

(a)

(b)

Fig. 4. Oxygen titration of the rate of H2 evolution by diKerentmutants, using the light-CE assay. (A) Mutants from H2-productionselective pressure. (B) Mutant from H2-uptake selective pressure.Open circles represent a 4rst-generation mutant and closed trianglesrepresent a second-generation mutant. The curves in each plot were4tted to a single 4rst-order exponential decay function, and thatpart of the curve to the right of the last data point represents anextrapolation of the data.

in the isolation of a variety of phenotypes with diKerent lev-els of tolerance to O2. These could be due to the diKerentgenotypes generated by the preceding EMS treatment.Chemochromic screening identi4ed 250 well-de4ned pos-

itive clones (about 2% of all screened colonies). Of these,41 were further tested and characterized in more detail. Theparameters used to characterize the strain phenotypes wereV0, the maximum rate of H2 evolution of samples that hadnot been inactivated by O2, and O2 I50, the O2 concentra-tion (%) that decreased the V0 by one-half. These param-eters were estimated by two diKerent assay methods, onemeasuring light-induced H2-evolution using a Clark elec-trode (light-CE), and the other using gas chromatographicmeasurements of H2-evolution catalyzed by reduced methylviologen in the dark (MV-GC).Fig. 4 shows characteristic O2 titration plots obtained

from the light-CE assay using the best strains isolated fromeach of the two rounds of the H2-production selection (Fig.4A) and the H2-uptake selection (Fig. 4B). Similar O2 titra-tions were done, using the MV-GC assay, as shown in

T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430 1427

20181614121086420

80

60

40

20

0

WT

104G5

155G6

1086420

100

80

60

40

20

0

Oxygen concentration,%

Rat

e of

H2

evol

utio

n, µ

mol

es H

2x

(mg

Chl

)-1 x

h-1

WT

76D4

141F2

(a)

Oxygen concentration,%

Rat

e of

H2

evol

utio

n, µ

mol

es H

2x

(mg

Chl

)-1 x

h-1

(b)

Fig. 5. Oxygen titration of the rate of H2 evolution by diKerentmutants, using the MV-GC assay. (A) Mutants from H2-productionselective pressure. (B) Mutant from H2-uptake selective pressure.Open circles represent a 4rst-generation mutant and closed trianglesrepresent a second-generation mutant. The curves in each plot were4tted to a single 4rst-order exponential decay function, and thatpart of the curve to the right of the last data point represents anextrapolation of the data.

Table 2Phenotypic characterization of selected mutants

Strain Mutagen Selective Light-Clark electrode Methyl viologen-GCPressure assay assay

V0a O2 I50b V0a O2 I50b

WT - - - - - - - - - - - - - - - - - - - - 79 0:37± 0:02 30 0:22± 0:0176D4 EMS H2-production 68 0:80± 0:04 78 0:82± 0:08141F2 EMS H2-production 170 2:20± 0:10 88 2:00± 0:15104G5 5BU H2-uptake 105 0:57± 0:05 82 2:80± 0:14155G6 5BU H2-uptake 162 1:12± 0:10 72 3:68± 0:15aThe rate of hydrogen evolution (�mol H2 mg Chl−1 h−1) in the absence of added O2.bThe concentration of O2 (%) that reduces V0 by 50% following a 2-min exposure in the dark, ± standard error (two

experiments).

Fig. 5. The decrease in H2 evolution activity as a functionof initial O2 concentration in each plot was 4tted to a sin-gle 4rst-order exponential decay function, and the V0 andO2 I50 values for each strain and assay were estimated fromthe 4tted curves. This analysis worked well for all strainswith the exception of the 155G6. The rates of H2 evolutionby this strain (Fig. 4B) did not 4t the mathematical functionused to estimate O2 I50 at higher O2 concentrations. Thisseems to indicate a relatively higher sensitivity of the strainto higher O2 concentrations. We currently do not have anexplanation for this apparent biphasic phenomenon. Table2 summarizes the phenotypic characteristics of the WT andeach isolate shown in Figs. 4 and 5. All titrations were re-peated at least twice on diKerent days, and yielded similarresults. The reported O2 I50 values were based on mean val-ues of at least two experiments. The V0 values were reportedas the maximum values obtained in each set of experiments.This is justi4ed because the V0 is not only a function of thehydrogenase’s a?nity for the substrate, but is also aKectedby variation in the levels of anaerobic induction on diKerentdays. Regardless of the assay method, it is apparent fromTable 2 that each successive generation within the same lin-eage displays both a higher V0 (except for the 76D4 strainwhen measured with the light-CE assay and the 155G6 strainwhen measured with the GC-MV assay) and a higher O2 I50with respect to H2 production. It is noteworthy to point out,however, that the O2 I50 values obtained from the H2-uptakemutants using the GC-MV assay (Fig. 5B) were much higherthan those estimated from the light-CE assay (Fig. 4B). Thisdisparity is probably caused by a chemical reactivation ofthe hydrogenase by the reduced MV used in the assay (seeDiscussion). Another observation is that the V0 values, ob-tained using the light-CE assay, method were consistentlyhigher than those obtained by the GC-MV method.A 4nal experiment (Table 3) was conducted to examine

the eKect of light on the inactivation of H2 production by O2

1428 T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430

Table 3EKect of the type of O2-inactivation treatment on the O2 I50 val-ues of mutants selected through application of diKerent selectivepressures

