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Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma
Accepted Manuscript Short Communication Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma Shidong Ban, Weitie Lin, Zhiwei Luo, Jianfei Luo PII: DOI: Reference:
S0960-8524(18)31723-1 https://doi.org/10.1016/j.biortech.2018.12.062 BITE 20819
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 November 2018 17 December 2018 18 December 2018
Please cite this article as: Ban, S., Lin, W., Luo, Z., Luo, J., Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.062
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma
Shidong Ban, Weitie Lin *, Zhiwei Luo, Jianfei Luo * Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, PR China * Corresponding author. [email protected] (W. Lin); [email protected] (J. Luo).
AB STRACT Reduction of chlorophyll size has great advantages on improving the photosynthesis efficiency as well as the photolysis algal H2 production. To promote the H2 production, Chlamydomonas reinhardtii was mutated by atmospheric and room temperature plasma (ARTP). After the selection, an algal mutant was observed to have 1.8-5.2 times (28.5-84.1 mL L-1) and 2.7-3.1 times (356.5-405.2 mL L-1) higher H2 production than wild-type during the algal subcultures grown in pure and co-cultures, respectively. In comparison with wild-type alga, the mutant grew as lighter green colonies on agar plate, with about 2 times larger cell diameter and 5.3-6.1 times lower chlorophyll content per unit cell volume. Results from the comparative transcriptomic analysis indicated that most of the genes relating to photosynthesis (photosystem I, II, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase) and LHC proteins were have higher expressions in mutant cells, suggesting the improvement of photosynthesis efficiency.
1
Keywords: Algal H2 production; ARTP; co-culture; chlorophyll size
1. Introduction The fossil fuel, as the primary energy resource of global economic growth, has resulted in serious damages to the earth’s environment and would be depleted in 50-100 years (Shih et al., 2018). Hydrogen’s versatilities including high energy density, clean, and renewable allow it to play an important role in future low-carbon energy supply (Hanley et al., 2018). However, as high as 96% of the hydrogen energy is generated from fossil fuels at the present time (Hanley et al., 2018), which makes it far from the demand for the reduction of greenhouse gas emission. The hydrogen production by green algae (Chlaymydomonas, Scenedesmus, Chlorella, etc) via the process of photosynthesis mediated water photolysis is carbon negative, less energy intensive and environmental friendly (Oey et al., 2016). During the algal hydrogen production, the highly sensitive of hydrogenase to O2 and low light conversion efficiency in photosynthesis are reported as the main bottlenecks (Melis, 2009; Oey et al., 2016). In our previous work, algal-bacterial cooperation was applied to remove the O2 toxicity out of the period of algal hydrogen production, as well as slow the reduction of chlorophyll content, enhance starch accumulation, and maintain algal protein content (Ban et al., 2018). Even though the hydrogen production was significantly improved, some methods are still needed to further enhance the algal hydrogen production to approach its ideal level. In green algae, the first step of photosynthesis is the capture of solar energy by the light-harvesting complex (LHC); the excitation energy transferred to photosystem II (PS II) by LHC II drives the water photolysis which converts H2O into H+, e- and O2 (Oey et al., 2016). However, up to 80% of the absorbed photons could be wasted because of large 2
chlorophyll antenna molecules (Melis, 2009). An efficient photosynthesis for algal culture could be reached after the reduction of chlorophyll antenna size; in this case, individual cells at the surface of algal culture would minimize light absorption, therefore permit greater transmittance of sunlight deeper into the culture, and thus enable more cells to contribute to photosynthetic productivity, as well as photolytic hydrogen production (Melis, 2009; Eroglu and Melis, 2016; Jeong et al., 2017). Atmospheric and room-temperature plasma (ARTP) is a newly developed whole-cell mutagenesis tool based on helium radio-frequency atmospheric-pressure glow discharge plasma which shows greater advantages than traditional UV radiation or chemical mutagens (Ottenheim et al., 2018). The ARTP could effectively cause the diversified damages to DNA and then increase the mutation rate, with no toxic and harmful matters generation (Cao et al., 2017; Zhang et al., 2014). It has been successfully used to improve the productivities of biomasses and metabolites for many bacterial, fungal, algal species (Fang et al., 2013; Cao et al., 2017; Ottenheim et al., 2018). In this study, we aimed to improve the algal hydrogen production by using ARTP mutagenesis to truncate the chlorophyll antenna of Chlamydomonas reinhardtii and analyzed potential mechanism for the improvement by comparing the wild-type with mutant on transcriptomic level.
