Bioresource Technology 121 (2012) 454–457

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Bioresource Technology

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Short Communication

Methane production from glycolate excreting algae as a new conceptin the production of biofuels

Anja Günther a, Torsten Jakob a, Reimund Goss a, Swetlana König b, Daniel Spindler b, Norbert Räbiger c,Saskia John c, Susanne Heithoff c, Mark Fresewinkel d, Clemens Posten d, Christian Wilhelm a,⇑a University of Leipzig, Institute of Biology, Department of Plant Physiology, Johannisallee 23, D-04103 Leipzig, Germanyb Saxon Institute of Applied Biotechnology, Permoserstr. 15, D-04318 Leipzig, Germanyc University of Bremen, Institute for Environmental Process Engineering, Leobener Str. 33, D-28359 Bremen, Germanyd Karlsruhe Institute of Technology, Institute of Life Science Engineering, Bioprocess Engineering, Fritz-Haber-Weg 2, D-76131 Karlsruhe, Germany

h i g h l i g h t s

" We show the feasibility of a photo-bioreactor concept to generate bio-methane." The concept is based on the direct fermentation of algal exudates." We show the metabolic engineering strategy to improve productivity." We show a concept for the technical realization of the bio-reactor.

a r t i c l e i n f o

Article history:Received 3 April 2012Received in revised form 14 June 2012Accepted 15 June 2012Available online 9 July 2012

Keywords:BiofuelBiomethaneBioreactorChlamydomonas

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.06.120

⇑ Corresponding author. Tel.: +49 341 9736874.E-mail address: cwilhelm@rz.uni-leipzig.de (C. Wi

a b s t r a c t

It is the aim of the present work to introduce a new concept for methane production by the interaction ofa glycolate-excreting alga (Chlamydomonas reinhardtii) and methanogenic microbes operating in separatecompartments within one photobioreactor. This approach requires a minimum number of metabolicsteps to convert light energy to methane thereby reducing the energetic and financial costs of biomassformation, harvest and refinement. In this feasibility study it is shown that the physiological limitationsfor sustained glycolate production can be circumvented by the use of C. reinhardtii mutants whose carbonconcentrating mechanisms or glycolate dehydrogenase are suppressed. The results also demonstrate thatmethanogenic microbes are able to thrive on glycolate as single carbon source for a long time period,delivering biogas composed of CO2/methane with only very minor contamination.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgal biofuel technologies have been reported as a poten-tial approach to displace fossil fuels in significant amounts in thepost-fossil carbon era (Stephens et al., 2010). Recent progress inreactor design (Posten, 2009) and genetically improved algalstrains (Beer et al., 2009) raise the hope that the costs of algal pro-duction and product processing can be greatly improved. However,the major constraints of microalgal biomass-based biofuel produc-tion include the energetic and financial costs of mixing, harvestingand refinement to the final product oil, ethanol or biogas (Whitakeret al., 2010). Therefore, strategies that bypass these productioncosts will have the greatest potential to increase the efficiencyfrom the absorbed photon to the final product. One such strategycould be algal biohydrogen production. However, at present the

ll rights reserved.

lhelm).

major drawback of this technology is the discontinuity of itssub-processes and the low photosynthetic conversion efficiency(Hallenbeck et al., 2012).

In the present study, a new concept of algal based biogas pro-duction is introduced which aims to minimize the metabolic costsof the algal cells and the energetic costs of the entire process. Thisis achieved by the conversion of photosynthetic energy not intocellular biomass, but into glycolate. This metabolite is producedvia the process of photorespiration and is easily excreted by algalcells. In a second step, glycolate is refined into methane by meth-anogenic bacteria under anaerobic conditions. To reduce the ener-getic costs of the process, a new reactor design is required and isdescribed in detail here.

Since in this new concept it is not intended to produce and har-vest cellular biomass but to convert the cells into ‘‘glycolate pro-ducing units’’, nutrients are needed only in minimal amounts forcellular maintenance. An elimination of nitrogen, sulfur and saltsduring refinement is not required. In addition, metabolic costs

A. Günther et al. / Bioresource Technology 121 (2012) 454–457 455

(e.g. respiratory losses) which are associated with biomass produc-tion will be largely prevented. The feasibility of the concept is dem-onstrated by a set of experimental data. Furthermore, it isdemonstrated that physiological limitations of short-term andlong-term excretion of glycolate can be circumvented by the useof genetically modified algal cells, i.e. specific mutants of Chla-mydomonas reinhardtii.

