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Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e6
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Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis ski, A. Nowicka, P. Rusanowska M. Dudek*, M. De˛bowski, M. Zielin University of Warmia and Mazury in Olsztyn, Faculty of Environmental Science, Department of Environment Engineering, Warszawska 117, 10-720 Olsztyn, Poland
article info
abstract
Article history:
Platymonas subcordiformis is a marine microalgae that under favorable environmental
Received 5 December 2017
conditions change metabolism pathways to hydrogen production in direct biophotolysis.
Received in revised form
Effective hydrogen production by Platymonas subcordiformis depends on application of
4 April 2018
efficient and economically viable biomass production technologies. In the study, the nat-
Accepted 5 April 2018
ural water from Vistula Lagoon was used for microalgae cultivation. No statistically sig-
Available online xxx
nificant differences were found regarding the biomass production in the natural water and synthetic medium. The influence of mixotrophic conditions on growth rate and biomass
Keywords:
production of Platymonas subcordiformis was also examined. The highest biogas production
Green algae
of 138.45 ± 3.39 mL with the rate of 1.15 ± 0.03 mL/h was noted by the biomass cultivated on
Mixotrophic growth
synthetic medium with glucose supplementation. Similarly high biogas production was
Photo-hydrogen production
observed by the biomass cultivated on natural water with glucose addition (1.11 ± 0.14 mL/
Fresh water
h). The use of the waters from Vistula Lagoon resulted in high yields of hydrogen production, which might reduce costs of biofuel production. © 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.
Introduction Microalgae biomass is a source of many valuable substances such as saccharides, proteins, fatty acids, vitamins, antibiotics and pigments (b-carotene, astaxanthin) [1,2]. Microalgae biomass might be also utilized for the energy purposes, since their liquid and gas metabolic products are used as a third generation fuels. Hydrogen energy is a clean and alternative energy that has been suggested as the energy carrier of the future. Solardriven microalgal hydrogen production is both, a promising and challenging biotechnology, which play an important role
in the global drive to reduce GHG emissions. The potential of hydrogen production in microalgae depends on strain specific capacity to synthesize different enzymes responsible for hydrogen metabolism and environmental conditions providing required energy [3e7]. The production of H2 in green algae is associated with the direct biophotolysis, which usually occurs when algal cultures are exposed to light after a period of dark with anaerobic adaptation. Microalgae species used for biohydrogen production are Chlamydomonas reinhardti, Chlorella sp. and Scenedesmus obtiguus etc.. Chlamydomonas reinhardti is the most widely used microorganism for hydrogen production studies in the laboratory due to its quality as a model organism [8]. However, it might not be the
* Corresponding author. E-mail address: [email protected] (M. Dudek). https://doi.org/10.1016/j.ijhydene.2018.04.039 0360-3199/© 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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most suitable organism for efficient production. Platymonas subcordiformis is a marine green alga demonstrated to produce hydrogen efficiently in various conditions [6,9,10]. One of the major barriers with regard to hydrogen economy is biomass production cost, which is related to composition of the cultivation medium. Thus, the utilization of the alternative media for biomass production is economically justified. The cultivation of microalgae Platymonas subcordiformis on the natural water from water reservoirs has not been widely applied in energy generation approach. In preliminary studies, the authors have observed that concentration of nutrients in natural waters is too low for intensive biomass production of Platymonas subcordiformis. Thus, in the study the authors supplemented the natural water with nitrogen and phosphorus compounds. Ran et al. [11] had similar observations in the study with Platymonas subcordiformis cultivation on natural water from Bohai Bay in China. The cell growth and microalgal biomass production might be enhanced by environmental factors (light, pH and temperature) and supplementation of macro and micronutrients in the medium [12]. Microalgae can survive in extreme environments as they can adapt their metabolism according to altering environmental conditions. Usually, microalgal biomass is produced through autotrophic cultivation in an open ponds or a photobioreactor using solar energy for fixing CO2. Alternatively, they are also cultivated in heterotrophic condition with organic compounds serving as energy and carbon sources [13e15]. Among the different modes of cultivation, mixotrophic operation offers several advantages. In mixotrophic conditions, both cell growth and biosynthesis of products are significantly influenced by the nutrients present in the medium and by the environmental factors. The syntrophic association between microalgae and its existing microenvironment facilitates higher biomass production. To stimulate mixotrophic growth glucose was chosen as a carbon source due to other carbon sources need more complicated inter-conversion metabolic process to provide energy for algal growth as well as bioenergy production microalgae growth [16,17]. In the study, the influence of mixotrophic conditions on the growth rate and biomass production by Platymonas subcordiformis was examined. Furthermore, the biomass of
Platymonas subcordiformis grown in the mixotrophic conditions was used for photosynthetic biohydrogen production.
