International Journal of Hydrogen Energy 27 (2002) 1239–1244www.elsevier.com/locate/ijhydene

Hydrogen photoproduction under continuous illumination bysulfur-deprived, synchronous Chlamydomonas reinhardtii

cultures

Anatoly Tsygankova;b, Sergey Kosourova;b, Michael Seibertb, Maria L. Ghirardib; ∗aInstitute of Basic Biological Problems RAS, Pushchino, Moscow Region, 142290, Russia

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

Abstract

UnsynchronizedChlamydomonas reinhardtii cells subsequently deprived of sulfur produce H2 under continuous illuminationin the laboratory for 3–4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronizetheir growth and cell division. While it is clear that only insigni6cant amounts of H2 can be produced by sulfur-deprived cellsduring the night period, little work has been done to examine the e7ects of the light=dark cycles preceding sulfur deprivationon subsequent H2 photoproduction. We show that (a) C. reinhardtii cells exhibit synchronized growth and cell division inthe presence of acetate, (b) cells with the highest speci6c rates of H2 photoproduction also have the highest rates of biomassaccumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetateaccumulation, but not with H2 photoproduction. This work shows that it is possible to maximize the production of H2 bysulfur-depriving synchronized cultures at about 4 h after the beginning of the light period.? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Green algae; Hydrogen; Photoproduction; Synchronous growth; Sulfur deprivation

1. Introduction

Synchronous growth and cell division of green algae areachieved by alternating light and dark cycles [1]. All thecells in a synchronized culture simultaneously increase insize during the light period, and undergo cell division dur-ing the dark period [1,2]. Most commonly, light and dark(L:D) periods of 14 h : 10 h or 12 h : 12 h have been usedto synchronize algal cells in a medium for photoautotrophs[1]. Several metabolic functions show periodicity in syn-chronous cultures. Respiratory O2 uptake peaks during theearly hours (¡ 4 h) of the light period [3], and photosynthe-sis, measured either as O2 evolution or CO2 6xation, peaksat about 6–8 h after the onset of the light period [4]. Thepeak of photosynthetic activity correlates with the peak of

∗ Corresponding author. Fax: +1-303-384-6150.E-mail address: maria ghirardi@nrel.gov (M.L. Ghirardi).

photosystem II (PSII) activity [5,6]. Starch accumulates inthe light and is consumed in the dark, so starch content ishighest at the end of the light period and lowest at the end ofthe dark period [7]. The periodicity of H2 photoproductionactivity in synchronized C. reinhardtii algal cells was stud-ied extensively by Yanyushin [7–10]. He observed a peak inanaerobically induced hydrogenase activity during the earlyhours of the dark period, coinciding with the beginning ofstarch degradation [8].

The periodicity of several cellular functions in synchro-nized cultures is dependent either on metabolic events thatare regulated by the cell cycle or on an internal circadianclock (see references in [2]). For example, the periodicity ofthe aHnity of the photosynthetic apparatus for CO2 can bemaintained even after synchronized cells are transferred tocontinuous light [11]. On the other hand, many metabolicinhibitors, nutrient-deprived conditions, or other physio-logical interferences prevent the normal operation of thecell cycle by interrupting it at di7erent control points [12].

0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.PII: S 0360 -3199(02)00108 -8

1240 A. Tsygankov et al. / International Journal of Hydrogen Energy 27 (2002) 1239–1244

Sulfur deprivation arrests the normal algal cell cycle beforethe cell division stage at the end of the light period, andgives rise to cultures enriched in large cells that do notdivide [13].

