Clean Energy With N. Crassa

Global mission :

To create a bioreactor and develop a protocol using the Neurospora Crassa fungi in order to transform and recycle cellulosic waste (paper, brewer spent grain, etc.) into ethanol. The aim of this project is to create the simplest protocole and machinery for small enterprises and breweries to implement the process in their facilities to be able to recycle cellulosic waste in a useful fuel.

Sub-objectives :

• To determine and obtain the most suitable species of mushroom for the implementation of our project ;
• Create the optimal protocole for optimal yield of bioethanol ;
• Create the necessary conditions for the fungi to transform cellulose to glucose and finally ethanol ;
• Build a low-cost bioreactor in which to conduct the process ;
• Simplify the protocole and the process for the project to be easily doable in other contexts.

We are a team of four students based in Paris : Anna Pierre, Antoine Cordoin, Cléo Perrin and Gaspard de Tournemire.

The recycling problem of cellulosic waste (paper, cotton, brewer spent grains...)

Cellulosic materials such as paper, journals, tissues are currently mostly recycled back into their original form [1]. In France, for instance, 3.6 million tons of packaging materials are recycled each year. However, after a dozen cycles, the cellulose fibers become damaged and can no longer be used [2]. Moreover, the recycling process takes an important amount of energy: Nature Sustainability published in 2020 a study demonstrating that producing recycled paper emits more greenhouse gases than producing non-recycled paper [3]. Furthermore, multiple cycles of paper recycling may potentially lead to accumulation or un-intended spreading of chemical substances contained in paper [4]. Also regarding paper, it is unfortunately more economically advantageous to produce paper than to recycle it : the Finnish giant UPM, owner of the plant of La Chapelle Darblay, the last paper recycling plant in France, decided in 2020 to close the latter, in favor of the establishment in Uruguay of an non-sustainable pulp mill. Considering that La Chapelle Darblay plant recycled 250,000 tonnes of paper waste per year, this once again underlines the need to find a new method to give paper waste a new life [5]. Moreover, we should consider another cellulosic material produced as waste by the beer industry, spent grain. Indeed, brewer spent grains, the leftover malt, and adjuncts after the beer production process has extracted most of the sugars, proteins, and nutrients, can constitute as much as 85 percent of a brewery’s total by-product [6]. Nowadays, agricultural uses of spent grain predominate (as a fertilizer or livestock feed), but new revolutionary uses of spent grains should be considered as sustainable recycling solution (baking bread, boiling it to create energy for the brewing process...) [7].

Starting from here, we wondered how we could make use of these cellulosic wastes... And we thought that ideally, the cellulosic compound could potentially be used to produce fuel.

The need for a new energy source

Nowadays, we rely mainly on fossil fuels to produce energy (heat in your home, electricity in large power stations, or to power engines such as cars). Fossil fuels are formed from the decomposition of buried carbon-based organisms, creating carbon-rich deposits that are extracted and burned for energy. Even though these fuels are highly needed (they currently supply around 80% of the world’s energy), there are non-renewable and their burning releases large amounts of carbon dioxide, a greenhouse gas [8]. Finding an alternative to the slow production process of fossil fuels could therefore be interesting. In that context, biofuels such as bioethanol could be considered as the ideal solution. However today, bioethanol relies on biomass exploitation, which to a certain extent can have a negative environmental impact like deforestation, soil degradation, and ecosystem endangerment [9].

Consequently, production of bioethanol using cellulosic waste could appear as a more sustainable production mode of biofuel... And could also help us recycle in an innovative way our cellulosic waste (paper, brewer spent grains, etc...). But how to do so in the best sustainable way ?

Using cellulosic waste to produce ethanol... Thanks to a fungus !