Materials and methods Cultivation conditions Microalgae Platymonas subcordiformis was obtained from culture collection UTEX (University of Texas at Austin). Microalgae were cultivated in two different media (series of the study). In the series 1 (S1), medium was deionized water, whereas in the series 2 (S2) medium was water collected from Vistula Lagoon (54 210 3000 N 19 400 5900 E). The water from Vistula Lagoon was characterized by: chemical oxygen demand (COD) 33.57 ± 7.72 mg O2/L, ammonium nitrogen 0.08 ± 0.01 mg NH4e N/L, total nitrogen 1.39 ± 0.32 mg Ntot/L, orthophosphates 30.14 ± 0.07 mg PO3 4 /L, total phosphorus 0.05 ± 0.01 mg PO4 2 Ptot/L, sulphureous 141 ± 1.53 mg SO4 /L, chlorine (I) 723 ± 4.10 mg Cl/L, chlorine (II) 99 ± 0.20 mg Cl/L, iron (II) 0.11 ± 0.01 mg Fe2þ/L, iron (III) 0.12 ± 0.02 mg Fe3þ/L, pH of 7.84 ± 0.25. Both media were supplemented with appropriate nutrients addition [18]. In each series of the study, one medium was additionally supplemented with glucose (variants of the experiments). Glucose was added to the medium to investigate the biomass and hydrogen production in the mixotrophic conditions. Based on the preliminary studies and data presented in the literature, the dose of glucose was 10 g/L (Xie et al., 2001, Guan et al., 2004) Thus, the study tested hydrogen production by Platymonas subcordiformis biomass cultivated on medium based on deionized water (S1) and fresh water from Vistula Lagoon (S2), both series were performed in two variants: without addition of glucose (V1) and with addition of glucose (V2). The characteristic of the media used for Platymonas subcordiformis cultivation are presented in Table 1. Microalgae were cultivated in bioreactor BioFlo 115 New Brunswick with active volume of 2 L. Temperature of cultivation was 25 ± 1 C. The cultures were illuminated at 5klux by cool-white light (14 h light, 10 h dark). The cultures were continuously stirred and aerated with air provided by pump Mistral 200 with intensity 200 L/h. Microalgae was cultivated for 11 days.
Table 1 e Characteristic of the media used for Platymonas subcordiformis cultivation. Parameter
COD Ammonium nitrogen Total nitrogen Orthophosphates Total phosphorus Sulphureous Chlorine (I) Chlorine (II) Iron (II) Iron (III) pH Salinity Glucose
Unit
mgO2/L mgNH4eN/L mg Nog./L mg PO34/L mg PO34Pog./L mg SO24/L mg Cl/L mg Cl/L mg Fe2þ/L mg Fe3þ/L e ppt g/L
Series 1
Series 2
Variant 1
Variant 2
Variant 1
Variant 2
55.5 ± 2.01 0.04 ± 0.01 21.13 ± 0.63 1.85 ± 0.24 5.00 ± 0.20 525 ± 1.00 7510 ± 10 16128 ± 55.2 0.09 ± 0.01 0.09 ± 0.01 8.09 ± 0.12 30 ± 0.7 e
11033 ± 57.73 0.039 ± 0.01 20.79 ± 0.56 13.53 ± 0.25 5.04 ± 0.14 521 ± 6.11 7318 ± 2.89 16113 ± 15.26 0.103 ± 0.06 0.102 ± 0.07 8.08 ± 0.09 30 ± .09 10
52.0 ± 1.28 0.65 ± 0.01 21.99 ± 1.35 5.11 ± 4.33 5.37 ± 0.03 548 ± 1.00 12466 ± 105 19623 ± 165 0.117 ± 0.03 0.048 ± 0.02 8.09 ± 0.23 31 ± 1.1 e
11014 ± 131 0.07 ± 0.01 22.11 ± 0.22 13.5 ± 0.26 5.25 ± 0.14 534 ± 5.51 7492 ± 10.4 17220 ± 60 0.114 ± 0.03 0.051 ± 0.01 8.10 ± 0.07 30 ± 0.4 10
Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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Hydrogen production procedure After cultivation, the biomass of Platymonas subcordiformis was placed in a respirometric bottles (0.5 L) filled with the medium of composition indicating hydrogen production (Guan et al., 2004). The concentration of P. subcordiformis biomass in hydrogen production procedure was about 3 g VS/L. To induce H2 production, the microalgal cells were subjected to a twophase incubation. In the first phase, in order to hydrogenase induction, the algal cells were placed in dark anaerobic conditions for 30 h. Anaerobiosis was achieved by continuous flushing of pure nitrogen through the culture suspension. The second phase of photobiological hydrogen production was then initiated by placing the cultures under continuous light illumination at 5 klux for 5 days. The conditions for indicating hydrogen production by Platymonas subcordiformis were based on the results obtained during the preliminary studies and the data presented in the literature [11,18]. During the experiments amount of produced gases were continuously monitored. After the incubations the composition of biogas was measured.