2. Materials and Methods 2.1. Algal H2 production by pure culture and co-culture Green alga Chlamydomonas reinhardtii FACHB-265 was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (http://algae.ihb.ac.cn/), Chinese Academy of Sciences. Algal cultivation was performed in 250-mL glass flasks filled up with 3
100 mL of Tris-Acetate-Phosphate (TAP) medium and maintained at 30 °C in an illumination incubator (ZQLY-180, Zhichu Instruments, Shanghai, China) under a light density of 50 μmol m-2 s-1 continuous light conditions. Bacterial strain Pseudomonas sp. D that isolated from a contaminated algal culture was grown in liquid TAP medium, performing on a rotary shaker with speed of 150 rpm and temperature of 30 °C in the dark. H2 production by algal-bacterial co-culture was carried out in accordance with our previous work (Ban et al., 2018). In brief, algal and bacterial cells in the late exponential phase were respectively harvested by the centrifugation at 1,500 and 8,000 g and the cell pellets were resuspended by using sulfur-deprived TAP (TAP-S) medium; algal and bacterial cells with a number of 1.0 × 107 cells mL-1 were transferred into a 100 mL Hypo-Vial bottle containing with 50 mL TAP-S medium; co-cultivations were continuously incubated at 30 °C under 50 μmol m-2 s-1 light density. The pure culture of algal cells grown in the TAP-S medium was used as control.
2.2. Algal mutagenesis by ARTP and mutant selection A number of 5×106 cells mL-1 of algal cells grown in the mid-log phase was collected and used for the ARTP mutagenesis according to a previous work (Cao et al., 2017). Briefly, 15 μL of algal culture was coated on the surface of a stainless steel slide and placed in the chamber of ARTP breeding machine (ARTP-M, Tmaxtree Biotechnology, Wuxi, China); the parameters of ARTP treatment was set as follows: radio-frequency power100 W, helium gas flow rate 10 L min-1, distance 2 mm, and treating time varied from 20 to 60 s; after the ARTP treatment, the algal cells were suspended with 1 mL of TAP liquid medium and spread on TAP solid agar plates after a serial dilution; algal growth on the plate was performed at 30 °C 4
under a continuous light density of 50 μmol m-2 s-1. After seven days incubation, algal colonies on the agar plates with light green color (compared with wild type) were picked as mutants and spread on new agar plates. The algal mutants were used for H2 production via pure culture or co-cultivation with bacterium.
2.3. Analytical methods The numbers of algal and bacterial cells were measured by cell counting on a hemocytometer and on TAP agar plates with serial dilution, respectively. Morphology of algal cells was captured using a polarized light microscope (BX51-P, Olympus Corporation, Japan). Chlorophyll content in algal cells was extracted by 95% (v/v) ethanol and determined by using spectrophotometry as previously reported (Ban et al., 2018). H2 and O2 concentrations in the headspace of Hypo-Vial bottles were measured by using GC9790 Plus gas chromatography (Fuli instruments, China) equipped with a thermal conductivity detector (TCD), and calculated by the external standard method (Ban et al., 2018). A 5 Å molecular sieve column (2 m × 1/8 mm) was used. Argon was used as carrier and reference gas. Temperatures of the injector, TCD and column were kept at 70 °C, 100 °C and 60 °C, respectively; flow rate of the column was 20 mL min-1 and injection volume was 1.0 mL.
2.4. RNA-Seq and transcriptomic analysis Algal cells from the wild-type and mutant of pure culture and co-culture grown for third days were collected and used for the total RNA extraction, and each sample was repeated for three times.. Twelve RNA samples were obtained after the extraction by TRIzol Reagent (Invitrogen, USA) and the purification by Plant RNA Purification Reagent (Invitrogen, USA). 5
RNA quality and concentration were assessed by using Agilent 2100 Bioanalyzer (Agilent Technolgies, USA) and NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA libraries were prepared using TruseqTM RNA Sample Preparation Kit (Illumina, USA) and sequenced on an Illumina Hiseq 4000 platform by Majorbio Bio-pharm Technology (Shanghai, China. http://www.majorbio.com). Raw sequences were quality-filtered by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle), and mapped to the reference genome Chlamydomonas reinhardtii v5.5 (http://plants.ensembl.org/Chlamydomonas reinhardtii/Info/Index) by using TopHat2 (http://ccb.jhu.edu/software/tophat/index.shtml). DESeq2 was used for the differential expression analysis of RNA-Seq expression profiles. Absolute values of log2 (fold change) >= 1 and p-adjust < 0.05 were set as thresholds. The genes that expressed at significantly different were subjected to KEGG and GO enrichment analysis. Hierarchical clustering analysis was performed using R (ver i386 3.3.1) to identity photosynthesis and photosynthesis-antenna proteins. 2.5 Statistical analysis Each experiment was carried out in triplicate. Mean and standard deviation values were analyzed using one-way analysis of variance (ANOVA). Significance of difference was determined as P < 0.05.