When left in a glucose lacking environment, some organisms will degrade cellulose into its simple sugars building bricks, for survival. The best documented and apparently most efficient fungus for this process found in our bibliographical study is a mould strand called Neurospora Crassa, which is a quite widespread model organism in a lot of domains. N. Crassa will not only break down cellulose, but also ferment the resulting glucose into ethanol in anaerobic conditions. As such, N. Crassa should be considered as the main actor in the conversion process of cellulosic waste to bioethanol [10][11]. However, as seen in the References section, we found less than 8 articles illustrating how N. Crassa could be used as a potential mean to reduce waste, and efficiently produce ethanol. More than that, not any of them mention how this natural process could be used on a larger scale to reduce our daily waste into a much needed energy source.

We therefore aim at implementing a literature-based efficient protocole using N. Crassa to produce bioethanol via degradation of some of our daily cellulosic waste (paper waste, brewer spent grains, cotton...).

A larger goal to our project would be to implement the cheapest and simplest protocole as possible for the process to be used at a larger scale. Indeed, we could imagine that the process could be used by breweries to recycle their spent grains, and use the resultant fuel to produce much needed energy in their brewing process. We could also think that small to medium size enterprises could use the process to recycle their paper and newspaper waste, and once again use the resultant ethanol to produce energy. Wanting to reduce as much as possible the cost of the process, we therefore aim to construct a “low-tech” bioreactor. A bioreactor is a device for growing organism in a controlled environment. Normally it is an high-tech apparatus but we found an open-sourced building instruction for building one using Arduino that could serve as an inspiration in our project context [12]. We could adapt it to a suitable co-culture for Neurospora Crassa. This would make our project accessible, and achievable by more individuals.

References

[1] « Paper Recycling ». Wikipedia, 6 avril 2021. Wikipedia, https://en.wikipedia.org/w/index.php?title=Paper_...

[2] « Le recyclage et la valorisation des déchets de cartons et emballages ». Ecodrop, 6 février 2019, https://www.ecodrop.net/le-recyclage-et-la-valori...

[3] van Ewijk, Stijn, et al. « Limited Climate Benefits of Global Recycling of Pulp and Paper ». Nature Sustainability, vol. 4, no 2, février 2021, p. 180‑87. www.nature.com, doi:10.1038/s41893-020-00624-z.

[4] Pivnenko, Kostyantyn, et al. « Waste Paper for Recycling: Overview and Identification of Potentially Critical Substances ». Waste Management (New York, N.Y.), vol. 45, novembre 2015, p. 134‑42. PubMed, doi:10.1016/j.wasman.2015.02.028.

[5] « Avec la fermeture de l’usine de La Chapelle Darblay, la France perd toute capacité de recycler du papier ». Basta !, https://www.bastamag.net/Fermeture-derniere-papet...

[6] « Brewer’s Spent Grain ». Wikipedia, 25 février 2021. Wikipedia, https://en.wikipedia.org/w/index.php?title=Brewer...

[7] Witkiewicz, Kay Kay. « Sustainable Uses of Spent Grain ». CraftBeer.Com, 27 juillet 2012, https://www.craftbeer.com/craft-beer-muses/sustai...

[8] « Concerns about Climate Change and the Role of Fossil Fuel Use ». Fuel Processing Technology, vol. 71, no 1‑3, juin 2001, p. 99‑119. www.sciencedirect.com, doi:10.1016/S0378-3820(01)00139-4.

[9] « Environmental impacts of biofuels». THE STATE OF FOOD AND AGRICULTURE 2008. FAO.org

[10] Xiros, Charilaos, et al. « Hydrolysis and Fermentation of Brewer’s Spent Grain by Neurospora Crassa ». Bioresource Technology, vol. 99, no 13, septembre 2008, p. 5427‑35. PubMed, doi:10.1016/j.biortech.2007.11.010

[11] Dogaris, I., Mamma, D., & Kekos, D. (2013). Biotechnological production of ethanol from renewable resources by Neurospora crassa: An alternative to conventional yeast fermentations? Applied Microbiology and Biotechnology, 97(4), 1457–1473. https://doi.org/10.1007/s00253-012-4655-2

[12] « Microbial Bioreactor ». Hackster.Io, https://www.hackster.io/open-bioeconomy-lab/micro...