We have used sulfur deprivation of green algae to obtaincultures that photoproduce H2 for several days before theyrequire a sulfur-replete, regenerative period [14,15]. Our ob-jective has been to develop a system for potential futureapplications under outdoor conditions, and the research hasconcentrated on optimizing H2 production rates and accu-mulated amounts in sulfur-deprived cultures. In our previ-ous studies, algal cells were grown photoheterotrophicallyto late-log phase, deprived of sulfate and then exposed tocontinuous light. We used 14 h : 10 h L:D synchronized cul-tures and observed that cells harvested 4 h after the start ofthe light period showed high yields of H2 photoproductionwhen subsequently deprived of sulfate [16]. In the work pre-sented here, we have expanded our investigations to includestudies of H2-photoproduction activity of cells harvested atother stages during the cell cycle.

2. Materials and methods

2.1. Cell growth

Chlamydomonas reinhardtii, strain cc124 (a wild-typestrain originally from Levine’s laboratory [1]), was grownphotoheterotrophically on Tris-acetate-phosphate (TAP)medium, pH = 7:2, in 1:2 l Lat glass bottles with stirringat about 28

◦C [16]. The synchronous growth and cell di-

vision of C. reinhardtii cells was achieved by alternatinglight (12 h) and dark (12 h) periods for at least 7 days andmaintained by daily dilution of the cultures to a startingdensity of about 0.5–1 × 106 cells ml−1. The achievementof synchronous cell division was checked by microscopicobservations, as before [16]. We chose a shorter light pe-riod in this work (12 h, compared to 14 h in Ref. [16]) inorder to conform with most of the previous synchronizationliterature. Synchronous and unsynchronous cultures wereaerated with 3% CO2 in air (approximately 90 ml min−1).Synchronous algal cells were grown to a 6nal concentrationof about 2–5× 106 cells ml−1, harvested at di7erent stagesduring the cell cycle by centrifugation at 2000 g for 5 min,washed 6ve times in TAP-minus-sulfur medium, and thenresuspended in the same medium at a 6nal concentration ofabout 9 �g Chl ml−1 (4–5× 106 cells ml−1).

2.2. Bioreactor system

Both synchronized and unsynchronized sulfur-deprivedcell suspensions were placed in each of four glass photo-bioreactors (4-cm optical path, 1:2 l culture volume). Thealgal cells were cultured under continuous two-sided illu-mination of ∼ 300 �E m−2 s−1 (12 40-W cool-white Luo-rescent lamps) at 28 ± 1:5

◦C for up to 140 h as described

before [16]. Four physical, biophysical and electrochemi-cal factors (pH, Eh, pO2, and temperature), as well as thevolume of gas produced, were computer-monitored simulta-neously, continuously and independently in each of the fourphotobioreactors, using the system described in Kosourovet al. [16]. The maximum speci6c rates of H2 produc-tion were calculated from the accumulated H2-productioncurves, usually at 20–24 h after the start of gas collection.The results were expressed on the basis of chlorophyllconcentrations in the cultures measured at the relevanttimes.

2.3. Biochemical assays

The chlorophyll a + b content was assayed spectropho-tometrically in 95% ethanol extracts by the method ofSpreitzer [1]. The samples for starch, protein, acetate andformate analysis were taken directly from the photobioreac-tors and centrifuged for 5 min at ∼ 2000 g. The pellets andsupernatants were separated and stored frozen at −80

◦C

until all the samples were ready for assay. The amount ofstarch and protein accumulated inside the cells was deter-mined in the pellet on a per ml of culture basis according tothe methods of Gfeller and Gibbs [17] and Lowry et al. [18],respectively. The levels of acetate and formate in themedium were determined by HPLC (Model 1050,Hewlett-Packard, Palo Alto, CA) using an ion-exchangecolumn (Aminex HPX-87H, Bio-Rad, Hercules CA) and4 mM H2SO4 as the mobile phase.

3. Results

Using the computer-monitored photobioreactor system,we have previously reported [16] that sulfur-deprivedC. reinhardtii cultures progress through 6ve well-de6nedphysiological phases: (a) an aerobic phase (during whichphotosynthetic O2 evolution activity is gradually inacti-vated); (b) an O2-consumption phase (when the photo-synthetic O2 evolution activity falls below the respiratoryactivity of the cells, leading to net O2 consumption); (c)an anaerobic phase (after all O2 in the culture medium isremoved by respiration); (d) a H2-production phase (afterestablishment of anaerobiosis and induction of hydrogenaseenzyme activity); and (e) a termination phase (when allcellular activity is gradually inactivated).