3. Results and discussion 3.1. Comparative study of wild-type and ARTP mutant algae After the mutation by ARTP treatment, 131 algal colonies showing lighter green color than wild-type C. reinhardtii were picked and transferred onto the fresh agar plates. After 6
seven days incubation, more than half of the mutants switched back to the wild-type alga whose colony shows dark green color. Ten colonies were randomly selected from the unswitched mutants and used for the H2 production. Among these mutants, three mutants were observed to have higher H2 production than the wild-type alga when co-cultivated with bacterium Pseudomonas sp. D (data not shown). One mutant, named A4, accumulated about 296 mL L-1 H2 after eleven days co-cultivation (data not shown). The colonies of mutant A4 grown on solid agar TAP medium were smaller than wild-type alga and with lighter colors. Light microscopic observation indicated that the cell diameter of mutant A4 was about 15 μm (mean value of ten cells), which is two times larger than wild-type cells (~7 μm). Though the colony color of mutant A4 was lighter than wild-type, the total chlorophyll content in one cell of mutant A4 grown in each subculture was 1.1-1.3 times higher than wild-type cell; however, during each subculture, the chlorophyll content of mutant A4 that calculated on per unit cell volume was 5.3-6.1 times lower than wild-type cell (Fig 1c). The observed H2 production of mutant A4 in each subculture was higher than that of wild-type either grown in pure culture or in co-culture. During the mutant A4 grown in pure culture, the maximum H2 accumulation was observed to be ranging from 28.5 to 84.1 mL L-1, which was 1.8 to 5.2 times higher than that of wild-type alga; similar result was observed in the algal-bacterial co-cultivation, the maximum H2 accumulation ranged from 356.5 to 405.2 mL L-1, which was 2.7 to 3.1 times higher than wild-type alga (Fig 1d). Apparently, the algal H2 production was significantly improved by the ARTP mutant whose chlorophyll content was largely reduced in unit cell volume. It is indicated that the chlorophyll reduced mutant A4 cells probably had a better light transmittance than wild-type cells, which resulted in higher solar conversion efficiency and then more H2 production. In 7
order to enhance algal H2 production, several studies concerning the chlorophyll antenna reduction have been carried out. A truncated antenna mutant of C. reinhardtii that isolated from a DNA-insertional mutagenesis library was reported to accumulate more H2 than the parental strain (Kosourov et al., 2011). A C. reinhardtii mutant with LHC proteins (LHCBM1, 2 and 3) simultaneously knock-down by RNAi was also reported to have significant increase of H2 production (Oey et al., 2013). Though there exist only several research articles relating to the reduction of chlorophyll antenna size for improving algal H2 production, a number of reviews have reported the advantages of it through replacing leucine, asparagine and arginine of D1 protein, transformating a permanently active variant NAB1 of the LHC translation repressor or knocking down Nac2 gene (Melis, 2009; Esquivel et al., 2011; Oey et al., 2016). With the development and application of new approaches (such as CRISPER/Cas gene editing tool) to algae genetic engineering, the process of chlorophyll antenna reduction would be easy to achieve.
3.2. Comparison of gene expression between wild-type and ARTP mutant algae To study the potential mechanism of algal mutant contributing to the improvement of H2 production, comparative transcriptome analyses of wild-type and mutant A4 grown in pure culture or co-culture were carried out. RNA-Seq generated about 596 million clean reads corresponding to 89 Gbp sequence data, and an average of 85% of reads were mapped to the referred genome Chlamydomonas reinhardtii v5.5 (data not shown). 23 genes involving in photosynthesis (photosystem I, II, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase) and 10 genes participating in protein synthesis of LHC antenna (LHC1 of photosystem I and LHC2 of photosystem II) were found to be differentially expressed (Fig 2). 8
Hierarchical clustering of these genes indicated the wild-type or mutant A4 cells grown in pure culture and co-culture have relatively similar gene expression profiles but significantly different from each other, suggesting the presence of different strategies of mutant A4 in respond to environmental stress. As results shown in Figure 2, the heatmap based comparison indicated the genes relating to PS II had higher expressions when algal cells (both of wild-type and mutant A4) grown in pure cultures than they grown in co-cultures. The proteins coded by psbO, psbP, psbQ and psbR are extrinsic proteins for the formation of oxygen-evolving complex in green algae, and usually released under environmental stresses (Thornton et al., 2004; Allahverdiyeva et al., 2013). During the algal H2 production under sulfur deprivation, approximate 75% PS II activity is reduced because of the reduction of repair rate of PS II center protein D1 in the absence of sulfur (Oey et al., 2016; Eroglu and Melis, 2016). Algal-bacterial cooperation during the H2 production was reported to have the potential to slow the reduction of chlorophyll and protein contents and in some case maintained the PS II activity (Ban et al., 2018). This protection probably resulted in the lower gene expression when alga grown in co-culture than that in pure culture. The expression profile of genes involving in PS II could not tell the difference between wild-type and mutant algae. Except for these genes, most of the genes relating to photosynthesis and LHC proteins were observed to have higher expressions in mutant A4 than wild-type either grown in pure culture or in co-culture (Fig 2). PS I is a multi-proteins based supercomplex consisting of 14 core subunits (PsaA to PsaO) and 9 peripheral LHC1 (LHCA1 to LHCA9); the interaction between core subunits have been regarded to play important roles in forming and stabilizing the PSI-LHC1 supercomplex, as well as in electron transfer 9
(Yadavalli et al., 2011; Winkler et al., 2011). As in parts of the photosynthetic electron transport (PET) chain, the genes coding for cytochrome b6f complex small subunits (PetC and PetN), plastocyanin (PetE), ferredoxin (PetF), ferredoxin-NADP+ reductase (PetH) and cytochrome c6 (PetJ) were also observed with higher expressions in mutant A4 (Fig 2). Upregulation of these genes involving in PET may provide more electrons for hydrogenase and result in more H2 production. As we discussed above, the chlorophyll content per unit cell volume was largely reduced by ARTP mutation (Fig 1c). However, the genes (except LHCA1 and LHCB7) coding for LHC1 and LHC2 were observed to have higher expressions in mutant A4 than in wild-type algal cells (Fig 2). It seems that the aforementioned result was denied by this observation. In fact, the average chlorophyll content in one cell of mutant A4 was about 1.1-1.3 times higher than that in wild-type cell (Fig 1c); only after the ARTP mutation, the cell size of mutant A4 became much larger than the wild-type cell, which resulted in the low chlorophyll content per unit cell volume in mutant A4. Then, the gene expressions relating to LHC syntheses were consistent with the chlorophyll contents in algal cells.