Analytical procedures Online detection of chlorophyll concentration, algae classes and photosynthetic activity was done with BBE AlgaeOnLine Analyser (Moldaenke). Biomass concentration was measured with a gravimetric method. Water from Vistula Lagoon was collected from May to October, before cultivation it was filtered on a filter paper (pore diameter 0.45 mm) and then sterilized at 121 C for 15 min (Tuttnauer 2840 ELeD). Light intensity was measured with Lux Meter (Hanna Hi 97500). Salinity and pH was measured with Marine Control Digital salinity meter (Aqua Medic) and multiparameter (Hach Lange HQ 440D), respectively. Nitrogen Ntot, ammonium nitrogen N3 NHþ 4 , total phosphorus Ptot, orthophosphate P-PO4 , sulfur 2þ 3þ SO2 , chloride Cl , iron Fe , Fe , and COD concentrations were measured with cuvette testes (Hach Lange) and spectrophotometer UV/VIS (DR 5000). The quality of biogas was measured by using a gas chromatograph connected with
Results and discussion The study revealed differences in the biomass and hydrogen production by Platymonas subcordiformis cultivated on tested media.
Biomass production The biomass concentration in all tested media did not differ statistically and was from 3203.33 ± 35.12 mg VS/L to 3790.33 ± 281.73 mg VS/L in S1V1 and S2V2, respectively (Fig. 1A). Similar results were obtained by other authors. The concentration of Platymonas subcordiformis cultivated on synthetic medium was from 3200 mg VS/L [6] to 3680 mg VS/L [19]. Chinnasamy et al. [20] cultivated species of Platymonas on carpet industry wastewaters. Growth of microalgae after 10 days of cultivation was estimated in terms of chlorophyll-a content. The authors obtained chlorophyll-a concentration of 2800 mg/L in Platymonas suecica cultivation and 7300 mg/L in Platymonas chuii cultivation. In the present study, the concentration of chlorophyll-a statistically differ depending on the medium used for microalgae cultivation (Fig. 1B). The chlorophyll-a concentration was almost twice times higher in the biomass cultivated on media without glucose addition than in the biomass cultivated on media with glucose addition, in the both series of the study. Xie et al. [19] also observed lower concentration of chlorophyll-a in cultures of Platymonas subcordiformis, when the microalgae growth was stimulated by addition of glucose to the medium. Similarly, the concentration of chlorophyll-a in the phototrophic culture (without glucose) was about twice times higher than in the mixotrophic culture (with glucose). Changes in concentration of the pigments responsible for photosynthesis might be observed in
B
5000
Chlorophyll-a concentration [μg/L-1]
Biomass concentration [mgVS/L-1]
A
thermal conductivity detector (GC-TCD) (Agilent 7890A). After testing for homogeneity of variance with Levene's test, the significance of differences between variants was tested with Tukey's HSD test. Differences were considered significant at p < 0.05.
4000
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5000
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0
0 1
3 S1V1
5 Time [d] S1V2 S2V1
7
9 S2V2
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1
3 S1V1
5 Time [d] S1V2 S2V1
7
9
11
S2V2
Fig. 1 e Changes in concentration of A) biomass and B) chlorophyll-a in variants of experiment. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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A
B
80
80
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60 [%]
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[%]
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40
40
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0
S1-V1
S1-V2
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Mean±stand.error
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S2-V2
Series-Variant
Mean±stand. dev.
Mean
Mean±stand. error
Mean±stand.dev
Fig. 2 e The efficiency of removal of A) Ntot and B) Ptot from the media in the variants of experiment.
relation to changes in microorganism's metabolism when they are grown in the phototrophic and mixotrophic conditions. The utilization of glucose weaker the photosynthesis process and reduce the number of chloroplasts.