2. Materials and methods

2.1. Culture conditions of algae

The green alga C. reinhardtii (SAG 11-32b, Culture Collection ofAlgae, Göttingen, Germany) was grown semi-continuously as air-lift cultures in Kuhl-medium (Kuhl and Lorenzen, 1964) at a tem-perature of 20 �C and an irradiance of 150 lmol photons m�2 s�1

(14:10 light/dark). The chlorophyll content of the cultures was ad-justed to a concentration of 5 mg Chl a L�1. Unless otherwise sta-ted, the semi-continuously grown cultures were aerated withCO2-enriched air (3% CO2). The following strains of mutants of C.reinhardtii were obtained from the Chlamydomonas Research Cen-ter (Minnesota): CC-1860 (CCM-mutant) and CC-4160 (GDH-mutant).

2.2. Experimental setup for glycolate production

For the measurements of glycolate excretion, 50 mL of algal cellsuspension were harvested. The cell suspension was aerated withambient air or oxygen-enriched air and illuminated as describedabove. The following O2/CO2 mixtures were used throughout theexperiments: 30/0.036, 40/0.03, 47/0.027, and 70/0.015 (%O2/%CO2). At different time points, samples (4 mL) were collected fromthe reaction vessel. Isoniazid (10 mM) was added to inhibit theinternal metabolization of glycolate. Algal cells were pelleted bycentrifugation and 3 � 1 mL of the supernatant were sterile-filtered (0.2 lm; Roth, Karlsruhe, Germany) and freeze-dried.These samples were used for the quantitative determination of gly-colate based on the colorimetric method with 2,7-dihydroxynaph-thalene (Takahashi, 1972).

2.3. Methanogenesis and gas measurement

The selection of glycolate-utilizing microorganisms was startedfrom an anaerobic culture obtained from a biogas plant. The single-stage cultivation was run at anaerobic conditions at 37 �C in the 5 Lvessel MD 5 (B. Braun Biotech International GmbH, Melsungen,Germany) with 3.5 L working volume. Glycolic acid was added tothe medium as the sole carbon source. Within the cultivation timeof 600 days, the amount of this carbon source was increased from1.12 to 2.35 mM. Gas volume production was measured by Milli-gascounter (Dr.-Ing. Ritter Apparatebau GmbH, Bochum, Ger-many). Oxygen, hydrogen, nitrogen, methane, carbon dioxide andhydrogen sulfide concentrations were estimated by gas chroma-tography (Chrompack CP 9001). The ammonia content was ana-lyzed with the ammonium cuvette test (LCK 303, Hach LangeGmbH, Duesseldorf, Germany).

2.4. Estimation of the expected area-based photo-production rate ofmethane

By taking into account the maximum production rate ofglycolate (PC2H3O�3 ; lmol (mg Chl a)�1 h�1) obtained from theexperiments of this study, the annual area-based photoproductionrate of methane (rCH4 ; g ha�1 a�1) was estimated by:

rCH4 ¼PC2H3O�3

yCH4

½Chl a�MC2H3O�3

Z Sept

Aprtd�2h ð1Þ

with [Chl a] being the maximum Chl a content (mg m�2), themolar mass of glycolate (MC2H3O�3 ; 75 g mol�1), td – 2 h (h) beingthe daily astronomic duration of solar insolation (minus 2 h forsunrise and sunset), and the glycolate to methane fermentationyield (yCH4

; gCH4g�1

C2H3O�3).

3. Results and discussion

3.1. The new concept of algal based biogas production

The basic design of the photo-bioreactor places the photosyn-thesizing algal cells and the methanogenic bacteria in neighboringcompartments (Supplemental Fig. 1). This design requires a sepa-ration by an interface providing a strong oxygen barrier betweenthe algal and bacterial compartment (see below). In the photosyn-thetic compartment, algae are embedded in a solid matrix coveredby a minimum volume of liquid culture medium to ensure the sup-ply of nutrients and a specific CO2/O2-gas mixture. The producedglycolate is transported through the separation unit (see below)into the anaerobic compartment, where methanogenic bacteriaconvert it to carbon dioxide and methane.