4. Conclusions In this study, an algal mutant showing several times higher H2 production than wild-type was obtained after the ARTP mutation. The hydrogen production of an algal mutant was 1.8-5.2 times and 2.7-3.1 times higher than wild-type in pure and co-culture, respectively. In comparison with wild-type alga, the mutant grew as lighter green colonies on agar plate, with much larger cell diameter and lower chlorophyll content per unit cell volume. The reduction of chlorophyll size was probably responsible for the enhancement of H2 production via the 10
improvement of photosynthesis, which was also proved by the comparative transcriptomic analysis.
E-supplementary data for this work can be found in e-version of this paper online.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 41473072).
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Cao S., Zhou X., Jin W., Wang F., Tu R., Han S., et al. 2017. Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma (ARTP). Bioresou. Technol. 244, 1400–1406.
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Melis A., 2009. Solar energy conversion efficiencies in photosynthesis: Minimizing the 11
chlorophyll antennae to maximize efficiency. Plant Sci. 177, 272-280. 7.
Jeong J., Baek K., Kirst H., Melis A., Jin E., 2017. Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii. Biochim. Biophy. Acta 1858, 45-55.
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Eroglu E., Melis A., 2016. Microalgal hydrogen production research. Int. J. Hyd. Ener. 41, 12772-12798.
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Ottenheim C., Nawrath M., Wu JC., 2018. Microbial mutagenesis by atmospheric and room-temperature plasma (ARTP): the latest development. Bioresour. Bioprocess. 5, 12.
10. Fang M., Jin L., Zhang C., Tan Y., Jiang P., Ge N., et al., 2013. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes. PLoS ONE 8, e77046. 11. Kosourov S.N., Ghirardi M.L., Seibert M., 2011. A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain. Int. J. Hyd. Ener. 36, 2044-2048. 12. Oey M., Ross IL., Stephens E., Steinbeck J., Wolf J., Radzun KA., et al. 2013. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii. PLoS ONE 8, e61375. 13. Esquivel MG., Amaro HM., Pinto TS., Fevereiro PS., Malcata FX., 2011. Efficient H2 production via Chlamydomonas reinhardtii. Trend. Biotechnol. 29, 595-600. 14. Thornton LE., Ohkawa H., Roose JL., Kashino Y., Keren N., Pakrasi HB., 2004. Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem II activity in the cyanobacterium Synechocystis 6803. Plant Cell 16, 2164-2175. 12
15. Allahverdiyeva Y., Suoras M., Rossi F., Pavesi A., Kater MM., Antonacci A., et al., 2013. Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms. Plant J. 75, 671-684. 16. Yadavalli V., Malleda C., Subramanyam R., 2011. Protein–protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii. Mol. BioSyst. 7, 3143-3151. 17. Winkler M., Kawelke S., Happe T., 2011. Light driven hydrogen production in protein based semi-artificial systems. Bioresou. Technol. 102, 8493-9500. 18. Zhang X., Zhang X., Li H., Wang L., Zhang C., Xing X., et al. 2014. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl Microbiol Biot, 98, 5387–5396. 19. Torzillo, G., Scoma, A., Faraloni, C., Ena, A., Johanningmeier, U. (2009). Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant. Int. J. Hydrogen Energy, 34(10): 4529-4536..
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Figure legend
Fig. 1. Comparative study of wild-type C. reinhardtii and ARTP mutant A4. The chlorophyll content per cell and per cell volume was extracted by 95% (v/v) ethanol and determined by using spectrophotometry (a) and H2 production of algae under sulfur deprivation in pure culture and co-culture (b) over the course of generations used TAP solid plate.
Fig. 2. Heatmap analysis of photosynthesis related genes expression of wild-type and ARTP mutated C. reinhardtii grown in pure or co-cultures. Data of gene expression were from transcriptomes by RNA-Seq (supplemental RNA-Seq data).