Nitrogen and phosphorus removal After 11 days of the biomass cultivation the concentration of Ntot and Ptot in the media was measured (Fig. 2). The concentration of nitrogen after Platymonas growth in the medium S1V1 was 0.61 ± 0.37 mg Ntot/L whereas in the medium S1V2 it was 5.11 ± 0.13 mg Ntot/L. Similar tendency was observed in the S2, the removal of nitrogen was about 95% and 75% in the V1 and V2, respectively (Fig. 2A). The concentration of phosphorus after Platymonas growth in the medium S1V1 was 0.04 ± 0.01 mg Ptot/L whereas in the medium S1V2 it was 0.66 ± 0.09 mg Ptot/L. Similar tendency was observed in the S2, the removal of phosphorus was 94% in the V1 and 86% in the V2 (Fig. 2B). It might be observed that in the variants without addition of glucose to the medium, removal of
A 10
nutrients was higher than in the variants with addition of glucose to the medium. The utilization of nutrient for the biomass growth was also calculated (Fig. 3). Higher amount of nutrients for the biomass growth was used in the variants without glucose addition than with glucose addition to the medium, in both series of experiments (efficiency of glucose removal was 98.55% and 98.77% in the S1V2 and S2V2, respectively). Zhai et al. [21] compared biomass production of Spirulina platensis in the mixotrophic conditions (with glucose addition) and photosynthetic conditions. The authors observed enhanced the microalgal production, nutrients removal and recovery in the mixotrophic conditions. It was opposite to the results obtained in this study. However, the authors used media with much lower concentrations of nutrients and used much lower dose of glucose than in this study. The authors observed that concentration of 150 mg/L of glucose increased nutrients removal of about 6%, when compared to the nutrients removal from the medium without glucose addition. When the authors increased concentration of glucose to 600 mg/L,
B
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2,0 [mg Ptot./gVS]
[mg Ntot./gVS]
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S1-V1 Mean
S1-V2
S2-V1
Series-Variant Mean±stand.error Mean±stand. dev.
S2-V2
0,0
S1-V1 Mean
S1-V2
S2-V1
S2-V2
Series-Variant Mean±stand. error Mean±stand. dev.
Fig. 3 e The utilization of A) Ntot and B) Ptot for biomass growth in the variants of experiment. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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160 140
Biogas [ml]
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Time [h] S1V1
S1V2
S2V1
S2V2
Fig. 4 e Biogas production after 11 days of biomass growth in the variants of experiment.
50
H2 [ml/g VS]
40
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S1V1 Mean
S1V2 Mean±stand. error
S2V1
5
1.11 ± 0.14 mL/h. Significantly lower biogas production was noted in variants without glucose addition to the medium (S1V1 and S2V1), the rate of biogas production of 0.91 ± 0.08 mL/h and 0.81 ± 0.05 mL/h was observed. Hydrogen content in the biogas was the highest in the S2V1 and it was about 71%. In the S1V2, hydrogen concentration was about 67%. Significantly lower concentration of hydrogen was noted in the S1V1 and S2V2, where it was 43% and 60%. Thus, hydrogen production per gram of VS was the highest in the S1V2, but similarly high result was observed in the S2V1 and S2V2 (Fig. 5). Significantly lower hydrogen production per gram VS was noted in the S1V1 (14.26 ± 1.20 mL/g VS). Microalgae Chlamydomonas reinhardtii also produced more H2 under the mixotrophic culture than under the phototrophic culture [23]. Organic carbon increased growth rate of C. reinhardtii leading to the accumulation of carbohydrates whose successive degradation can participate in H2 production. Probably the same mechanism occurs in microalgae P. subcordiformis. It is also worth noting on increased hydrogen production by microalgae cultivated on the natural water without glucose addition (Fig. 5). Enhanced biogas production was probably a result of the medium components deprivation. Natural water is a medium richer in compounds than the synthetic medium. The change of environmental conditions in case of microalgae cultivated on the natural water (S2V1) to medium for biogas production was more significant than that of the synthetic medium (S1V1). This dramatic change could result in the higher biogas production. Microalgae Chlamydomonas reinhardtii grown on a medium consisting of pretreated olive mill wastewater and TAP (tris-acetate-phosphate) (1:1 ratio) was found to exhibit a greater hydrogen production, compared to the control cells grown in TAP only [24].
S2V2
Mean±stand. dev
Fig. 5 e Hydrogen production per gram of VS from biomass cultivated in the variants of experiment.
no significant improvement was observed in nitrogen and phosphorus removal. The dose of glucose used in this study, for Platymonas subcordiformis cultivation was chosen on the basis of literature data [19]. Probably, this dose was too high for efficient nutrients removal by Platymonas subcordiformis, due to mixotrophic cultivation usually leads to enhanced biomass production and nutrients removal, which was not observed in this study. For Chlorella vulgaris the same observation was noted, a higher biomass and lipid accumulation was obtained under lower concentration of glucose and glycerol [22].
Biogas/hydrogen production Biogas production took place after 11 days of the biomass growth (Fig. 4). The highest biogas production of 138.45 ± 3.39 mL with the rate of 1.15 ± 0.03 mL/h was noted in the S1V2. Similarly high biogas production was observed in the S2V2, the rate was
Conclusions The cultivation of microalgae on the natural water did not significantly change the biomass production of Platymonas subcordiformis in comparison to cultivation on the synthetic medium. However, the biomass of Platymonas subcordiformis cultivated on the natural water produced higher hydrogen yield. Additionally, mixotrophic conditions in the bioreactor improved biogas production by Platymonas subcordiformis biomass.