The same system was used to study the inLuence of thecell cycle on H2 photoproduction by sulfur-deprived cells.Cells grown under 12 h : 12 h L:D cycles were harvested atdi7erent times during the cycle (4, 8, 12, 16, 20 and 24 h).These cells were transferred to fresh medium, which wasdeprived of sulfur, and exposed to continuous illumination.In the text, these will be designated as t=4; 8; 12; 16; 20 and24 h cells or cultures. Table 1 shows the average length ofeach of the 6rst four phases mentioned above. The length ofthe aerobic phase varied from 23–53 h. It was shortest when

A. Tsygankov et al. / International Journal of Hydrogen Energy 27 (2002) 1239–1244 1241

Table 1Length of the physiological phases resulting from sulfur deprivation, and the total yield of H2 production by cultures harvested at di7erentstages during a 12 h : 12 h L:D cycle. The cultures were harvested, sulfur-deprived and then exposed to continuous illumination

Type of culture Aerobic O2-consumption Anaerobic H2-production Total yield ofphase (h) phase (h) phase (h) phase (h) H2 (after 140 h) (ml)

Synchronized (h)4 35 14 11 79 1028 22.5 17 16.5 71 29

12 23 14.5 10.5 64 3316 25 11 14 60 4420 53 4 13 62 5024 47 8 13 72 74

Unsynchronized 23 13 9 63 86

the samples were taken at the 8 and 12 h time points (secondhalf of the light period), and longest with samples at the 20and 24 h time points (second half of the dark period). Thistrend reversed during the O2-consumption phase, where theshortest duration was seen with t = 20–24 h cells and thelongest duration with t=8–12 h cells. The longest anaerobicphase was also observed with t = 8 h cells, but the dura-tion of this phase varied little in cultures harvested at theother time points. Finally, the duration of the H2-productionphase peaked with t=4 h cells and was shortest with t=16and 20 h cells. The behavior of the unsynchronized con-trol cultures most closely resembled that of cells harvestedat t = 12 h, with short aerobic and long O2-consumptionphases, and short anaerobic and H2-production phases.However, in contrast with t = 12 h cultures, unsynchro-nized cultures produced larger volumes of H2 gas andexhibited higher maximum speci6c rates of H2 produc-tion (data not shown), which are characteristic of t = 4 hcultures.

Fig. 1 shows the accumulation of H2 as a function of timein the sulfur-deprived medium. Each panel in Fig. 1 showsthe total yield of H2 (in ml) obtained with samples har-vested at the indicated times. The upper panels correspondto cells that were harvested and sulfur-deprived during thelight period and the lower panels represent samples fromthe dark period. Vertical dashed lines in each panel indicatethe times at which the cultures became anaerobic. The totalamount of H2 produced by each culture and the maximumspeci6c H2-production rates were then plotted in Fig. 2Aas a function of the time during the cell cycle at which thecells were harvested and deprived of sulfur. The maximumspeci6c rates of H2 production and the total accumulatedamounts of H2 were highest at t=4 h, and declined sharplywith cells harvested at t=8 h. Fig. 1 also shows that proteinand starch concentrations increase during the early stagesof sulfur deprivation, as previously reported, and then de-crease, concomitantly with H2 production [16,19]. The con-centration of chlorophyll follows a less predictable course.An initial increase in concentration, followed by a decrease

concomitantly with H2 production is only observed with t=4and 8 h cells. Cells from t = 12 and 16 h show a decreasein chlorophyll concentration throughout sulfur deprivation,and very small variations in chlorophyll concentration wereseen during the experiment with t = 20 and 24 h cells.Fig. 2B shows that maximum protein and chlorophyll (butnot starch) accumulation occurs during the early stages ofsulfur deprivations in cells harvested at t=4 and 24 h. Thisindicates that the peak of H2 photoproduction and biomassaccumulation (measured by increases in chlorophyll andprotein contents) by sulfur-deprived cells coincides with thepreviously reported peak in photosynthetic activity of algalcells [4].