1. Pre-treatment of spent grains

• Obtaining and drying the brewer's spent grains : for a project in January, we had already borrowed spent grains to a local parisian brewer. Back then, it was dried for 2 days in an oven, and stored in a glass jar. We were therefore able to use it for our protocole.

• On Wednesday the 28th, we prepared the spent grain by reducing it to powder using a mixer of the MakerLab.

• On Thursday the 29th, we conducted the first step of the pre-treatment, the heating of spent grain with NaOH. As explained, we firstly wanted to prepare 3 times the same preparation. We first weighed (x3) in a 300 mL becher 60g of spent grain powder. Then, under a chemical hood, we added with a pipette 150 mL of NaOH, and mixed the resulting liquid-solid mixture using a metal stirrer. We obtained a dark slurry. As we firstly expected to have a more liquid mixture, we decided to only launch 2 times the process, in case it was not really the best.

• We then put the 2 bechers in an autoclave for 1h at 121°C, and the next day, we needed to lower the pH to 5, for it to be ideal for the fungi growth.

• To do so, we needed for the slurry to be more liquid. We firstly added 120 mL of distilled water to the 135 g mixture. We then added iteratively controlled amount of HCl to lower the pH to 5 in the slurry. We proceeded by adding 2 mL by 2 mL, stirring the slurry and controlling the pH with a pH meter. In order to lower the pH to 5,7 , we added 13,5 mL of HCl.

• We then replaced the final adjusted pre-treated spent grain back into the frigo.

• On Monday 3/05, we did the same readjustment of pH on our 2nd batch of pre-treated spent grains, this time with 150 g of pre-treated mixture, adding 120 mL of distilled water and this time 14,5 mL of HCl to adjust the pH to 5,7.

2. Fungi growth / inoculum

• We were able to obtain N. Crassa from a Pasteur team, Fungal Epigenomics (Lab Head : Eugene Gladyshev). On friday, they provided us with a tube containing a slant of medium and some N. Crassa.

• On monday, we prepared some YPD, autoclaved it, prepared some petri-dishes, and put some N. Crassa to grow on it. Unfortunately, part of the petri-dishes were not usable as the YPD was not good.

• We set the time-lapse on the one correct petri-dish we could obtain.

• On Thursday, we prepared 18 petri dishes containing Hortense YPD and using the N. Crassa grown in the Pasteur tube

• We then let them grow for 3 days. We put 11 petri-dishes at 37°C and 7 at ambiant temperature.

No new growth can be seen by eyes, the fungi became whiter and do not seem in good shape. 37°C is not the suited temperature for fungi growth.

3. Grow the fungi in a mineral medium

• On thursday 29/04, we prepared the mineral medium. Considering that we wanted to 300 mL, we added in an erlenmeyer :
  • 0.54 g of KH2PO4
  • 2 g (NH4)2SO4 (instead of 3g (NH4)2HPO4 as not available in the lab, replace it with still a source of ammonium, and just sulfate which we know is not too dangerous as in MgSO4. We decided to add 2 instead of 3 to not put too much SO4)
  • 0.09 g of MgSO4 , 7H2O
  • 0.09 g of CaCl2 , 2H2O
  • 12 g of spent-grains

• The pH was at 5.7 so not needed to adjust it.

• We then used the 4 days old N. Crassa, in which petri-dish we added 15 mL of distilled water but no Tween (Hortense told us that it was toxic for the fungi). We detached all N. Crassa sticked to the edges of the petri-dish.

• We then used a pipette to take the water and the fungi from the petri-dish and put it in a 200 mL erlenmeyer containing 100 mL of the prepared liquid medium.

• The flask was incubated at 37 °C for 2 days in an orbital shaker (37° and 150 rpm, not 30°C and 250 rpm as explained in Xiros article, as we could only use one of CRI, and was already working for other experiment at 150 rpm) for mycelium production.

We can see that it is less clear, an indication of fungi development.