Acknowledgements "The project was financed by the National (Polish) Science Center, the project number DEC-2011/03/N/ST8/06027.
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Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis ski, A. Nowicka, P. Rusanowska M. Dudek*, M. De˛bowski, M. Zielin University of Warmia and Mazury in Olsztyn, Faculty of Environmental Science, Department of Environment Engineering, Warszawska 117, 10-720 Olsztyn, Poland
article info
abstract
Article history:
Platymonas subcordiformis is a marine microalgae that under favorable environmental
Received 5 December 2017
conditions change metabolism pathways to hydrogen production in direct biophotolysis.
Received in revised form
Effective hydrogen production by Platymonas subcordiformis depends on application of
4 April 2018
efficient and economically viable biomass production technologies. In the study, the nat-
Accepted 5 April 2018
ural water from Vistula Lagoon was used for microalgae cultivation. No statistically sig-
Available online xxx
nificant differences were found regarding the biomass production in the natural water and synthetic medium. The influence of mixotrophic conditions on growth rate and biomass
Keywords:
production of Platymonas subcordiformis was also examined. The highest biogas production
Green algae
of 138.45 ± 3.39 mL with the rate of 1.15 ± 0.03 mL/h was noted by the biomass cultivated on
Mixotrophic growth
synthetic medium with glucose supplementation. Similarly high biogas production was
Photo-hydrogen production
observed by the biomass cultivated on natural water with glucose addition (1.11 ± 0.14 mL/
Fresh water
h). The use of the waters from Vistula Lagoon resulted in high yields of hydrogen production, which might reduce costs of biofuel production. © 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.
Introduction Microalgae biomass is a source of many valuable substances such as saccharides, proteins, fatty acids, vitamins, antibiotics and pigments (b-carotene, astaxanthin) [1,2]. Microalgae biomass might be also utilized for the energy purposes, since their liquid and gas metabolic products are used as a third generation fuels. Hydrogen energy is a clean and alternative energy that has been suggested as the energy carrier of the future. Solardriven microalgal hydrogen production is both, a promising and challenging biotechnology, which play an important role
in the global drive to reduce GHG emissions. The potential of hydrogen production in microalgae depends on strain specific capacity to synthesize different enzymes responsible for hydrogen metabolism and environmental conditions providing required energy [3e7]. The production of H2 in green algae is associated with the direct biophotolysis, which usually occurs when algal cultures are exposed to light after a period of dark with anaerobic adaptation. Microalgae species used for biohydrogen production are Chlamydomonas reinhardti, Chlorella sp. and Scenedesmus obtiguus etc.. Chlamydomonas reinhardti is the most widely used microorganism for hydrogen production studies in the laboratory due to its quality as a model organism [8]. However, it might not be the
* Corresponding author. E-mail address: [email protected] (M. Dudek). https://doi.org/10.1016/j.ijhydene.2018.04.039 0360-3199/© 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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most suitable organism for efficient production. Platymonas subcordiformis is a marine green alga demonstrated to produce hydrogen efficiently in various conditions [6,9,10]. One of the major barriers with regard to hydrogen economy is biomass production cost, which is related to composition of the cultivation medium. Thus, the utilization of the alternative media for biomass production is economically justified. The cultivation of microalgae Platymonas subcordiformis on the natural water from water reservoirs has not been widely applied in energy generation approach. In preliminary studies, the authors have observed that concentration of nutrients in natural waters is too low for intensive biomass production of Platymonas subcordiformis. Thus, in the study the authors supplemented the natural water with nitrogen and phosphorus compounds. Ran et al. [11] had similar observations in the study with Platymonas subcordiformis cultivation on natural water from Bohai Bay in China. The cell growth and microalgal biomass production might be enhanced by environmental factors (light, pH and temperature) and supplementation of macro and micronutrients in the medium [12]. Microalgae can survive in extreme environments as they can adapt their metabolism according to altering environmental conditions. Usually, microalgal biomass is produced through autotrophic cultivation in an open ponds or a photobioreactor using solar energy for fixing CO2. Alternatively, they are also cultivated in heterotrophic condition with organic compounds serving as energy and carbon sources [13e15]. Among the different modes of cultivation, mixotrophic operation offers several advantages. In mixotrophic conditions, both cell growth and biosynthesis of products are significantly influenced by the nutrients present in the medium and by the environmental factors. The syntrophic association between microalgae and its existing microenvironment facilitates higher biomass production. To stimulate mixotrophic growth glucose was chosen as a carbon source due to other carbon sources need more complicated inter-conversion metabolic process to provide energy for algal growth as well as bioenergy production microalgae growth [16,17]. In the study, the influence of mixotrophic conditions on the growth rate and biomass production by Platymonas subcordiformis was examined. Furthermore, the biomass of
Platymonas subcordiformis grown in the mixotrophic conditions was used for photosynthetic biohydrogen production.