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Fig. 1. 6.0
a Chlorophyll content per cell (pg cell-1) Chlorophyll content per cell volume (fg m-3)
Chlorophyll content
5.5
a
5.0
a ab
2.0
bc
b
b
b
b
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b
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c
c
c
c
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13th
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3th
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9th
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400 350 300 250 200
WT pure WT co-culture 1st pure 1st co-culture 3th pure 3th co-culture 5th pure 5th co-culture 7th pure 7th co-culture 9th pure 9th co-culture 11th pure 11th co-culture 13th pure 13th co-culture
b
150 100 50 0 0
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8
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Fig. 2.
16
H I GH LI GHTS
Chlorophyll size was reduced by atmospheric and room temperature plasma.
Algal mutant grew as lighter green colonies on agar plate.
Algal mutant had much lower chlorophyll content per unit cell volume.
Algal mutants produced 356.5-405.2 mL L-1H2 when grown in co-cultures.
Genes relating to photosynthesis had higher expressions in mutant cells.
17
S0960-8524(18)31723-1 https://doi.org/10.1016/j.biortech.2018.12.062 BITE 20819
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
8 November 2018 17 December 2018 18 December 2018
Please cite this article as: Ban, S., Lin, W., Luo, Z., Luo, J., Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.062
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Improving hydrogen production of Chlamydomonas reinhardtii by reducing chlorophyll content via atmospheric and room temperature plasma
Shidong Ban, Weitie Lin *, Zhiwei Luo, Jianfei Luo * Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, PR China * Corresponding author. [email protected] (W. Lin); [email protected] (J. Luo).
AB STRACT Reduction of chlorophyll size has great advantages on improving the photosynthesis efficiency as well as the photolysis algal H2 production. To promote the H2 production, Chlamydomonas reinhardtii was mutated by atmospheric and room temperature plasma (ARTP). After the selection, an algal mutant was observed to have 1.8-5.2 times (28.5-84.1 mL L-1) and 2.7-3.1 times (356.5-405.2 mL L-1) higher H2 production than wild-type during the algal subcultures grown in pure and co-cultures, respectively. In comparison with wild-type alga, the mutant grew as lighter green colonies on agar plate, with about 2 times larger cell diameter and 5.3-6.1 times lower chlorophyll content per unit cell volume. Results from the comparative transcriptomic analysis indicated that most of the genes relating to photosynthesis (photosystem I, II, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase) and LHC proteins were have higher expressions in mutant cells, suggesting the improvement of photosynthesis efficiency.
1
Keywords: Algal H2 production; ARTP; co-culture; chlorophyll size
1. Introduction The fossil fuel, as the primary energy resource of global economic growth, has resulted in serious damages to the earth’s environment and would be depleted in 50-100 years (Shih et al., 2018). Hydrogen’s versatilities including high energy density, clean, and renewable allow it to play an important role in future low-carbon energy supply (Hanley et al., 2018). However, as high as 96% of the hydrogen energy is generated from fossil fuels at the present time (Hanley et al., 2018), which makes it far from the demand for the reduction of greenhouse gas emission. The hydrogen production by green algae (Chlaymydomonas, Scenedesmus, Chlorella, etc) via the process of photosynthesis mediated water photolysis is carbon negative, less energy intensive and environmental friendly (Oey et al., 2016). During the algal hydrogen production, the highly sensitive of hydrogenase to O2 and low light conversion efficiency in photosynthesis are reported as the main bottlenecks (Melis, 2009; Oey et al., 2016). In our previous work, algal-bacterial cooperation was applied to remove the O2 toxicity out of the period of algal hydrogen production, as well as slow the reduction of chlorophyll content, enhance starch accumulation, and maintain algal protein content (Ban et al., 2018). Even though the hydrogen production was significantly improved, some methods are still needed to further enhance the algal hydrogen production to approach its ideal level. In green algae, the first step of photosynthesis is the capture of solar energy by the light-harvesting complex (LHC); the excitation energy transferred to photosystem II (PS II) by LHC II drives the water photolysis which converts H2O into H+, e- and O2 (Oey et al., 2016). However, up to 80% of the absorbed photons could be wasted because of large 2
chlorophyll antenna molecules (Melis, 2009). An efficient photosynthesis for algal culture could be reached after the reduction of chlorophyll antenna size; in this case, individual cells at the surface of algal culture would minimize light absorption, therefore permit greater transmittance of sunlight deeper into the culture, and thus enable more cells to contribute to photosynthetic productivity, as well as photolytic hydrogen production (Melis, 2009; Eroglu and Melis, 2016; Jeong et al., 2017). Atmospheric and room-temperature plasma (ARTP) is a newly developed whole-cell mutagenesis tool based on helium radio-frequency atmospheric-pressure glow discharge plasma which shows greater advantages than traditional UV radiation or chemical mutagens (Ottenheim et al., 2018). The ARTP could effectively cause the diversified damages to DNA and then increase the mutation rate, with no toxic and harmful matters generation (Cao et al., 2017; Zhang et al., 2014). It has been successfully used to improve the productivities of biomasses and metabolites for many bacterial, fungal, algal species (Fang et al., 2013; Cao et al., 2017; Ottenheim et al., 2018). In this study, we aimed to improve the algal hydrogen production by using ARTP mutagenesis to truncate the chlorophyll antenna of Chlamydomonas reinhardtii and analyzed potential mechanism for the improvement by comparing the wild-type with mutant on transcriptomic level.
