<p>The Netherlands has been facing a pressing <strong>nitrogen crisis</strong> for several years. This crisis is largely attributed to the <strong>agriculture sector</strong>, with over 80% of ammonia (a nitrogenous compound) emissions coming from manure <ahref="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm"style="color: #185A4F;">[NitrogenWUR]</a> and chemical fertilizers <ahref="https://www.government.nl/topics/nitrogen-crisis"style="color: #185A4F;">[TheGovernment.nl]</a>.</p>
<p>The over-use of fertilizers has a detrimental effect on the environment through the deposition of excess nitrogen oxides and ammonia in the ground, excessively enriching the environment with nutrients promoting uncontrolled plant and algal growth, or eutrophication, a form of nutrient imbalance <ahref="https://oceanservice.noaa.gov/facts/eutrophication.html"style="color: #185A4F;">[US Department of Commerce What is Eutrophication]</a> that negatively impacts the local biodiversity. This highlights the need of the hour: <strong>tackle the nitrogen crisis without negatively affecting food production</strong>, which still depends highly on fertilizers.</p>
<p>The Netherlands has been facing a pressing <strong>nitrogen crisis</strong> for several years. This crisis is largely attributed to the <strong>agriculture sector</strong>, with over 80% of ammonia (a nitrogenous compound) emissions coming from manure <ahref="#cite1"style="color: #185A4F;">[1]</a> and chemical fertilizers <ahref="#cite2"style="color: #185A4F;">[2]</a>.</p>
<p>The over-use of fertilizers has a detrimental effect on the environment through the deposition of excess nitrogen oxides and ammonia in the ground, excessively enriching the environment with nutrients promoting uncontrolled plant and algal growth, or eutrophication, a form of nutrient imbalance <ahref="#cite3"style="color: #185A4F;">[3]</a> that negatively impacts the local biodiversity. This highlights the need of the hour: <strong>tackle the nitrogen crisis without negatively affecting food production</strong>, which still depends highly on fertilizers.</p>
<p>The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed to reduce nitrogen deposition, particularly in agriculture due to fertilizer use and ammonia emissions. However, in 2019, the Council of State deemed the programme <strong>insufficient</strong>, highlighting that nitrogen emissions were not just affecting rural ecosystems but also impacting urban development. As a result, new residential construction projects were halted until nitrogen emissions could be adequately compensated for, exacerbating the already critical housing shortage in the Netherlands <ahref="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm"style="color: #185A4F;">[NitrogenWUR]</a>. This demonstrates how agricultural nitrogen management has far-reaching effects beyond the environment, directly influencing urban issues like the housing crisis, thereby emphasizing the urgency of addressing both challenges in tandem.</p>
<p>The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed to reduce nitrogen deposition, particularly in agriculture due to fertilizer use and ammonia emissions. However, in 2019, the Council of State deemed the programme <strong>insufficient</strong>, highlighting that nitrogen emissions were not just affecting rural ecosystems but also impacting urban development. As a result, new residential construction projects were halted until nitrogen emissions could be adequately compensated for, exacerbating the already critical housing shortage in the Netherlands <ahref="#cite1"style="color: #185A4F;">[1]</a>. This demonstrates how agricultural nitrogen management has far-reaching effects beyond the environment, directly influencing urban issues like the housing crisis, thereby emphasizing the urgency of addressing both challenges in tandem.</p>
<p>To combat global hunger and feed a growing population, an increase in global food production is crucial. This can be at least partially addressed through increasing crop yields, for which fertilizers are needed. Production of fertilizer is possible due to the Haber-Bosch process, where elemental nitrogen is converted into ammonia. <strong>Over-fertilization</strong> and its direct and indirect impact on the environment make agriculture the second leading contributor to short-term <strong>increases in global surface temperature</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #185A4F;">[Elhai2023 Engineering Neoplasts]</a>.</p>
<p>To combat global hunger and feed a growing population, an increase in global food production is crucial. This can be at least partially addressed through increasing crop yields, for which fertilizers are needed. Production of fertilizer is possible due to the Haber-Bosch process, where elemental nitrogen is converted into ammonia. <strong>Over-fertilization</strong> and its direct and indirect impact on the environment make agriculture the second leading contributor to short-term <strong>increases in global surface temperature</strong><ahref="#cite4"style="color: #185A4F;">[4]</a>.</p>
