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Komaplastics: A Cellulose-Based Agricultural Plastic
Komaplastics: A Cellulose-Based Agricultural Plastic
Melody Azimi1, Sophia Sneddon1, Joshua Elkins1, Kyra Eyerman2, Alicia Jorgenson1, Navdeep Kalkat2, T. Conor Kensok1, Rachel Mace2, Claudia Paz Flores2,
Gabriel Sanchez Jr.1, Neil Smith1, Faith Williams1, Taylor Ziccardi1

1 Department of Biomolecular Engineering, UC Santa Cruz
2 Department of Molecular, Cell, & Developmental Biology, UC Santa Cruz

Abstract


Plastic bed mulches are vital in the agricultural industry for providing UV and water resistance, limiting fumigant emissions, preventing weed growth, and overall increasing crop yields. Most bed mulches on the market are made of polyethylene (PE) plastic and end up in landfills where they pollute local land as they degrade into harmful chemicals. Some degradable mulches are currently on the market, but leave behind microplastics that compromise soil integrity. The Komaplastics project pioneers a design for a biodegradable bed mulch made from bacterial cellulose (BC) produced by Komagataeibacter rhaeticus in order to tackle plastic waste in the agricultural industry. The main obstacle presented in this project is overcoming the high degree of crystallinity of BC that makes the material brittle. To add plasticity and elasticity to the material, we investigated the use of carbohydrate binding modules (CBMs) as an additive to disrupt the hydrogen bonding between BC strands. Additionally, we chemically decrystallized BC and studied the use of various chemical plasticizers as methods of adding plasticity to the material. Our research and design provides stepping stones to the creation of a fully biodegradable bed mulch made from cellulose.
Goals
Project Goals
The goals of our project are to:
  • Express Cellulose Binding Modules (CBM) in E. coli to decrystallize bacterial cellulose (BC)
  • Design a method to plasticize cellulose to add elasticity and flexibility
  • Design a co-culture for easy integration of our plasticizing-agent and CBM into BC
Inspiration
Inspiration
The United States agricultural industry produces 816 million pounds of plastic waste each year [1]. Unfortunately, there are no good options for growers to switch to sustainable plastics. Current biodegradable options are expensive and leave microplastics behind that contaminate the soil. Due to this concern, many growers avoid using biodegradable plastic mulches, leaving little to no options for moving towards sustainable farming practices [2].

The University of California, Santa Cruz (UCSC) is located in an agriculturally rich area, making this project especially important to our team. This proximity has allowed us to connect with growers who have struggled to find a biodegradable solution to plastic mulches.

Our team was motivated to design a biodegradable plastic bed mulch that growers can till into the soil without leaving microplastics and help to reduce overall plastic accumulation in landfills.
Methodology
Methodology
Please click on one of the three buttons labeled Overview, CBMs, or Plasticizers for more information!
Overview
Overview
Cellulose, a structural component found in plants, consists of repeating glucose monomers which are connected by extensive inter- and intra-hydrogen bonding. These bonds result in a high degree of crystallinity and rigidity within the cellulose structure. To make a cellulose-based plastic, cellulose must be decrystallized before adding non-toxic chemicals that impart plastic-like qualities.

Figure 1: A CBM binding to the negative regions of cellulose and disrupting the strong hydrogen bonds between the cellulose strands.

CBMs
CBMs
CBMs decrease the crystallinity of cellulose by disrupting hydrogen bonds between BC strands. We utilized two CBM genes from separate CBM families to understand their different binding affinities to cellulose:
  1. The Sirius part from the 2018 Toulouse iGEM team - CBM3a gene
  2. A new part we created called CBM2a (part: BBa_K3426000)
Part Name Part Number Part Type Description Designer Length
Sirius: CBM3a - mRFP1 fusion (Sirius) BBa_K2668020 Coding region CBM3a, mRFP, N- and C-Terminus linkers. Younes Bouchiba 1,349 bp
Cellulose-Binding Modules of Type 2a (CBM2a) Fused to mRFP1 BBa_K3426000 Coding region CBM2a, mRFP, N- and C-Terminus linkers. Kyra Eyerman 2,371 bp
Sirius CBM3a gene was used because it is highly characterized and is also present in a thermostable bacterium, C. thermocellum, potentially giving thermostable qualities to our film.

Our team created a part called CBM2a (part: BBa_K3426000), because this specific CBM has slightly lower binding affinity to cellulose than CBM3a. We theorize that this will allow for greater plasticizer incorporation [3]. Golden Gate Assembly was used to construct the pET28a backbone with the different gene blocks, including the mRFP gene.

Improving on a Part


We want to improve the Sirius CBM3a part by removing the mRFP gene. The mRFP is a large protein that can inhibit the binding of CBMs. Rather than using the mRFP as a selection method for CBM-cellulose binding, we are creating a new selection and quantification. This method will allow us to quantify the amount of cellulose bound to reach our desired crystallinity.
Plasticizers
Plasticizers
Our team tested various chemical plasticizers in order to add desired plastic qualities to BC that bed mulches must have, including increased tensile strength and flexibility, gas impermeability, and water resistance.

