Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.
The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.
Population and economic growth have caused an increasing demand for food, water and fuels. Much of these supplies are produced, processed and transported using finite fossil-based means, such as petroleum. It is, however, clear that this practice is not sustainable, and the development of alternative resources will therefore be of great importance1. Many renewable resources are, to varying degrees, currently being exploited, including wind, water motion, solar, geothermal, and wave based energy sources. Another sustainable and largely untapped resource is the biomass from plants. This resource also offers a very cost efficient way to convert solar derived energy into fuels2. Apart from providing bio-based fuel, the plant biomass also offers unique opportunities for alternative products, including plastics, detergents, and valuable chemicals.
The plant cell wall, which largely consists of sugar based polymers, makes up the main bulk of the plant's biomass and much effort is currently being invested in its efficient conversion into bioethanol. The remaining biomass may subsequently be processed into biogas and oil related products3. Much of the perennial plant species, including grasses and trees, that produce large amounts of cellulosic biomass typically grow best in the temperate zones. However, approximately 20% of the land area is semi-arid, and is therefore also prone to droughts4. Obviously, it would be of interest to also cultivate these arid lands with plants that could effectively contribute to the sustainable production of energy and material. These plants need to have an optimal water use efficiency and drought resistance and would include the tobacco tree (Nicotiana glauca) and species from the Agave genus.
The MultiBioPro consortium aims to implement an integrated biorefinery pipeline, using the two important crop species, poplar and tobacco tree. Poplar has emerged as a promising biofuel crop as it is fast growing, easily clonally propagated and highly adaptable to a wide range of climatic and soil conditions. It also provides a wide range of wood, fiber, fuel wood, and other forest products5. The tobacco tree has also emerged as a suitable plant for biofuel and biorefinery purposes. It typically produces substantial quantities of biomass, contains high amounts of nonstructural carbohydrates6, and also has the rare ability to accumulate large quantities of readily extractable nonfood oils (including long chain C29-C31 saturated hydrocarbons and triterpenoids) that are suitable for biodiesel production. The tobacco tree is, moreover, amenable to genetic improvement, has high sprouting capacity, and grows happily on semi-arid soils not used for food production. It therefore appears that both poplar and tobacco tree have intrinsic potential for multipurpose crops, i.e. as new high value feedstocks for an integrative bio-based industry. In this paper we focus on diverse set of approaches to discern how tobacco tree deposits long chain hydrocarbons.
In an attempt to identify the underlying molecular machinery responsible for the production and secretion of the saturated long-chain hydrocarbons on tobacco leaves, we apply modern “omics” based technologies. This includes RNA seq of a developmental leaf series (ten stages), and multiplatform metabolite profiling approaches using LC- and GC-MS (for polar and nonpolar metabolites and lipidomics). These data will be used to mine for gene expression that correlates with, or precedes, the onset of biosynthesis of the molecules indicated above. Genes and pathways that appear promising from these endeavors will be used for functional testing in the model species Arabidopsis and could ultimately be amenable for biotechnological engineering in tobacco tree.
1. Plant Material
Grow Nicotiana glauca plants in 30 cm diameter pots containing M2 professional growing medium. Grow plants in glasshouse, with a daytime temperature of 20-25 °C and nocturnal temperature of 15 °C. Use a 16 hr light and 8 hr darkness cycle as supplementary light regime.
2. Sample Preparation
3. Extraction Protocol for Metabolite Profiling for Primary Metabolites by GC-MS
4. Extraction for Metabolite Profiling for Secondary Metabolites by LC-MS
5. Data Analysis
6. Hydrocarbon Extraction13
7. Analysis of Isoprenoids14
8. RNA Extraction for RNA Seq
This protocol combines Trizol RNA extraction with a RNeasy Kit to obtain high quality RNA.
The HPLC profile in Figure 1 shows a representative result of the isoprenoid analysis of N. glauca leaf extracts. The different isoprenoids of C40 and above were detected using a Photo Diode Array (PDA) detector. The peaks were annotated based on co-chromatography and spectral comparison between authentic standards, Beta carotene (provitamin A), phytoene, lycopene, lutein and zeaxanthin. The two MS chromatograms in Figure 2 show the result of primary metabolite analysis from N. glauca leaf and stem material, respectively. The MS spectrum of a peak corresponding to serine (indicated by an arrow) is also given as an example. Figure 3 shows the Bioanalyzer used for determining the quality of the RNA and a representative output from the device. The two main peaks in the chromatogram corresponding to 18 S and 25 S ribosomal RNA, indicating intact RNA in the sample. Additional peaks of fragmented ribosomal RNA would appear in case of partially or heavily degraded RNA.