In comparison to conventional bioreactors, the design of thisnew reactor is expected to reduce the energetic costs significantly.There is no demand for auxiliary energy input for (i) a completemixing of the algal suspension or (ii) harvesting the algal biomass.It is also expected that (iii) only low pressure gradients are neededfor the transport of glycolate from the photosynthetic to the meth-anogenic compartment.

3.2. Proof of concept

3.2.1. The photosynthetic compartmentThe green alga C. reinhardtii was used for the feasibility study

because it provides the best algal experimental system for furthergenetic engineering. The results of the present study demonstratethat the glycolate production of C. reinhardtii cells can be con-trolled by the gas composition and that the excretion potential ofthe cells can be improved significantly. Cultivation of C. reinhardtiiunder optimal growth conditions with a low O2/CO2-ratio (20/3%)did not lead to excretion of glycolate. A transfer of the cells back toambient air conditions (21% O2, 0.04% CO2), however, resulted insignificant glycolate production at a rate of 9 lmol(mg Chl a)�1 h�1 (data not shown). This glycolate excretion wasfurther enhanced to a rate of 13 lmol (mg Chl a)�1 h�1 by an in-crease of the O2/CO2-ratio to 30/0.03% and 40/0.024%, respectively.

Two cellular processes have a strong influence on the rate ofglycolate excretion and provide important targets for the meta-bolic engineering of the algal cells: (i) the carbon concentratingmechanisms (CCMs), and (ii) the metabolization of glycolate.Numerous algal species possess CCMs to increase the internalCO2 concentration and to favor carbohydrate production via thecarboxylating reaction of RubisCO (Kaplan and Reinhold, 1999). Itis known that CCMs can be suppressed by a high CO2 concentrationduring the cultivation of algae. In line with these data, the resultsof the present study show that the increase of the CO2 concentra-tion from 0.04% (ambient air) to 1% during cell cultivation inducedstimulation of the oxygenating reaction of RubisCO and thus, astrong increase of the glycolate production (Fig. 1a). However,long-term experiments indicated a stagnation of glycolate excre-tion in WT cells of C. reinhardtii after 4 h under photorespiratoryconditions (Fig. 1b). It is likely that the suppression of glycolateexcretion was caused by the activation of CCMs. This means thatsustained glycolate excretion is only possible when cells with

0.04 % 0.5 % 1 % 2 % 3 %0

3

6

9

12

15

18

Gly

cola

te [µ

mol

(mg

Chl

a)-1

h-1

]

CO2 concentration

control10 mM NIA

a

time [h]

Gly

cola

te [µ

mol

(mg

Chl

a)-1

]

0 1 2 3 4 5 6 7 80

15

30

45

60

75

90

CCM mutant

GDH mutant

+ NIA

WT

b

Fig. 1. Rates of glycolate excretion of illuminated cells of C. reinhardtii underdifferent experimental conditions. (a) Comparison of the rate of glycolate excretionduring a 4 h incubation period at an O2/CO2 ratio of 40/0.02% in cells which werepreviously acclimated to different concentration levels of CO2. Data are shown asmean values (n = 3, ±SD) for control samples and samples in the presence of theinhibitor of glycolate metabolism (Isoniazid, NIA). (b) Representative examples oftime-dependent changes in the amount of excreted glycolate in the comparison WTcells, the CCM mutant, and the GDH mutant (±NIA) during incubation with a O2/CO2

ratio of 40/0.02%.

456 A. Günther et al. / Bioresource Technology 121 (2012) 454–457

constitutively suppressed CCM activity are used. This assumptionwas proven by using the CCM mutant CC-1860 of C. reinhardtii.Fig. 1b illustrates that, in contrast to WT cells of C. reinhardtii,the excretion rate of glycolate was stable for at least 7 h. However,the suppression of CCMs in CC-1860 mutants did not change themaximum rate of glycolate excretion, which was dependent onthe rate of glycolate metabolization.