Fig. 1 also shows that signi6cant starch and proteindegradation takes place during the H2-production phaseof the sulfur-deprivation process. Both starch and proteindegradation are important for H2 photoproduction, becausethey are involved in the establishment and maintenance ofanaerobic conditions in the photobioreactor vessel followingthe decrease in photosynthetic O2-evolution activity of thecultures [16,19], and also because they contribute a smallfraction of the reductants used for H2 photoproduction [20].The total amounts of starch and protein degraded duringthe later phases of the sulfur-deprivation process, estimatedfrom Fig. 1, are shown in Fig. 2C. In contrast to chlorophylland protein accumulation and H2 photoproduction, starchand protein degradation under sulfur-deprived conditionsare slightly higher with cells harvested during the darkperiod. We also measured the extracellular accumulationof fermentation products during sulfur deprivation that re-sulted from anaerobic starch and protein degradation. Fig.3 shows that the acetate content of the medium decreasesduring the early phases of sulfur deprivation, due to acetateconsumption when PSII inactivation is occurring. After theestablishment of anaerobiosis, however, both acetate andformate are produced and excreted into the growth medium.Fig. 2C shows that the total production of formate andacetate also depends on the stage in the cell cycle at whichthe cells were sulfur-deprived and, as expected, the depen-

1242 A. Tsygankov et al. / International Journal of Hydrogen Energy 27 (2002) 1239–1244

24h

0 20 40 60 80 100 120 140

Tot

alH

ydro

gen

accu

mul

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,ml

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120

Chl

orop

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Time after sulfur deprivation, h0 20 40 60 80 100 120 140

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0 20 40 60 80 100 120 140

Sta

rch

and

prot

ein

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ent,

µ mol

es/m

l

0.0

0.5

1.0

1.5

2.0

ChlorophyllProteinStarchHydrogen

4h

0.0

0.5

1.0

1.5

2.0

Fig. 1. Concentrations of chlorophyll a + b (�g ml−1), protein and starch (�moles ml−1) and accumulated volume of H2 (ml) producedduring continuous illumination under sulfur-deprived conditions. Cells were harvested at indicated times during a 12 h : 12 h L:D cycle.Upper panels: cultures of cells harvested during the light period (at 4, 8 and 12 h); lower panels: cells harvested during the dark period (16,20 and 24 h). Vertical dashed lines indicate the times at which the cultures became anaerobic.

dence on the cell cycle resembles that for starch and proteindegradation.

4. Discussion

In the past, researchers claimed that synchronization ofC. reinhartdtii cell growth and division required photoau-totrophic conditions [1], and if acetate were present in themedium, cell division occurred unsynchronously [21]. ForH2 photoproduction from sulfur-deprived algal cultures, ac-etate and light are necessary for timely inactivation of O2

evolution [20]. In our experiments, however, the presence ofacetate in the growth medium did not prevent the establish-ment of synchronous growth in C. reinhardtii cell cultures[16]. Photomicrographs taken at three di7erent points dur-ing the L:D cell cycle, at t = 4; 9 and 23 h, clearly showedthe well-de6ned di7erences in cell structure [16] that cor-relate with previously-described changes occurring as thecells went through the cycle [1].