2. Materials and Methods 2.1. Algal H2 production by pure culture and co-culture Green alga Chlamydomonas reinhardtii FACHB-265 was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (http://algae.ihb.ac.cn/), Chinese Academy of Sciences. Algal cultivation was performed in 250-mL glass flasks filled up with 3
100 mL of Tris-Acetate-Phosphate (TAP) medium and maintained at 30 °C in an illumination incubator (ZQLY-180, Zhichu Instruments, Shanghai, China) under a light density of 50 μmol m-2 s-1 continuous light conditions. Bacterial strain Pseudomonas sp. D that isolated from a contaminated algal culture was grown in liquid TAP medium, performing on a rotary shaker with speed of 150 rpm and temperature of 30 °C in the dark. H2 production by algal-bacterial co-culture was carried out in accordance with our previous work (Ban et al., 2018). In brief, algal and bacterial cells in the late exponential phase were respectively harvested by the centrifugation at 1,500 and 8,000 g and the cell pellets were resuspended by using sulfur-deprived TAP (TAP-S) medium; algal and bacterial cells with a number of 1.0 × 107 cells mL-1 were transferred into a 100 mL Hypo-Vial bottle containing with 50 mL TAP-S medium; co-cultivations were continuously incubated at 30 °C under 50 μmol m-2 s-1 light density. The pure culture of algal cells grown in the TAP-S medium was used as control.
2.2. Algal mutagenesis by ARTP and mutant selection A number of 5×106 cells mL-1 of algal cells grown in the mid-log phase was collected and used for the ARTP mutagenesis according to a previous work (Cao et al., 2017). Briefly, 15 μL of algal culture was coated on the surface of a stainless steel slide and placed in the chamber of ARTP breeding machine (ARTP-M, Tmaxtree Biotechnology, Wuxi, China); the parameters of ARTP treatment was set as follows: radio-frequency power100 W, helium gas flow rate 10 L min-1, distance 2 mm, and treating time varied from 20 to 60 s; after the ARTP treatment, the algal cells were suspended with 1 mL of TAP liquid medium and spread on TAP solid agar plates after a serial dilution; algal growth on the plate was performed at 30 °C 4
under a continuous light density of 50 μmol m-2 s-1. After seven days incubation, algal colonies on the agar plates with light green color (compared with wild type) were picked as mutants and spread on new agar plates. The algal mutants were used for H2 production via pure culture or co-cultivation with bacterium.
2.3. Analytical methods The numbers of algal and bacterial cells were measured by cell counting on a hemocytometer and on TAP agar plates with serial dilution, respectively. Morphology of algal cells was captured using a polarized light microscope (BX51-P, Olympus Corporation, Japan). Chlorophyll content in algal cells was extracted by 95% (v/v) ethanol and determined by using spectrophotometry as previously reported (Ban et al., 2018). H2 and O2 concentrations in the headspace of Hypo-Vial bottles were measured by using GC9790 Plus gas chromatography (Fuli instruments, China) equipped with a thermal conductivity detector (TCD), and calculated by the external standard method (Ban et al., 2018). A 5 Å molecular sieve column (2 m × 1/8 mm) was used. Argon was used as carrier and reference gas. Temperatures of the injector, TCD and column were kept at 70 °C, 100 °C and 60 °C, respectively; flow rate of the column was 20 mL min-1 and injection volume was 1.0 mL.
2.4. RNA-Seq and transcriptomic analysis Algal cells from the wild-type and mutant of pure culture and co-culture grown for third days were collected and used for the total RNA extraction, and each sample was repeated for three times.. Twelve RNA samples were obtained after the extraction by TRIzol Reagent (Invitrogen, USA) and the purification by Plant RNA Purification Reagent (Invitrogen, USA). 5
RNA quality and concentration were assessed by using Agilent 2100 Bioanalyzer (Agilent Technolgies, USA) and NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA libraries were prepared using TruseqTM RNA Sample Preparation Kit (Illumina, USA) and sequenced on an Illumina Hiseq 4000 platform by Majorbio Bio-pharm Technology (Shanghai, China. http://www.majorbio.com). Raw sequences were quality-filtered by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle), and mapped to the reference genome Chlamydomonas reinhardtii v5.5 (http://plants.ensembl.org/Chlamydomonas reinhardtii/Info/Index) by using TopHat2 (http://ccb.jhu.edu/software/tophat/index.shtml). DESeq2 was used for the differential expression analysis of RNA-Seq expression profiles. Absolute values of log2 (fold change) >= 1 and p-adjust < 0.05 were set as thresholds. The genes that expressed at significantly different were subjected to KEGG and GO enrichment analysis. Hierarchical clustering analysis was performed using R (ver i386 3.3.1) to identity photosynthesis and photosynthesis-antenna proteins. 2.5 Statistical analysis Each experiment was carried out in triplicate. Mean and standard deviation values were analyzed using one-way analysis of variance (ANOVA). Significance of difference was determined as P < 0.05.