To evaluate the maximum potential of glycolate excretion, theinhibition of glycolate metabolization is required. In the presentexperiments, Isoniazid (NIA) was used to inhibit the C2-pathway(Nelson and Tolbert, 1970) and glycolate oxidation by the glycolatedehydrogenase (GDH; Colman et al., 1974). In the presence of NIAthe glycolate excretion rates in WT cells of C. reinhardtii were en-hanced by a factor of at least three (Fig. 1a). Prolonged incubationwith NIA, however, exerted toxic effects on the algal cells, prevent-ing the long-term application of this inhibitor. Thus, the use oftransgenic cells with a suppressed expression of the GDH is theonly alternative to achieve a high glycolate excretion rate. In thepresent study, the GDH mutant CC-4160 was tested for its glyco-late excretion rates. Within 4.5 h of incubation under photorespira-tory conditions, a comparable rate of glycolate excretion wasobserved for both the GDH mutant and the C. reinhardtii WT cells

in the presence of NIA (Fig. 1b). However, like in the WT cells, gly-colate excretion of the GDH mutant started to stagnate after 4–5 hunder photorespiratory conditions, again due to the induction ofCCMs.

The data from the present study clearly demonstrate that themetabolic limitations for a high and long-term stable excretion ofglycolate can be circumvented by the use of respective mutants.

3.2.2. The methanogenic compartmentIn the present study, methanogenic bacteria were isolated from

a commercial biogas fermentation facility and fed with glycolicacid as the single source of carbon. After an adaptation phase,the gas composition changed from values of a conventional biogasplant to the optimum values of 40% methane and 60% CO2 (Supple-mental Table 1), which are close to the CH4/CO2-ratio given byFriedrich et al. (1991). Hydrogen sulfide and ammonia as contam-inants could only be found in concentrations below 0.1%, and oxy-gen and nitrogen were detected as minor impurities. Thefermentation process was observed to be stable for 600 days at arate of 77 mL biogas per day and liter working volume and a meth-ane yield of 0.173 m3 CH4 kg�1 glycolate was calculated.

3.2.3. Realization of technical aspectsThe conversion of the presented concept into a technical appli-

cation requires the establishment of an algal biofilm in the photo-synthetic compartment. The aeration of this reactor compartmentcan be realized by a membrane integrated into the reactor-inside.This will reduce the energy consumption for aeration and providegreater contact areas for mass transport in comparison to bubble orsurface aeration. The immobilization of algal cells in a biofilm canbe realized by different techniques, such as inclusion or adhesion/adsorption of the cells on appropriate materials (Mallick, 2002).Vílchez et al. (1991) showed that long-term excretion of glycolatecan be achieved with immobilized cells of C. reinhardtii, which is inline with our first results with C. reinhardtii cells embedded in algi-nate (data not shown). With respect to the illumination of the algalcells, it could be necessary to find a technical solution for an opti-mal vertical distribution of light in the biofilm. To realize a densepackage of algal cells in a biofilm while still maintaining a highlight absorption of the individual cells, the use of transgenic cellswith a decreased absorptivity of light may be promising (Melis,2009). Since the results from laboratory experiments may not bedirectly applicable to outdoor conditions (Scoma et al., 2012), nat-ural aerophytic algae have also been investigated in the presentstudy (data not shown).

The fermentation of glycolate to methane requires anaerobicconditions in the methanogenic compartment. Thus, the transportof oxygen from the photosynthetic biofilm into the bacterial com-partment has to be restricted. Consequently, a separation unit(Supplemental Fig. 1) must fulfill two functions: (i) the provisionof a high transport rate of glycolate and (ii) the retention of oxygen.These two requirements can be realized by two different technicalapproaches. The first approach combines a membrane, which al-lows the transfer of glycolate, with a gas stripping facility that re-moves oxygen from the glycolate containing medium. A selectivediffusion of glycolate can be obtained by e.g. micro-filtration mem-branes (RM MV 020, Microdyn-Nadir GmbH, Wiesbaden, Ger-many). The first experiments with such a membranedemonstrate the applicability of microfiltration membranes to al-low the diffusion of glycolate between two separated compart-ments and a gas stripping unit (data not shown). A secondapproach could employ a specific semi-permeable membrane thatallows a maximum passage of glycolate molecules while simulta-neously providing the retention of oxygen.

