Team:UCSC/Proof Of Concept

Proof of Concept

Building prototypes and experiments to prove the viability of our project!


Our team used prototypes and experiments to better understand the importance of decrystallizing cellulose to make a biodegradable bed mulch for agricultural practices. Due to restrictions with COVID-19, it was crucial to use time in the lab effectively. Biology is a time consuming process, possibly taking months to get good results. Chemistry can take from minutes to just a few days to see progress. With our time crunch in mind and chemical compounds that mimic biological experiments at our disposal, our team believed prototyping to be the best way to demonstrate the importance of amorphous cellulose to build our desired film.


Chemical Modeling to Demonstrate Project Efficacy

Our project aims to utilize select carbohydrate binding modules (CBMs) for their ability to bind cellulose, hopefully creating amorphous, or non-crystalline, cellulose. As we have not yet reached the stage of testing CBMs, we simulated the anticipated effects of CBMs decrystallization of cellulose using ionic liquids. Similarly to CBMs, ionic liquids are theorized to disrupt inter and intramolecular hydrogen bonding (through various methods expanded on below) resulting in decreased crystallinity and formation of amorphous cellulose. Our work with chemical modeling allowed us to create an applicable system to test various plasticizers, cross-linking agents, and copolymer inclusion with amorphous cellulose before we even expressed any CBMs. Once we get to the stage of testing CBMs with cellulose, we will have a short list of plasticizers, copolymers, and cross-linking agents on hand to include that have been proven to impart beneficial properties on our bacterial cellulose.


Microcrystalline Cellulose Versus Bacterial Cellulose

Since the production of bacterial cellulose (BC) from K. rhaeticus can take up to 10 days to grow, an alternate source of cellulose was used for testing during the early stages of our project [1]. Microcrystalline cellulose (MCC) is a readily available, refined source of cellulose, so many of our initial tests were conducted with this material. BC and MCC are both over 99% pure, but MCC has a degree of polymerization 10 times smaller than BC meaning the fibers of MCC are much shorter [2]. This results in longer regions of crystallinity within strands of BC and a higher crystallinity index [3]. Though our project focuses on decreasing the crystallinity of BC, MCC was an easily accessible source of cellulose that allowed us to expedite our research while modeling a less crystalline version of BC.


Cellulose Dissolution

The first step to making a cellulose-based plastic is the dissolution of highly crystalline cellulose fibrils. Cellulose fibrils are densely packed due to extensive inter and intramolecular hydrogen bonding making it difficult to integrate plasticizers, copolymers, and cross-linking agents. To create amorphous cellulose, we treated MCC with aqueous NaOH, ZnCl2, diisopropyl imidazolium, diisobutyl imidazolium, and BMIMCl in an attempt to separate or dissolve cellulose fibrils allowing for addition of plasticizing molecules. To achieve desired plastic-like qualities, it is necessary to fully dissociate fibrils for a homogenous incorporation of plasticizer.

Treatment of cellulose with NaOH, known as Mercerisation, causes cellulose fibrils to swell irreversibly as sodium hydroxide penetrates between fibrils. The sodium ions help to break the intermolecular bonds between the hydrogens and oxygens of cellulose monomers. This process doesn’t dissolve the cellulose, but does decrystallize it. We used this method to create amorphous cellulose to allow for plasticizer testing. We noticed little incorporation of plasticizers/cross linking agents using NaOH as a dissolution agent and began to explore the use of ionic liquids.

We continued to explore more methods of cellulose dissolution, and came across the use of Zinc Chloride hydrate. The Zn2+ and Cl- ions of ZnCl2 bind with the hydroxyl groups of cellulose, interrupting the hydrogen bonding between strands, causing cellulose to dissolve [4]. It is critical, however, that the Zinc Chloride Hydrate be at a hydration level of three (3 moles water to 1 moles Zinc Chloride) or dissolution cannot occur [5]. This method allowed us to create another version of amorphous cellulose to begin plasticizer testing. Because this amorphous cellulose was in a liquid-form, it was not viable for the next steps of plasticizer testing, as it did not provide properties we needed in our cellulose even after being subjected to the curing process. This discovery led us to look for more methods of cellulose dissolution that would result in a solid-amorphous cellulose, which would be more effective in developing a plastic that needs to be heated and cured.

Figure 1: Dissolution of cellulose with a zinc chloride hydrate[5].

Ionic liquids such as BMIMCl are very effective chemicals in the dissolution of biomass. The asymmetry of the BMIMCl molecule as well as the chloride ion aids in the interruption of the extensive cellulosic hydrogen bonding network and allows for attachment of chloride ions to hydroxyl groups of cellulose strands. This breaks intermolecular hydrogen bonds between cellulose strands and results in dissolution [6].

Diisopropyl Imidazolium (DIPI) should follow a similar mechanism to BMIMCl due to the presence of chloride ions in its molecular structure. However, DIPI is a more symmetric molecule so it may have a weaker effect than BMIMCl on the dissolution cellulose. Diisobutyl Imidazolium(DIBU) lacks chloride ions but has a bromide ion which mimics the chloride ion by binding hydroxyl hydrogens thus dissociating cellulose strands. Both DIPI and DIBU as ionic liquids resulted in a low dissolution of cellulose due to absence of asymmetry and low concentrations of chloride ions, which affected the ability of xanthan gum to fully integrate into the cellulose strands as a copolymer.



While experimenting with plasticization of MCC, we began to explore the important qualities of BC to understand the necessary modifications to create a film.

The next step was to develop assays to quantify and compare this product to plastics we hope to emulate. The basis of our project is to decrystallize cellulose in order to manipulate it to create elasticity and other desired qualities. We designed assays to understand the baseline measurement of crystallinity for our BC sample. We worked to produce a film by using cleaning and drying protocols [7]. For our first assay, we used X-ray diffraction to calculate the crystallinity of pure BC samples, with no plasticizer or CBM incorporporation, with the help of Jerah Barnett, a graduate student in the Scott Oliver lab at UCSC. We will use the crystallinity results of these pure BC samples for comparison with BC samples post CBM and plasticizer addition. Our pure BC sample tested to be around 76% crystallinity. We are aiming to decrease this percentage after the incorporation of a CBM and plasticizer, resulting in the formation of amorphous cellulose [8].

Expoly Green/TIF Raven™ Reflective Film Sample Average
Crystallinity Index 60% 65% 70% 69% 66%

Table 1: Crystallinity index results produced using an XDR machine for the bed-mulch films provided by Dr. Hussein Ajwa.

Crystallinity tests were performed using an X-Ray Diffraction (XDR) machine which produced graphs. Further information on these tests is on our Results page. The pure BC film sample served as a prototype that helped to conduct the crystallinity index assay, and will provide a reference for completion of future assays such as measurement of biodegradation rate and tensile strength. Completion of these assays will allow us to accurately compare our film to other commercially available agricultural films, to assure our product meets the standard growers are looking for.