Materials and methods Cultivation conditions Microalgae Platymonas subcordiformis was obtained from culture collection UTEX (University of Texas at Austin). Microalgae were cultivated in two different media (series of the study). In the series 1 (S1), medium was deionized water, whereas in the series 2 (S2) medium was water collected from Vistula Lagoon (54 210 3000 N 19 400 5900 E). The water from Vistula Lagoon was characterized by: chemical oxygen demand (COD) 33.57 ± 7.72 mg O2/L, ammonium nitrogen 0.08 ± 0.01 mg NH4e N/L, total nitrogen 1.39 ± 0.32 mg Ntot/L, orthophosphates 30.14 ± 0.07 mg PO3 4 /L, total phosphorus 0.05 ± 0.01 mg PO4 2 Ptot/L, sulphureous 141 ± 1.53 mg SO4 /L, chlorine (I) 723 ± 4.10 mg Cl/L, chlorine (II) 99 ± 0.20 mg Cl/L, iron (II) 0.11 ± 0.01 mg Fe2þ/L, iron (III) 0.12 ± 0.02 mg Fe3þ/L, pH of 7.84 ± 0.25. Both media were supplemented with appropriate nutrients addition [18]. In each series of the study, one medium was additionally supplemented with glucose (variants of the experiments). Glucose was added to the medium to investigate the biomass and hydrogen production in the mixotrophic conditions. Based on the preliminary studies and data presented in the literature, the dose of glucose was 10 g/L (Xie et al., 2001, Guan et al., 2004) Thus, the study tested hydrogen production by Platymonas subcordiformis biomass cultivated on medium based on deionized water (S1) and fresh water from Vistula Lagoon (S2), both series were performed in two variants: without addition of glucose (V1) and with addition of glucose (V2). The characteristic of the media used for Platymonas subcordiformis cultivation are presented in Table 1. Microalgae were cultivated in bioreactor BioFlo 115 New Brunswick with active volume of 2 L. Temperature of cultivation was 25 ± 1 C. The cultures were illuminated at 5klux by cool-white light (14 h light, 10 h dark). The cultures were continuously stirred and aerated with air provided by pump Mistral 200 with intensity 200 L/h. Microalgae was cultivated for 11 days.
Table 1 e Characteristic of the media used for Platymonas subcordiformis cultivation. Parameter
COD Ammonium nitrogen Total nitrogen Orthophosphates Total phosphorus Sulphureous Chlorine (I) Chlorine (II) Iron (II) Iron (III) pH Salinity Glucose
Unit
mgO2/L mgNH4eN/L mg Nog./L mg PO34/L mg PO34Pog./L mg SO24/L mg Cl/L mg Cl/L mg Fe2þ/L mg Fe3þ/L e ppt g/L
Series 1
Series 2
Variant 1
Variant 2
Variant 1
Variant 2
55.5 ± 2.01 0.04 ± 0.01 21.13 ± 0.63 1.85 ± 0.24 5.00 ± 0.20 525 ± 1.00 7510 ± 10 16128 ± 55.2 0.09 ± 0.01 0.09 ± 0.01 8.09 ± 0.12 30 ± 0.7 e
11033 ± 57.73 0.039 ± 0.01 20.79 ± 0.56 13.53 ± 0.25 5.04 ± 0.14 521 ± 6.11 7318 ± 2.89 16113 ± 15.26 0.103 ± 0.06 0.102 ± 0.07 8.08 ± 0.09 30 ± .09 10
52.0 ± 1.28 0.65 ± 0.01 21.99 ± 1.35 5.11 ± 4.33 5.37 ± 0.03 548 ± 1.00 12466 ± 105 19623 ± 165 0.117 ± 0.03 0.048 ± 0.02 8.09 ± 0.23 31 ± 1.1 e
11014 ± 131 0.07 ± 0.01 22.11 ± 0.22 13.5 ± 0.26 5.25 ± 0.14 534 ± 5.51 7492 ± 10.4 17220 ± 60 0.114 ± 0.03 0.051 ± 0.01 8.10 ± 0.07 30 ± 0.4 10
Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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Hydrogen production procedure After cultivation, the biomass of Platymonas subcordiformis was placed in a respirometric bottles (0.5 L) filled with the medium of composition indicating hydrogen production (Guan et al., 2004). The concentration of P. subcordiformis biomass in hydrogen production procedure was about 3 g VS/L. To induce H2 production, the microalgal cells were subjected to a twophase incubation. In the first phase, in order to hydrogenase induction, the algal cells were placed in dark anaerobic conditions for 30 h. Anaerobiosis was achieved by continuous flushing of pure nitrogen through the culture suspension. The second phase of photobiological hydrogen production was then initiated by placing the cultures under continuous light illumination at 5 klux for 5 days. The conditions for indicating hydrogen production by Platymonas subcordiformis were based on the results obtained during the preliminary studies and the data presented in the literature [11,18]. During the experiments amount of produced gases were continuously monitored. After the incubations the composition of biogas was measured.