Oxygen deactivation O2 I50 valuea

treatmentb

WTc 104G5c 141F2c

Dark 0.28 0.46 2.7Light 1.35 2.60 3.5

aThe concentration of O2 (%) that reduces the maximum anaer-obic rate of hydrogen evolution (V0) to 50% following a 2 minpre-exposure to O2; measurements were done with a Clark elec-trode.bExposure to O2 for 2 min in the dark (our normal assay) or in

the light.cParental WT strain, 4rst-generation H2-uptake selection mu-

tant 104G5, and second-generation H2-production selection mutant141F2.

in order to determine whether the pathway is more resistantto O2 in the presence of the hydrogenase’s substrate, reducedferredoxin [11]. In this experiment, light-CE assays wereperformed following pre-exposure of the cells for 2 min toO2 either in the dark or in the light. For all of the selectedstrains, the O2 I50 values were greater when the cells wereexposed to O2 in the light rather than the dark. This suggeststhat the hydrogenase is less O2 labile when it is activelycatalyzing the reduction of protons.

4. Discussion

The application of classical mutagenesis, selection, andscreening procedures has allowed us to isolate two gen-erations of C. reinhardtii strains that evolve H2 withsigni4cantly higher tolerances to O2 than the parentalWT, and this demonstrates clearly that the WT pheno-type can be improved by genetic means. The phenotypesof the diKerent mutant strains were preserved along thetwo lineages displayed in Fig. 1. The procedures usedto measure the tolerance of the H2-evolving activity ofthe diKerent strains to O2 were based on two diKerentassays, light-Clark electrode (light-CE) and dark methylviologen-gas chromatography (MV-GC). In general, theH2-production capability (V0) of the strains increased insubsequent generations along each lineage. This is notunexpected since the selective pressures were based on hy-drogenase activity. The two exceptions mentioned in theResults section (Table 2) can be explained by diKerencesin cell vigor and the level of anaerobic induction.When comparing the O2 I50 values of the isolates from the

H2-production selection lineage, we observed that both as-say methods yielded essentially the same values (Table 2).However, there were signi4cant diKerences in the O2 I50

values obtained from the two assays with isolates from theH2-uptake selection lineage (Table 2); the MV-GC assayyielded much higher values than the light-CE assay. Theseresults suggest that the isolates from each lineage have sig-ni4cantly diKerent phenotypes. The detection of the diKer-ent phenotypes was possible due to the diKerent nature ofthe two assays. The light-CE assay uses the physiologicalpathway from water through the two photosystems to re-duce ferredoxin. Ferredoxin then donates electrons to thereversible hydrogenase that in turn releases H2gas. The hy-drogenase O2 inactivation pre-treatment lasts for 2 min inthe dark. The MV-GC assay, on the other hand, is an invitro assay that uses reduced methyl viologen as a ferre-doxin analog [11] to donate electrons in the dark directly tothe reversible hydrogenase for H2 production. In this assay,the O2-inactivation treatment must be separated from theH2-evolution assay, because the presence of O2 during theassay will reoxidize reduced MV and stop the generation ofH2. The main diKerence between this assay and the light-CEassay is that the reducing environment of the GC-MV assaymay reactivate some hydrogenase that had been inactivatedby the O2 pre-treatment, thus leading to higher apparent O2I50 values in strains that have the capacity to more quicklyreactivate hydrogenase activity.The isolation of diKerent O2-tolerant phenotypes by the

application of either H2-uptake or H2-evolution selectivepressures may also be explained by the nature of the twoselective pressures. The H2-production selective pressureincludes a period of dark anaerobic induction of the hy-drogenase, followed by a dark O2-inactivation pre-treatmentprior to short illumination in the presence of MNZ. Duringthe short illumination, there is very little time for new proteinsynthesis to occur to replace the hydrogenase inactivated byO2. Under these conditions, survival will depend only on theability of the cells to maintain hydrogenase activity follow-ing exposure to O2 in the dark. With the H2-uptake selection,cells are also induced anaerobically before the introductionof O2. In order to survive the subsequent 2 weeks of selec-tive pressure in the presence of O2, speci4c clones wouldhave not only have to maintain an active hydrogenase, butalso to resynthesize hydrogenase in the presence of O2. Ifthe cells were not completely starved before application ofthe selective pressure, one can envision a situation in whichO2 would initially inactivate all hydrogenases. However, O2would then be consumed in the closed vessel by respiration.The resulting anaerobic conditions should then favor the sur-vival of those clones that could more rapidly resynthesize orreactivate the hydrogenase, and they would survive prefer-entially under the resulting photoreductive conditions untilO2 was added to the vessel, again.In summary, the phenotypes of the O2-tolerant isolates