Interestingly, we wanted to try if there was any chance that as we already moved N. Crassa to a mineral medium with spent grains and without any glucose source, we could already have obtained ethanol. We carefully opened the erlenmeyer, and used the Arduino MQ3 ethanol captor : the basal value of 47 went to a value of 259. When putting the captor above a purified ethanol drop, approximately 3 cm above, we can obtain a value of 255. It seemed that we already obtained some ethanol !

To check if this was not due to the spent grain fermentation, a phenomenon that we already observed before, we decided to create a control and launch the exact same protocole, but this time with just the mineral medium in which we did not put any N. Crassa.

On monday, we did the exact same protocole this time using petri dish that stayed at 37°C (curiously, seemed to have grown less, and was whiter + one that stayed at room temperature → this time, as we needed to change incubator for laboratory reasons, we moved the erlenmeyer and put it in the incubator at 30°C at 180 rpm.

4. Fungi with spent-grains

• Considering that we did not build yet the bioreactor, we launched the process in a rotatory incubator of the CRI, at 30°C at 180 rpm, as indicated in previous papers. To create an hermetic environment, we closed an erlenmeyer with aluminum paper.

• We proceeded under the biological hood in a 500 mL erlenmeyer.

• We poured 150 mL of treated spent grains in the erlenmeyer, and after shaking the erlenmeyer containing the inoculum (mineral medium + fungi grown during 50h in the rotating incubator at 37°C at 150 rpm), we poured 100 mL of the inoculum.

• Then, we launched the incubation at 30°C and 180 rpm in the student lab.

On Wednesday, we decided to launch multiple trials by changing certain parameters, to see how we could adapt the protocole. The first line of the table is for our first trial described above.

[Table in the images above]

At the end of the 3 weeks, for the incubator to not work without utility, we decided to remove all 4 erlenmeyers from the incubator, and put them in the refrigerator to stop the reaction.

5. Distillation process

During the 3 weeks of project's pause, we conducted the distillation process with the help of David Jung, on our first and second trial (line 1 and 4 of the above table). Considering the outcome and the amount of ethanol obtained, we decided to conduct the protocole on our two ideal conditions.

Interestingly, when we took the erlenmeyers out of the refregerator, we found that N.Crassa had grown in whitish filaments, but also that an other fungi grew on the medium, as seen in the picture below. This means that our erlenmeyer content was not completely sterile, either when we prepared it, or when it was settling in the refrigerator.

To conduct the distillation process, we firstly poured the content of the erlenmeyer inside a ballon. The consistance was that of a slurry, a semi-liquid and solid content. The fungi had formed a viscous aggregate at the bottom of the flask. We therefore tried not to put too much of this part in the ballon for the distillation process, because it could burn during the heating, and also because we knew that the ethanol was contained in the liquid part of the mixture.

We then conducted the protocole described in the "Ideal protocole".

• Wearing a blouse, a protective glasses and gloves, we firstly placed the mixture contained in the erlenmeyer in the ballon (after 3 days in the refrigerator, any ethanol vapors present have liquefied, and we therefore hoped to minimize losses when transferring from the Erlenmeyer flask)

• Then, we prepared the montage :
  • We placed the elevator support and put the heater on it.
  • We then placed the balloon support, attached the balloon using the 2-finger pliers, checked that it was raised relative to the heating, but also that we would be able to raise it fast enough if any problem occurred.
  • Then, we placed the Vigreux column on top of the ballon. The Vigreux column is directly linked to the condensation column and a tip arriving in the collection flask, as seen in the scheme below. We adjusted the distillation column above the flask and placed the thermometer.
  • We then placed the collection flask just below the tip, and checked that every junctions were sealed.
  • Then, we placed the water inlet pipes, water is coming from the bottom and is discharged from the top, to allow it to fill entirely the cooling colonne (réfrigérant). Starting the water, we finally ensured that the flow was sufficient to allow cooling.

• We then started heating our solution by setting the heater at 120°C. We can remind that the Vigreux column makes it possible to separate liquids according to their difference in volatility. The inside of the tube is bristling with spikes, oriented downwards, which allow, by successive condensation of the various components, a more precise separation of the various bodies present.

• We covered the ballon with aluminium folding and cotton to build a isolating coat to avoid any heat loss.