<p>We expected, as the results of the experiment, that the light emitted by the primitive lux operon was a very faint blue light (similar to a schematic diagram of PV), which is very technological, but far from being called "LAMPS". Our project first set out to make our enzymes emit brighter and more colorful light for practical applications. We use both <b>protein engineering</b> and <b>metabolic engineering</b> to achieve the design goal of <b>increasing light intensity</b>.</p>
<p>In 2022, Dutch agriculture lost 74% (312,000 tons) of the nitrogen it spread as manure and synthetic fertilizer to the air and soil. Synthetic fertilizer production alone is also the cause of nearly <strong>2% of global CO<sub>2</sub> emissions</strong><ahref="https://www.cbs.nl/en-gb/news/2023/increase-in-agriculture-s-nitrogen-emissions"style="color: #185A4F;">[ToenameCBS]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion of nitrates to N<sub>2</sub>O leads to a global greenhouse effect equivalent to 10% of that caused by the increase in atmospheric CO<sub>2</sub><ahref="https://www.ipcc.ch/report/ar4/wg1/"style="color: #185A4F;">[AR4IPCC]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #185A4F;">[Elhai2023 Engineering Neoplasts]</a>. Rising prices for fertilizer have been one of the problems leading to farmers' protests in Europe, and efforts to reduce nitrogen emissions in the Netherlands have been met with its own wave of protests <ahref="https://dutchnews.nl/news/2023/farmers-protests-in-the-netherlands/"style="color: #185A4F;">[ProtestingDutchNews.nl]</a>.</p>
<p>In 2022, Dutch agriculture lost 74% (312,000 tons) of the nitrogen it spread as manure and synthetic fertilizer to the air and soil. Synthetic fertilizer production alone is also the cause of nearly <strong>2% of global CO<sub>2</sub> emissions</strong><ahref="#cite5"style="color: #185A4F;">[5]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion of nitrates to N<sub>2</sub>O leads to a global greenhouse effect equivalent to 10% of that caused by the increase in atmospheric CO<sub>2</sub><ahref="#cite6"style="color: #185A4F;">[6]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong><ahref="#cite4"style="color: #185A4F;">[4]</a>. Rising prices for fertilizer have been one of the problems leading to farmers' protests in Europe, and efforts to reduce nitrogen emissions in the Netherlands have been met with its own wave of protests <ahref="#cite7"style="color: #185A4F;">[7]</a>.</p>
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="https://www.government.nl/topics/agriculture-and-food/agricultural-exports"style="color: #185A4F;">[Netherlands2024 Dutch2023]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <ahref="https://2021.igem.org/Team:Wageningen"style="color: #185A4F;">[TeamHomepage]</a> and Stony-Brook 2023 <ahref="https://2023.igem.org/Team:Stony-Brook"style="color: #185A4F;">[Team2023]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="https://www.nature.com/articles/nature2024"style="color: #185A4F;">[Coale2024 Nitrogen-fixing Alga]</a>.</p>
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="#cite8"style="color: #185A4F;">[8]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <ahref="#cite9"style="color: #185A4F;">[9]</a> and Stony-Brook 2023 <ahref="#cite10"style="color: #185A4F;">[10]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
<p>The Nature publication by Coale <em>et al.</em> examines UCYN-A, which evolved from a cyanobacterial species capable of converting N<sub>2</sub> into organic nitrogenous compounds, and its relationship with the marine alga <em>Braadurosphaera bigelowii</em>. It has already been established that UCYN-A and <em>B. bigelowii</em> have a symbiotic relationship, where <em>B. bigelowii</em> functions as a so-called host, and has taken up the UCYN-A bacteria into its cell in a process known as endosymbiosis. <strong>The symbiont, UCYN-A, fixes nitrogen for the host</strong> whereas <em>B. bigelowii</em> supplies organic carbon and a conducive living environment. This paper proved that UCYN-A is not simply a symbiont, but has instead evolved into an organelle for the eukaryotic alga for nitrogen fixation, and is now called the <strong>"nitroplast"</strong><ahref="https://www.nature.com/articles/nature2024"style="color: #185A4F;">[Coale2024 Nitrogen-fixing Alga]</a>.</p>
</div> Insert bigelowii pic with magnified view of UCYN-A -->
<p>The Nature publication by Coale <em>et al.</em> examines UCYN-A, which evolved from a cyanobacterial species capable of converting N<sub>2</sub> into organic nitrogenous compounds, and its relationship with the marine alga <em>Braadurosphaera bigelowii</em>. It has already been established that UCYN-A and <em>B. bigelowii</em> have a symbiotic relationship, where <em>B. bigelowii</em> functions as a so-called host, and has taken up the UCYN-A bacteria into its cell in a process known as endosymbiosis. <strong>The symbiont, UCYN-A, fixes nitrogen for the host</strong> whereas <em>B. bigelowii</em> supplies organic carbon and a conducive living environment. This paper proved that UCYN-A is not simply a symbiont, but has instead evolved into an organelle for the eukaryotic alga for nitrogen fixation, and is now called the <strong>"nitroplast"</strong><ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