from the two lineages are diKerent, probably due to the se-lection of diKerent types of mutants by each of the selec-tive pressures. The H2-production selective pressure favorsthe isolation of mutants that exhibit a higher tolerance toO2 inactivation. The H2-uptake selective pressure, on the

T. Flynn et al. / International Journal of Hydrogen Energy 27 (2002) 1421–1430 1429

other hand, also allows for the isolation of mutants that canquickly reactive O2-inactivated hydrogenases or resynthe-size the enzyme in the presence of O2.The most interesting conclusion from the work is that

both selective pressures yield mutants with increasedO2-tolerance compared to their parental strains. The dataalso suggest that all 4ve strains reported in Fig. 2 (includ-ing the WT) have unique phenotypes. Given the range ofincreased O2-tolerance estimated by the two assay methodsamong the selected strains, one could argue that there ismore than one genotype resulting in an O2-tolerant pheno-type. Three possible genetic alterations could explain theexistence of diKerent phenotypes among our mutants: (a)diKerent amino acid substitutions at a single critical residuein the hydrogenase enzyme, (b) random substitutions dis-tributed throughout the O2-sensitive domain of the hydroge-nase, or (c) mutations of genes other than the hydrogenasethat cause a decrease in intracellular O2 concentration.Examples of (c) are increased rates of respiration, over ex-pression of O2-scavenging systems in the algal chloroplast,or decreases in the cell (or chloroplast) membrane perme-ability to O2. However, variations in O2 consumption donot occur in our mutants. The rate of dark O2 consumptionby anaerobically induced cell suspensions (measured inthe presence of O2 concentrations below 3%) is roughly20 �mol O2 mg Chl−1 h−1 in all the strains tested. Since in-creased dark O2 consumption would be observed if mutantcells had increased rates of respiration or of O2-scavengingactivity, our data suggest that category (c) mutants are un-likely. Previous work by McTavish, et al. [28] demonstratesthat it is possible to improve the O2-tolerance of the Ni–Fehydrogenase from Azotobacter vinelandii, a heterotrophicbacterium, by means of single amino acid substitutions inthe enzyme itself. Although we cannot conclude de4nitivelythat the multiple observed O2-tolerant phenotypes resultedonly from mutations of the hydrogenase enzyme, the datapresented in Table 2 is consistent with this supposition.Finally, using the light-CE assay, we observed that the O2

tolerance of the hydrogenase in theWT and in both classes ofmutants is higher in light (Table 3).We have not pursued thisobservation any further at this point. Perhaps, the binding ofthe electron donor, ferredoxin, to the hydrogenase, acts asprotective mechanism against O2 inactivation. Protection bybinding of electron donors has been proposed previously [2],based on the observation that the O2-tolerance of the E. colihydrogenase was signi4cantly enhanced by the binding ofarti4cial electron carriers such as methyl or benzyl viologen[29]. However, to our knowledge, protection against O2 bybinding of natural electron carriers has not been reported.The ultimate goal of our research is to create an organ-

ism that uses solar energy to photoproduce H2 from wa-ter at atmospheric O2 levels. Hydrogen is an energy-richmolecule that can be stored and used as a chemical energysource. Despite the fact that the strains isolated thus far arenot useful yet, our current results are important when placedin the following perspective. Clearly, the O2 sensitivity of

hydrogen-production activity in algae can be improved, andour work has provided important strains that will serve asvaluable tools in understanding the structure and=or bio-chemistry that confers or inhibits the sensitivity of algal hy-drogen production to O2. The recent cloning of the C. rein-hardtii hydrogenase hydA1 and hydA2 genes in our labora-tory [30] and that of Happe [31] has opened the door to ge-netic manipulation to achieve the generation of O2-tolerantH2 production. The above-described mutants, when com-pared to the WT on the hydrogenase gene nucleotide level,will provide insight that will help us understand, manipu-late, and improve aerobic H2 metabolism by genetic means.

Acknowledgements

This work was supported by the US DOE Hydrogen Pro-gram. We would like to thank Dr. Robert K. Togasaki for hishelp during the early stages of this project, useful sugges-tions on the manuscript, and continued interest in the work.

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