We used ionic liquids to quickly and efficiently decrystallize cellulose in order to test plasticizers. However, these chemicals are not biodegradable and therefore cannot be a component in our final mulch film. Ionic liquids allowed us to create decrystallized cellulose to test different chemical plasticizers before we can express CBMs.
Human Practices
Human Practices
Our team connected with stakeholders in the agricultural industry to address the concerns of current biodegradable plastics. We want to build a product that growers will be comfortable with and proud to use on their farms.

Current biodegradable mulches do not pass fumigation protocols, making many farmers hesitant to move towards sustainable options. To understand these regulations further, we talked to multiple experts about fumigation and biodegradable testing practices that need to be passed in order for these plastics to be used on farms. We had the oppertunity to visit the Ajwa Analytical Lab to understand what goes into fumigation testing and what it takes to pass the protocols. Many growers are concerned with the breakdown products of biodegradable plastics. To address these concerns, we changed the direction of our project to make sure all products we are using to create the plastic only break down to natural components. This would ensure our product doesn't leave behind harmful microplastics.

We adapted our product after every interview and every stage of our research to evolve our project and make a product we are proud of.
Results
Results

CBMs

Figure 1: Colony PCR gel of the Golden Gate Construct using T7 primers. A band around 1.3 kb is shown for colonies 2 and 4. This proves the successful construction of the CBM3a plasmid.

Our team was able to successfully amplify the CBM2a and mRFP genes with the corresponding Bsa1 sites needed for Golden Gate Assembly. Work for the construction of this plasmid is still in progress.

Plasticizers


We tested the following ionic liquids: NaOH, ZnCl2, and variations of BMIMCl: diisopropyl imidazolium (DIIP) and diisobutyl imidazolium (DIBU). Sodium hydroxide worked best as a decrystallization-agent in producing amorphous cellulose, which was used throughout multiple experiments.

A 70% to 30% ratio of glycerol and citric acid plasticizer mixture to amorphous cellulose proved to be the most promising mixture to create a thick cellulose-based gel.

Figure 2: 70:30 ratio of plasticizer mixture (glycerol/citric acid) to amorphous cellulose after being baked.

Assays

Figure 3: X-ray diffraction of the room-dried BC sample. Crystallinity index of 76% was derived from the graph.

X-ray diffraction (XRD) was also perfomed on current bed mulch films. These tests gave an crystallinity index of 66% from the films. This proved we must decrease the crystallinity of pure BC in order to get a crystallinity similar to that of commercial films.
Future Work
Future Work
Our future work consists of:
  1. Constructing the plasmid for the CBM2a gene block using Golden Gate Assembly
  2. Continuing testing of plasticizers and copolymers to achieve the discovery of optimum concentrations
  3. Finding optimal curing and baking conditions to create a cellulose-based film that works to test multiple plasticizers
  4. Performing protein expression of CBM3a in electrocompetent BL21 E. coli cells
  5. Removing the mRFP gene from the existing Sirius part to quantify and control the amount of CBMs attached to BC
  6. Creating a co-culture consisting of CBM-producing E.coli, plasticizer-producing E. Coli, and BC-producing K.rhaeticus
  7. Testing integration of desired plasticizers with BC-CBM composite
  8. Performing Long Term Compostability (Mesh Bag) Assays of our film
References and Acknowledgements
References and Acknowledgements

References


[1] Jones, Gene. “Recovering Agricultural Plastics: Obstacles and Opportunities.” Waste Advantage Magazine, 1 September 2018, www.wasteadvantagemag.com/recovering-agricultural-plastics-obstacles-and-opportunities/

[2] Charles, Dan. “The Secret Life Of California's World-Class Strawberries.” NPR, NPR, 17 May 2012, www.npr.org/sections/thesalt/2012/05/17/152522900/the-secret-life-of-californias-world-class-strawberries

[3] Zhang, Mengmeng et al. “Measurements of single molecular affinity interactions between carbohydrate-binding modules and crystalline cellulose fibrils.” Physical chemistry chemical physics : PCCP vol. 15,17 (2013): 6508-15. doi:10.1039/c3cp51072g

Acknowledgements


We would like to thank our PI, Dr. David Bernick for providing us with guidance and advice throughout our project. Thank you to our TA, Ryan Modlin, who provided us with advice on our project and trained us to use the lab equipment. We are thankful for Dr. Husein Ajwa who provided us insight into fumigation practices and allowed us to visit his fumigation lab. We would like to thank Dave Peck and Mark Bolda for giving us a growers perspective to making our product. Thank you to all the stakeholders who took the time to talk to us and guided our project towards success. Finally, we would like to thank those who funded us through our crowdfunding and other avenues. Without all of you, we would not be able to perform our research.