• The slurry then started to heat and we observed the temperature of the vapors increasing in temperature, thanks to the thermometer.

• We knew that the boiling point of ethanol is 78 ° C and the one of water is 100 ° C. We therefore expected to collect our distillation head (the first liquid that evaporates, with the lowest boiling temperature) at a temperature around 78/80 °C. But we actually observed that the first vapors we collected as a liquid in our collection flask were at a temperature of 90°C. The second temperature level was reached at 100°C, which we hypothesized was containing water and partly ethanol. Note that when conducting the protocole, special care should be taken to change the collection flask tube for recovering the distillate as soon as the thermometer indicates that the temperature of the vapors begins to rise again.

• We placed the content of the collection flask in a falcon tube. For our ideal protocole, we obtained 5 mL of the distillation head. When using a simple Arduino ethanol sensor (MQ5) set above the tube, we obtained a value of 414 (the sensor was calibrated to 0 in ambiant air), indicating that the tube was actually containing ethanol. However, to estimate the amount of ethanol contained in the solution, we had to use a refractometer. The principle is simple : calibrated with distilled water, the alcohol percentage of a solution can be determined through a measurement of a refraction index.

The use of the refractometer lead us to determine the percentage of alcohol in our final 5mL solution... 4%. Disappointed at first, we realized that it was correlated with the fact that the we collected the distillation head at 90°C, which is more elevated than the vaporisation temperature of ethanol. We can therefore say that we also have water and maybe some other solutions (such as NaOH or HCl) in our distillat.

Bioreactors provide a controlled environment for culturing microorganisms or cells.
Conditions that can be optimized for cell growth are:

• Medium in which the cells are grown
• Temperature
• Oxygen, Nitrogen
• Agitation
• pH

In our case, the regulation that felt the most important was temperature. In litterature, it didn't appear that the pH was regulated at any point. A part of the process requires anaerobic conditions, or at least with controlled airflow, but this can be modulated depending on the shape of the container. Agitation could also be added using small motors connected to the container in the water bath. We chose not to monitor the concentration and nature of substances inside the reactor.

As such, we focused on a building a thermostatic environment for our fungus to work in best conditions.

Materials :

• an Arduino Mega
• an Anycubic Mega X Heatbed
• a RAMPS (here v1.4) for the heatbed management
• a 1-Wire DS18B20 temperature sensor
• a breadboard, jumpers and a 4.7 kΩ resistor for the temperature sensor hookup
• a PC power supply (here the Enermax EG365AX-VE(G) 12V)
• a pot for the waterbath, as thermostatic as possible, and a container for the fungus and spent grain to be processed

Methods :

We found an old bioreactor prototype in our makerlab's storerooms, originally intended for yeast-based fermentation. This old project allowed us to learn about all the elements we needed to build our own bioreactor. In addition, there are many common elements that are useful for our project.

Namely, we found in it an Arduino Mega which was already assembled to a RAMPS, and which was easily enough connected to a temperature sensor (as shown in the first image), as well as the heat plate. The assembly was supplied power by an ATX PSU, which had to be "softstarted" by connecting the green PS ON wire to ground.

We bought a jar of about 1.5L closed with a wooden cap, to act as a container, and used pots from an old rice cooker for the waterbath. The temperature sensor was placed inside the container, which was itself placed and stabilised inside the waterbath.

Code :

The code for the thermostat can be found linked here.

It uses the OneWire library to fetch the data from the temperature sensor, and performs a "proportional bang-bang" algorithm to regulate the temperature.

Basically, the heatplate is set to max until the temperature sensor placed inside the container reaches 28.6°C (1.5 degrees beneath the set ideal value of 30.1°C), at which point the heatplate is set proportionally to the temperature measured. This proportionality is regulated by a constant called KP, and which was calibrated in our case to 200 for ideal results. Once the temperature has gone for one cycle higher than the set temperature, the ensemble is stabilized at this set temperature efficiently.

The mean power consumption measured entering the system was of 30W (0.08 kWh in 2:41 hours).