<!-- Insert bigelowii pic with magnified view of UCYN-A -->
<p>The discovery of the nitroplast captured our interest - we had considered a project on nitrogen fixation before but failed to see a way in which we could innovate or propose new solutions to the problems previous teams faced. All diazotrophs (bacteria and archaea that fix atmospheric N<sub>2</sub>) use the <strong>enzyme nitrogenase</strong> to fix nitrogen, but the expression of this enzyme presents great difficulties: it is <strong>irreversibly damaged by reacting with oxygen</strong>, while at the same time catalyzing an energetically demanding reaction. Due to this, diazotrophs have evolved very complex mechanisms to couple nitrogen fixation with respiration and/or photosynthesis, which so far has been beyond reach in terms of reproduction by synthetic biologists. The <strong>nitroplast solves this problem, acting as a fully contained compartment within a eukaryote where nitrogen fixation takes place</strong>, utilizing several years of evolutionary optimization.</p>
<p><strong>Symbiotic relationships</strong> between diazotrophs and plants already exist in nature, specifically in crops - legumes have a relationship with rhizobia (bacteria living around the plant root), as do some grass species with other nitrogen-fixers. However, most <strong>other crops do not have anything of the sort</strong>. Besides transgenic nitrogenase expression, the other main avenue currently being explored in nitrogen fixation is the <strong>engineering of external symbiosis</strong> between diazotrophs and other plants. However, this poses a challenge in replicating fragile extracellular signaling pathways and physical conditions that are dependent on the plant species' roots, as well as potential containment issues.</p>
<p>One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. <strong>To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="https://www.nature.com/articles/nature2024"style="color: #185A4F;">[Coale2024 Nitrogen-fixing Alga]</a>. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified <ahref="https://www.nature.com/articles/nature2024"style="color: #185A4F;">[Coale2024 Nitrogen-fixing Alga]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="#cite11"style="color: #185A4F;">[11]</a>. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified <ahref="#cite11"style="color: #185A4F;">[11]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>To understand the functioning of the UCYN-A import mechanism, we attempted to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from <em>Paulinella chromatophora</em> (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by <strong>obtaining the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and used <strong>docking</strong> tools to select candidate proteins likely to bind the previously identified transit motifs.</p>
<p>In addition to <em>in silico</em> experiments, we also aimed to investigate the transport mechanisms of UCYN-A <em>in vivo</em>. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast <em>S. cerevisiae</em> and the green alga <em>C. reinhardtii</em>. The initial <em>in vivo</em> characterization of the UCYN-A transport system involved <strong>examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms</strong> to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into <em>S. cerevisiae</em> and <em>C. reinhardtii</em>. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.</p>
<p>Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <em>S. cerevisiae</em> using either <em>E. coli</em> or <em>S. elongatus</em><ahref="https://link.springer.com/article/10.1007/s00438-018-1448-1"style="color: #185A4F;">[Mehta2018 EngineeringMitochondria]</a>. Another study successfully inserted <em>Azotobacter</em> strains into <em>C. reinhardtii</em><ahref="https://www.jstor.org/stable/24358669"style="color: #185A4F;">[Nghia1986 UptakeReinhardii]</a>. Building on this research, we initially aimed to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <em>C. reinhardtii</em> and <em>S. cerevisiae</em> as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, <em>E. coli</em>, and refined a protocol for its fusion with <em>S. cerevisiae</em>.</p>
<p>Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <em>S. cerevisiae</em> using either <em>E. coli</em> or <em>S. elongatus</em><ahref="#cite12"style="color: #185A4F;">[12]</a>. Another study successfully inserted <em>Azotobacter</em> strains into <em>C. reinhardtii</em><ahref="#cite13"style="color: #185A4F;">[13]</a>. Building on this research, we initially aimed to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <em>C. reinhardtii</em> and <em>S. cerevisiae</em> as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, <em>E. coli</em>, and refined a protocol for its fusion with <em>S. cerevisiae</em>.</p>