Analytical procedures Online detection of chlorophyll concentration, algae classes and photosynthetic activity was done with BBE AlgaeOnLine Analyser (Moldaenke). Biomass concentration was measured with a gravimetric method. Water from Vistula Lagoon was collected from May to October, before cultivation it was filtered on a filter paper (pore diameter 0.45 mm) and then sterilized at 121 C for 15 min (Tuttnauer 2840 ELeD). Light intensity was measured with Lux Meter (Hanna Hi 97500). Salinity and pH was measured with Marine Control Digital salinity meter (Aqua Medic) and multiparameter (Hach Lange HQ 440D), respectively. Nitrogen Ntot, ammonium nitrogen N3 NHþ 4 , total phosphorus Ptot, orthophosphate P-PO4 , sulfur 2þ 3þ SO2 , chloride Cl , iron Fe , Fe , and COD concentrations were measured with cuvette testes (Hach Lange) and spectrophotometer UV/VIS (DR 5000). The quality of biogas was measured by using a gas chromatograph connected with
Results and discussion The study revealed differences in the biomass and hydrogen production by Platymonas subcordiformis cultivated on tested media.
Biomass production The biomass concentration in all tested media did not differ statistically and was from 3203.33 ± 35.12 mg VS/L to 3790.33 ± 281.73 mg VS/L in S1V1 and S2V2, respectively (Fig. 1A). Similar results were obtained by other authors. The concentration of Platymonas subcordiformis cultivated on synthetic medium was from 3200 mg VS/L [6] to 3680 mg VS/L [19]. Chinnasamy et al. [20] cultivated species of Platymonas on carpet industry wastewaters. Growth of microalgae after 10 days of cultivation was estimated in terms of chlorophyll-a content. The authors obtained chlorophyll-a concentration of 2800 mg/L in Platymonas suecica cultivation and 7300 mg/L in Platymonas chuii cultivation. In the present study, the concentration of chlorophyll-a statistically differ depending on the medium used for microalgae cultivation (Fig. 1B). The chlorophyll-a concentration was almost twice times higher in the biomass cultivated on media without glucose addition than in the biomass cultivated on media with glucose addition, in the both series of the study. Xie et al. [19] also observed lower concentration of chlorophyll-a in cultures of Platymonas subcordiformis, when the microalgae growth was stimulated by addition of glucose to the medium. Similarly, the concentration of chlorophyll-a in the phototrophic culture (without glucose) was about twice times higher than in the mixotrophic culture (with glucose). Changes in concentration of the pigments responsible for photosynthesis might be observed in
B
5000
Chlorophyll-a concentration [μg/L-1]
Biomass concentration [mgVS/L-1]
A
thermal conductivity detector (GC-TCD) (Agilent 7890A). After testing for homogeneity of variance with Levene's test, the significance of differences between variants was tested with Tukey's HSD test. Differences were considered significant at p < 0.05.
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Fig. 1 e Changes in concentration of A) biomass and B) chlorophyll-a in variants of experiment. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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Fig. 2 e The efficiency of removal of A) Ntot and B) Ptot from the media in the variants of experiment.
relation to changes in microorganism's metabolism when they are grown in the phototrophic and mixotrophic conditions. The utilization of glucose weaker the photosynthesis process and reduce the number of chloroplasts.