Following sulfur deprivation, synchronous cultures ofanother green alga, Chlorella ellipsoidea, continue to gothrough the cell cycle, until they are arrested at the stageof cell division [13]. At the cell division stage, early duringthe dark phase, C. reinhardtii cultures exhibit the highestinducible hydrogenase activity and lowest rate of photo-

synthetic O2 evolution [7]. One might expect, then, thatcell cultures sampled at di7erent times during the cell cyclewould all eventually show the same high H2-productionactivity after sulfur deprivation. In reality, sulfur-deprivedcultures harvested at di7erent intervals in the cell cycleexhibited marked di7erences in H2-production activity. Cul-tures from 4 and 24 h cells produced the highest amountsof H2 and showed the highest speci6c rates of H2 photopro-duction after sulfur deprivation, and they also exhibited themaximum rates of growth (protein and chlorophyll accu-mulation) during the aerobic and O2-consumption phases.In contrast, cultures harvested at 8 and 12 h produced thelowest amounts of H2 and had the lowest rates of proteinaccumulation. While it is diHcult to explain this observa-tion, it may be that an initial high rate of growth allowssulfur-deprived cells to undergo more than one cell cycle,and thus to build up more biomass (measured as protein)before growth is arrested by the lack of sulfate. This wouldexplain the higher quantity of biomass at t = 4 and 24 h,but would not explain the increased speci6c H2-productionactivity of the same cultures. We must thus conclude thatthe level of cell growth and photosynthetic activity of thecultures during the early stages of sulfur deprivation (theaerobic phase) determines the speci6c H2-production ca-pacity of the cultures during the subsequent H2-productionphase.

A. Tsygankov et al. / International Journal of Hydrogen Energy 27 (2002) 1239–1244 1243

Point in the cell cycle (h) at which cells were harvested

0 4 8 12 16 20 24

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3.5Protein degradedStarch degradedAcetate producedFormate produced

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mg

Chl

xh)

0

1

2

3

4

5

6

7

Total yield of H2

H2 production rate

(A)

(B)

(C)

Fig. 2. Total production or degradation of di7erent products during sulfur deprivation in 1:2 l of cells harvested at di7erent times duringthe cell cycle: (A) amount of H2 (mmoles) and maximum speci6c rate of H2 produced (�moles (mg Chl h)−1); (B) changes in the totalamount of chlorophyll a+ b (mg), starch and protein (mmoles) during the early phases of sulfur deprivation; and (C) amount of starch andprotein degraded and formate and acetate produced (mmoles) during the late phases of sulfur deprivation. The conversion of H2 from ml(Fig. 1) to mmoles at 28

◦C and 1600 m altitude is 1 ml = 0:033 mmoles.

We also observed that the amounts of starch and pro-tein degraded during the sulfur-deprivation process arehigher when the cells are harvested at t = 16–24 h, whichcoincides with the maximum accumulation of fermen-tation products. This con6rms our observation that H2

photoproduction is directly related to the photosyntheticwater oxidation activity, and not to endogenous sub-strate degradation ([20]; S. Kosourov et al., unpublishedresults).

In summary, the rates and accumulated amounts of H2

photoproduction by sulfur-deprived C. reinhardtii culturesis inLuenced by synchronous L:D growth and division of thecultures prior to sulfur deprivation. Furthermore, in contrastto previous reports, synchronous growth and cell divisionof C. reinhardtii can be achieved under photoheterotrophicconditions with acetate in the medium. Sulfur deprivationand subsequent continuous illumination of cells harvestedduring the early hours of the light cycle yield higher

1244 A. Tsygankov et al. / International Journal of Hydrogen Energy 27 (2002) 1239–1244

Time after sulfur deprivation, h

0 20 40 60 80 100 120 140

Ace

tate

cont

ent,

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es

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2.4

4 h8 h12 h16 h20 h24 h

(A)

(B)

Fig. 3. Changes in the amount of formate (A) and acetate (B)in the extracellular medium (mmoles) during sulfur deprivation,using cells harvested at di7erent times in the cell cycle.

production rates and volumes of H2. This information maybe useful in the design of algal H2-production photobioreac-tor systems, which will have to utilize cells grown outdoorsunder day=night cycles.

Acknowledgements

This work was supported by the US DOE HydrogenProgram.

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