A. Günther et al. / Bioresource Technology 121 (2012) 454–457 457

3.3. Estimation of the current and future potential of methaneproduction

The present study demonstrates the suitability of the principleprocesses and selected organisms to produce methane on the basisof glycolate excretion. Fig. 2 summarizes the process componentsand the key parameters in order to estimate a glycolate productionrate per unit area. The initial process of photosynthesis requireslight, water and CO2. The glycolate production rate (k1) dependson the presence of high concentrations of oxygen, the daily insola-tion hours, the amount of biomass per area (based on Chlorophyllor cell number), and the oxygenation rate of RubisCO. A reasonableassumption for the biomass concentration is a value of 300 mgChlorophyll per m2, which corresponds to an absorption efficiencyof the photosynthetically available radiation (PAR) of 90%. Assum-ing a light saturation point of 100 lmol photons m�2 s�1 for thephotorespiratory glycolate production, the daily light period,which is able to promote maximum glycolate production, can bederived from the duration of daily solar insolation (minus 2 h forsunrise and sunset). With the additional assumption that algal cellscan be physiologically active at a minimum temperature of 10 �C,this results in a ‘vegetation period’ from April to September witha total of 2240 h of solar insolation. Based on the maximum glyco-late excretion rates (Fig. 1), an annual, area-based glycolate pro-duction rate of 664 g m�2 a�1 is estimated.

The rate of methane production (k2) by methanogenic bacteriabasically depends on their fermentation yield (y). Using the exper-imentally measured glycolate production and fermentation yield, amaximum annual production rate of approximately 1100 m3

methane per hectare is estimated. With the presumption of verylow energy costs during the operation of the proposed bioreactor(10% of gross energy production), an annual net energy gain of40 GJ per hectare is calculated. It should be emphasized that thecurrently measured glycolate excretion rates are well below theexpected physiological maximum value. Due to the fact that for

Fig. 2. Process diagram of the photosynthetic conversion of solar energy intoglycolate and its subsequent fermentation to methane. The glycolate productionrate (k1) results from the duration of daily solar insolation, the oxygenation rate ofRubisCO (POx), and the biomass concentration (BM). The oxygenation rate willdepend on the recycling of the primary acceptor of the Calvin cycle (ribulose 1,5-bisphosphate) by the carboxylation reaction of RubisCO. The kinetics of methaneproduction will result from k1, the fermentation yield of methane formation fromglycolate (yCH4

), and the volume (V; or respective amount of biomass concentra-tion). It is assumed that the diffusion of glycolate from the photosynthetic into themethanogenic compartment is not a rate-limiting step. It is further assumed thatCO2 produced by the fermentation of glycolate can be re-used in the photosyntheticreaction.

every molecule of excreted glycolate, two molecules of CO2 mustbe refixed, the maximum glycolate excretion rate can be as highas one-third of the total CO2 fixation rate (appr. 230 lmolCO2 mg Chl a�1 h�1 according to Wagner et al., 2006). Thus, basedupon optimized conditions for photorespiration and glycolateexcretion, a maximum annual net energy gain of 240 GJ per hectareis estimated. This is about 2.6 times higher than the net energy ofmethane production based on maize. Work is under progess to in-crease the glycolate production under high light conditions whichwould strongly improve the annual net gain.

4. Conclusions

The concept for biofuel production on the basis of glycolate fer-mentation has several synergistic advantages: (i) the bio-methaneproduction via glycolate excretion can be much higher comparedto biofermentation of biomass, because metabolic losses can bestrongly reduced. (ii) The energetic costs for harvesting, extractionand refinement can be decreased to a minimum. (iii) Nutrients areneeded only in minimal amounts, because cells consume themonly for cellular maintenance but not for growth. (iv) The biofilmtechnology does not require heavy hardware. The ideal conceptof a photo-bioreactor incorporates a thin biofilm enclosed in atransparent robust foil.

Acknowledgements

The financial support from the German Ministry of Science andEducation (VIP16V0001) is greatly acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2012.06.120.

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