3. Results and discussion 3.1. Comparative study of wild-type and ARTP mutant algae After the mutation by ARTP treatment, 131 algal colonies showing lighter green color than wild-type C. reinhardtii were picked and transferred onto the fresh agar plates. After 6
seven days incubation, more than half of the mutants switched back to the wild-type alga whose colony shows dark green color. Ten colonies were randomly selected from the unswitched mutants and used for the H2 production. Among these mutants, three mutants were observed to have higher H2 production than the wild-type alga when co-cultivated with bacterium Pseudomonas sp. D (data not shown). One mutant, named A4, accumulated about 296 mL L-1 H2 after eleven days co-cultivation (data not shown). The colonies of mutant A4 grown on solid agar TAP medium were smaller than wild-type alga and with lighter colors. Light microscopic observation indicated that the cell diameter of mutant A4 was about 15 μm (mean value of ten cells), which is two times larger than wild-type cells (~7 μm). Though the colony color of mutant A4 was lighter than wild-type, the total chlorophyll content in one cell of mutant A4 grown in each subculture was 1.1-1.3 times higher than wild-type cell; however, during each subculture, the chlorophyll content of mutant A4 that calculated on per unit cell volume was 5.3-6.1 times lower than wild-type cell (Fig 1c). The observed H2 production of mutant A4 in each subculture was higher than that of wild-type either grown in pure culture or in co-culture. During the mutant A4 grown in pure culture, the maximum H2 accumulation was observed to be ranging from 28.5 to 84.1 mL L-1, which was 1.8 to 5.2 times higher than that of wild-type alga; similar result was observed in the algal-bacterial co-cultivation, the maximum H2 accumulation ranged from 356.5 to 405.2 mL L-1, which was 2.7 to 3.1 times higher than wild-type alga (Fig 1d). Apparently, the algal H2 production was significantly improved by the ARTP mutant whose chlorophyll content was largely reduced in unit cell volume. It is indicated that the chlorophyll reduced mutant A4 cells probably had a better light transmittance than wild-type cells, which resulted in higher solar conversion efficiency and then more H2 production. In 7
order to enhance algal H2 production, several studies concerning the chlorophyll antenna reduction have been carried out. A truncated antenna mutant of C. reinhardtii that isolated from a DNA-insertional mutagenesis library was reported to accumulate more H2 than the parental strain (Kosourov et al., 2011). A C. reinhardtii mutant with LHC proteins (LHCBM1, 2 and 3) simultaneously knock-down by RNAi was also reported to have significant increase of H2 production (Oey et al., 2013). Though there exist only several research articles relating to the reduction of chlorophyll antenna size for improving algal H2 production, a number of reviews have reported the advantages of it through replacing leucine, asparagine and arginine of D1 protein, transformating a permanently active variant NAB1 of the LHC translation repressor or knocking down Nac2 gene (Melis, 2009; Esquivel et al., 2011; Oey et al., 2016). With the development and application of new approaches (such as CRISPER/Cas gene editing tool) to algae genetic engineering, the process of chlorophyll antenna reduction would be easy to achieve.
3.2. Comparison of gene expression between wild-type and ARTP mutant algae To study the potential mechanism of algal mutant contributing to the improvement of H2 production, comparative transcriptome analyses of wild-type and mutant A4 grown in pure culture or co-culture were carried out. RNA-Seq generated about 596 million clean reads corresponding to 89 Gbp sequence data, and an average of 85% of reads were mapped to the referred genome Chlamydomonas reinhardtii v5.5 (data not shown). 23 genes involving in photosynthesis (photosystem I, II, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase) and 10 genes participating in protein synthesis of LHC antenna (LHC1 of photosystem I and LHC2 of photosystem II) were found to be differentially expressed (Fig 2). 8
Hierarchical clustering of these genes indicated the wild-type or mutant A4 cells grown in pure culture and co-culture have relatively similar gene expression profiles but significantly different from each other, suggesting the presence of different strategies of mutant A4 in respond to environmental stress. As results shown in Figure 2, the heatmap based comparison indicated the genes relating to PS II had higher expressions when algal cells (both of wild-type and mutant A4) grown in pure cultures than they grown in co-cultures. The proteins coded by psbO, psbP, psbQ and psbR are extrinsic proteins for the formation of oxygen-evolving complex in green algae, and usually released under environmental stresses (Thornton et al., 2004; Allahverdiyeva et al., 2013). During the algal H2 production under sulfur deprivation, approximate 75% PS II activity is reduced because of the reduction of repair rate of PS II center protein D1 in the absence of sulfur (Oey et al., 2016; Eroglu and Melis, 2016). Algal-bacterial cooperation during the H2 production was reported to have the potential to slow the reduction of chlorophyll and protein contents and in some case maintained the PS II activity (Ban et al., 2018). This protection probably resulted in the lower gene expression when alga grown in co-culture than that in pure culture. The expression profile of genes involving in PS II could not tell the difference between wild-type and mutant algae. Except for these genes, most of the genes relating to photosynthesis and LHC proteins were observed to have higher expressions in mutant A4 than wild-type either grown in pure culture or in co-culture (Fig 2). PS I is a multi-proteins based supercomplex consisting of 14 core subunits (PsaA to PsaO) and 9 peripheral LHC1 (LHCA1 to LHCA9); the interaction between core subunits have been regarded to play important roles in forming and stabilizing the PSI-LHC1 supercomplex, as well as in electron transfer 9