Nitrogen and phosphorus removal After 11 days of the biomass cultivation the concentration of Ntot and Ptot in the media was measured (Fig. 2). The concentration of nitrogen after Platymonas growth in the medium S1V1 was 0.61 ± 0.37 mg Ntot/L whereas in the medium S1V2 it was 5.11 ± 0.13 mg Ntot/L. Similar tendency was observed in the S2, the removal of nitrogen was about 95% and 75% in the V1 and V2, respectively (Fig. 2A). The concentration of phosphorus after Platymonas growth in the medium S1V1 was 0.04 ± 0.01 mg Ptot/L whereas in the medium S1V2 it was 0.66 ± 0.09 mg Ptot/L. Similar tendency was observed in the S2, the removal of phosphorus was 94% in the V1 and 86% in the V2 (Fig. 2B). It might be observed that in the variants without addition of glucose to the medium, removal of
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nutrients was higher than in the variants with addition of glucose to the medium. The utilization of nutrient for the biomass growth was also calculated (Fig. 3). Higher amount of nutrients for the biomass growth was used in the variants without glucose addition than with glucose addition to the medium, in both series of experiments (efficiency of glucose removal was 98.55% and 98.77% in the S1V2 and S2V2, respectively). Zhai et al. [21] compared biomass production of Spirulina platensis in the mixotrophic conditions (with glucose addition) and photosynthetic conditions. The authors observed enhanced the microalgal production, nutrients removal and recovery in the mixotrophic conditions. It was opposite to the results obtained in this study. However, the authors used media with much lower concentrations of nutrients and used much lower dose of glucose than in this study. The authors observed that concentration of 150 mg/L of glucose increased nutrients removal of about 6%, when compared to the nutrients removal from the medium without glucose addition. When the authors increased concentration of glucose to 600 mg/L,
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S1-V2
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Series-Variant Mean±stand. error Mean±stand. dev.
Fig. 3 e The utilization of A) Ntot and B) Ptot for biomass growth in the variants of experiment. Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039
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Fig. 4 e Biogas production after 11 days of biomass growth in the variants of experiment.
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1.11 ± 0.14 mL/h. Significantly lower biogas production was noted in variants without glucose addition to the medium (S1V1 and S2V1), the rate of biogas production of 0.91 ± 0.08 mL/h and 0.81 ± 0.05 mL/h was observed. Hydrogen content in the biogas was the highest in the S2V1 and it was about 71%. In the S1V2, hydrogen concentration was about 67%. Significantly lower concentration of hydrogen was noted in the S1V1 and S2V2, where it was 43% and 60%. Thus, hydrogen production per gram of VS was the highest in the S1V2, but similarly high result was observed in the S2V1 and S2V2 (Fig. 5). Significantly lower hydrogen production per gram VS was noted in the S1V1 (14.26 ± 1.20 mL/g VS). Microalgae Chlamydomonas reinhardtii also produced more H2 under the mixotrophic culture than under the phototrophic culture [23]. Organic carbon increased growth rate of C. reinhardtii leading to the accumulation of carbohydrates whose successive degradation can participate in H2 production. Probably the same mechanism occurs in microalgae P. subcordiformis. It is also worth noting on increased hydrogen production by microalgae cultivated on the natural water without glucose addition (Fig. 5). Enhanced biogas production was probably a result of the medium components deprivation. Natural water is a medium richer in compounds than the synthetic medium. The change of environmental conditions in case of microalgae cultivated on the natural water (S2V1) to medium for biogas production was more significant than that of the synthetic medium (S1V1). This dramatic change could result in the higher biogas production. Microalgae Chlamydomonas reinhardtii grown on a medium consisting of pretreated olive mill wastewater and TAP (tris-acetate-phosphate) (1:1 ratio) was found to exhibit a greater hydrogen production, compared to the control cells grown in TAP only [24].
S2V2
Mean±stand. dev
Fig. 5 e Hydrogen production per gram of VS from biomass cultivated in the variants of experiment.
no significant improvement was observed in nitrogen and phosphorus removal. The dose of glucose used in this study, for Platymonas subcordiformis cultivation was chosen on the basis of literature data [19]. Probably, this dose was too high for efficient nutrients removal by Platymonas subcordiformis, due to mixotrophic cultivation usually leads to enhanced biomass production and nutrients removal, which was not observed in this study. For Chlorella vulgaris the same observation was noted, a higher biomass and lipid accumulation was obtained under lower concentration of glucose and glycerol [22].
Biogas/hydrogen production Biogas production took place after 11 days of the biomass growth (Fig. 4). The highest biogas production of 138.45 ± 3.39 mL with the rate of 1.15 ± 0.03 mL/h was noted in the S1V2. Similarly high biogas production was observed in the S2V2, the rate was
Conclusions The cultivation of microalgae on the natural water did not significantly change the biomass production of Platymonas subcordiformis in comparison to cultivation on the synthetic medium. However, the biomass of Platymonas subcordiformis cultivated on the natural water produced higher hydrogen yield. Additionally, mixotrophic conditions in the bioreactor improved biogas production by Platymonas subcordiformis biomass.
Acknowledgements "The project was financed by the National (Polish) Science Center, the project number DEC-2011/03/N/ST8/06027.
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Please cite this article in press as: Dudek M, et al., Water from the Vistula Lagoon as a medium in mixotrophic growth and hydrogen production by Platymonas subcordiformis, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.04.039