Figure 1. HPLC profile showing isoprenoids present in leaf extracts from N. glauca. Most isoprenoids of C40 and above are not amenable to GC-MS analysis. We therefore used HPLC separations with photodiode array detection. A typical chromatogram recorded at 450 nm is shown. The carotenoid pigments are typical of photosynthetic tissues with lutein predominating. Also present is zeaxanthin, which is rarely found in leaf tissue unless placed under high light stress. The levels of zeaxanthin make it a good source of this high-value compound. Please click here to view a larger version of this figure.
Figure 2. MS chromatogram and spectrum of N. glauca tissue extracts. Total ion MS chromatogram (TIC) of leaf and stem extracts measured by GC-MS is presented (70-600 m/z). GC-MS analysis was performed as described previously in15. Detected peaks were annotated using the mass spectral tags library. MS spectrum of serine (2TMS) is shown as an example. MS chromatogram: X axis and Y axis indicate retention time (min) and the intensity (abundance) of the signal, MS spectrum: X axis and Y axis indicate the M/Z ratios and the intensity (abundance) of the signal, respectively. Click here to view larger image.
Figure 3. Bioanalyzer measurement of RNA prepared for RNA seq of N. glauca leaf material. To obtain highly pure RNA needed for RNA seq we extracted RNA using Trizol reagent and subsequently purified the RNA using the columns from the RNeasy Mini Kit (Qiagen, Hilden, Germany). We determined the RNA quality using the Bioanalyzer (Agilent, Waldbronn, Germany) displayed on the left. An example of a Bioanalyzer output is given on the right. The two main peaks in the sample represent 18 S and 25 S ribosomal RNA. Our sample showed a RNA integrity number (RIN) of 9.2, which is well above the required value of 8. Click here to view larger image.
The protocols presented here provide a comprehensive framework to analyze tobacco tree leaves for metabolites and transcripts. It is envisaged that these combined efforts should provide us with new insights into the processes underlying the synthesis and extrusion of the hydrocarbons and the high value compounds present in this tissue. These approaches should therefore give us a better understanding for how the compounds are being synthesized. In addition to the tobacco tree aspects of the work, it is also aimed to improve poplar biomass, especially targeting lignification of the secondary wall structure, but also to explore whether we can use the bark for extraction of valuable compounds.
The methods presented in this paper are slight modifications of standardized methods for metabolite profiling. These methods are of course limited to known metabolic profiles, and it is possible that several new metabolic peaks may be obtained for which no compound is known. We hope to put these compounds in context to other metabolites by combining the behavior of metabolites and transcripts over the developmental time series.
None of the methods presented here are significantly changed from methods typically used for plant materials. The interesting aspect lies in the combination of methods to understand the underlying framework for mainly long chain hydrocarbon production and modification in the tobacco tree leaves. One of the critical steps for obtaining this information is the subsequent combination of the different data types. We envision that the data as a first evaluation will be divided into different clusters based on the behavior of the metabolites/transcripts over the development and that these data will be used to infer transcript vs metabolite behaviors, and also to potentially assign certain metabolites to pathways. In addition, more elaborate network-based analyses are then envisioned to exploit causal relationships.
The analytical protocols presented here will also provide a basis for field-trials and industrial exploitation of the biomass. To accomplish this, the MultiBioPro consortium contains several industrial partners that have the abilities to further explore the biomass, with the aim to deliver biodiesel, bioethanol and other high-value compounds. These types of biomass exploitation will be assessed based on; (1) testing the robustness and quality of the bio products produced (typical industry standard tests will be carried out to ensure the products generated have good market value), (2), an economic, social and environmental evaluation of the technologies will be performed using literature sources, interviews and material that is generated during field trials and pilot plant biorefinery assessments. These activities will include cost benefit and life cycle analysis, the generation of an environmental dossier and market and business strategies. We believe that this pipeline will become a useful blend of academia, applied science and industrial exploitation to further poplar and tobacco tree biomass for consumer end-products.
The authors have nothing to disclose.
MultiBioPro would like to thank the following people who also contribute to the project: Dominic Swinton (Green Fuels), Thomas Lowery (Green Fuels), Sam Buekenhout (Capax), and Sylvia Drouven (Capax).
Name | Company | Catalog number | Yorumlar |
Trizol reagent | Invitrogen | 15596-026 | |
Chloroform | Merck | 102445 | |
Ethanol | Merck | 101986 | |
Rneasy Mini Kit | Qiagen | 74104 | |
TURBO Dnase | Invitrogen | AM2238 | |
RNA 6000 Nano Kit | Agilent | G2938-90034 | |
2100 Electrophoresis Bioanalyzer | Agilent | G2939AA | |
1.5 ml and 2 ml safe-lock tubes | Eppendorf | 0030 120.086, 0030 120.094 | |
Steel balls | Geyer Berlin GmbH | VA2mm | |
Mixer mill MM 300 | Retsch | YO-04182-09 | |
Microcentrifuge | Eppendorf | 5424 |