(Yadavalli et al., 2011; Winkler et al., 2011). As in parts of the photosynthetic electron transport (PET) chain, the genes coding for cytochrome b6f complex small subunits (PetC and PetN), plastocyanin (PetE), ferredoxin (PetF), ferredoxin-NADP+ reductase (PetH) and cytochrome c6 (PetJ) were also observed with higher expressions in mutant A4 (Fig 2). Upregulation of these genes involving in PET may provide more electrons for hydrogenase and result in more H2 production. As we discussed above, the chlorophyll content per unit cell volume was largely reduced by ARTP mutation (Fig 1c). However, the genes (except LHCA1 and LHCB7) coding for LHC1 and LHC2 were observed to have higher expressions in mutant A4 than in wild-type algal cells (Fig 2). It seems that the aforementioned result was denied by this observation. In fact, the average chlorophyll content in one cell of mutant A4 was about 1.1-1.3 times higher than that in wild-type cell (Fig 1c); only after the ARTP mutation, the cell size of mutant A4 became much larger than the wild-type cell, which resulted in the low chlorophyll content per unit cell volume in mutant A4. Then, the gene expressions relating to LHC syntheses were consistent with the chlorophyll contents in algal cells.
4. Conclusions In this study, an algal mutant showing several times higher H2 production than wild-type was obtained after the ARTP mutation. The hydrogen production of an algal mutant was 1.8-5.2 times and 2.7-3.1 times higher than wild-type in pure and co-culture, respectively. In comparison with wild-type alga, the mutant grew as lighter green colonies on agar plate, with much larger cell diameter and lower chlorophyll content per unit cell volume. The reduction of chlorophyll size was probably responsible for the enhancement of H2 production via the 10
improvement of photosynthesis, which was also proved by the comparative transcriptomic analysis.
E-supplementary data for this work can be found in e-version of this paper online.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 41473072).
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15. Allahverdiyeva Y., Suoras M., Rossi F., Pavesi A., Kater MM., Antonacci A., et al., 2013. Arabidopsis plants lacking PsbQ and PsbR subunits of the oxygen-evolving complex show altered PSII super-complex organization and short-term adaptive mechanisms. Plant J. 75, 671-684. 16. Yadavalli V., Malleda C., Subramanyam R., 2011. Protein–protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii. Mol. BioSyst. 7, 3143-3151. 17. Winkler M., Kawelke S., Happe T., 2011. Light driven hydrogen production in protein based semi-artificial systems. Bioresou. Technol. 102, 8493-9500. 18. Zhang X., Zhang X., Li H., Wang L., Zhang C., Xing X., et al. 2014. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl Microbiol Biot, 98, 5387–5396. 19. Torzillo, G., Scoma, A., Faraloni, C., Ena, A., Johanningmeier, U. (2009). Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant. Int. J. Hydrogen Energy, 34(10): 4529-4536..
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Figure legend
Fig. 1. Comparative study of wild-type C. reinhardtii and ARTP mutant A4. The chlorophyll content per cell and per cell volume was extracted by 95% (v/v) ethanol and determined by using spectrophotometry (a) and H2 production of algae under sulfur deprivation in pure culture and co-culture (b) over the course of generations used TAP solid plate.
Fig. 2. Heatmap analysis of photosynthesis related genes expression of wild-type and ARTP mutated C. reinhardtii grown in pure or co-cultures. Data of gene expression were from transcriptomes by RNA-Seq (supplemental RNA-Seq data).
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Fig. 1. 6.0
a Chlorophyll content per cell (pg cell-1) Chlorophyll content per cell volume (fg m-3)
Chlorophyll content
5.5
a
5.0
a ab
2.0
bc
b
b
b
b
d 1.5
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c
c
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0.5 0.0 WT
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450
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400 350 300 250 200
WT pure WT co-culture 1st pure 1st co-culture 3th pure 3th co-culture 5th pure 5th co-culture 7th pure 7th co-culture 9th pure 9th co-culture 11th pure 11th co-culture 13th pure 13th co-culture
b
150 100 50 0 0
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Fig. 2.
16
H I GH LI GHTS
Chlorophyll size was reduced by atmospheric and room temperature plasma.
Algal mutant grew as lighter green colonies on agar plate.
Algal mutant had much lower chlorophyll content per unit cell volume.
Algal mutants produced 356.5-405.2 mL L-1H2 when grown in co-cultures.
Genes relating to photosynthesis had higher expressions in mutant cells.
17