<p>Our project <strong>lays the foundation for the transplantation of nitroplast</strong> into eukaryotic hosts. The emergence of nitrogen-fixing plants could lead to a significant drop in the demand for fertilizers, and consequently in both carbon emissions and nitrogen pollution.</p>
</div>
<!-- <div class="h" id="five">
<div class="h1">Reference</div>
<p style="text-align:left;">[1] Kaku T, Sugiura K, Entani T, Osabe K, Nagai T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci Rep. 2021;11(1):14994. Published 2021 Jul 22. doi:10.1038/s41598-021-94551-4</p>
<p style="text-align:left;">[2] Brodl, Eveline et al. “The impact of LuxF on light intensity in bacterial bioluminescence.” Journal of photochemistry and photobiology. B, Biology vol. 207 (2020): 111881. doi:10.1016/j.jphotobiol.2020.111881</p>
<p style="text-align:left;">[3] Nijvipakul, Sarayut et al. “LuxG is a functioning flavin reductase for bacterial luminescence.” Journal of bacteriology vol. 190,5 (2008): 1531-8. doi:10.1128/JB.01660-07</p>
<p style="text-align:left;">[4] Snijder J, Axmann IM. The Kai-Protein Clock-Keeping Track of Cyanobacteria's Daily Life. Subcell Biochem. 2019;93:359-391. doi: 10.1007/978-3-030-28151-9_12. PMID: 31939158.</p>
<p style="text-align:left;">[5] Chavan AG, Swan JA, Heisler J, Sancar C, Ernst DC, Fang M, Palacios JG, Spangler RK, Bagshaw CR, Tripathi S, Crosby P, Golden SS, Partch CL, LiWang A. Reconstitution of an intact clock reveals mechanisms of circadian timekeeping. Science. 2021 Oct 8;374(6564):eabd4453. doi: 10.1126/science.abd4453. Epub 2021 Oct 8. PMID: 34618577; PMCID: PMC8686788.</p>
<p style="text-align:left;">[6] Li, S., Sun, T., Chen, L., and Zhang, W. (2021). Designing and Constructing Artificial Small RNAs for Gene Regulation and Carbon Flux Redirection in Photosynthetic Cyanobacteria. Methods Mol Biol 2290, 229-252.</p>
<p style="text-align:left;">[7] Hirokawa Y, Kubo T, Soma Y, Saruta F, Hanai T. Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942. Metab Eng. 2020 Jan;57:23-30. doi: 10.1016/j.ymben.2019.07.012. Epub 2019 Aug 1. PMID: 31377410.</p>
<p style="text-align:left;">[8] Čelešnik H, Tanšek A, Tahirović A, Vižintin A, Mustar J, Vidmar V, Dolinar M. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biol Open. 2016 Apr 15;5(4):519-28. doi: 10.1242/bio.017129. PMID: 27029902; PMCID: PMC4890671.</p>
<liid="cite2">The nitrogen strategy and the transformation of the rural areas — Nature and biodiversity — Government.nl. url: https://www.government.nl/topics/nature-and-biodiversity/the-nitrogen-strategy-and-the-transformation-of-the-rural-areas.</li>
<liid="cite3">National Oceanic US Department of Commerce and Atmospheric Administration. “What is eutrophication?”.</li>
<liid="cite4">Jeff Elhai. “Engineering of crop plants to facilitate bottom-up innovation: A possible role for broad host-range nitroplasts and neoplasts”. In: (Apr. 2023). doi: 10.31219/OSF.IO/NY2RC. url: https://osf.io/ny2rc.</li>
<liid="cite5">Toename stikstofoverschot in landbouw door droge zomer 2022 — CBS. url: https://www.cbs.nl/nl-nl/nieuws/2024/05/toename-stikstofoverschot-in-landbouw-door-droge-zomer-2022..</li>
<liid="cite7">Protesting farmers close roads and borders in nationwide campaign - DutchNews.nl. url:https://www.dutchnews.nl/2019/12/protesting-farmers-close-roads-and-borders-in-nationwide-campaign/.</li>
<liid="cite11">Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale T.H. and T. Nishimura. “Nitrogen-fixing organelle in a marine alga”. In: Science 384 (2024), pp. 217–222.</li>
<liid="cite12">Angad P. Mehta et al. “Engineering yeast endosymbionts as a step toward the evolution of mitochondria”. In: Proceedings of the National Academy of Sciences of the United States of America 115.46 (Nov. 2018), pp. 11796–11801. issn: 10916490. doi: 10.1073/PNAS.1813143115/SUPPL{\ _ }FILE/PNAS .1813143115 . SM02 . MP4. url: https://www.pnas.org/doi/abs/10.1073/pnas.1813143115.</li>
<liid="cite14">Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale, T.H. and T. Nishimura. Nitrogen-fixing organelle in a marine alga. Science, 384:217–222, 2024. 6</li>
<liid="cite15">Angad P. Mehta, Lubica Supekova, Jian Hua Chen, Kersi Pestonjamasp, Paul Webster, Yeonjin Ko, Scott C. Henderson, Gerry McDermott, Frantisek Supek, and Peter G. Schultz. Engineering yeast endosymbionts as a step toward the evolution of mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 115(46):11796–11801, 11 2018.</li>
<liid="cite16">N.H. Nghia et al. “Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii”. In: Biochemie und Physiologie der Pflanzen 181.5 (Jan. 1986), pp. 347–357. issn: 0015-3796. doi: 10.1016/S0015-3796(86)80008-7.</li>
