Deforestation is one of the largest factors that play into the ongoing climate crisis.  Around 1 billion acres of forest, primarily in South America and Africa, have been lost since 1990, and deforestation rates continue to climb, increasing by 21% in 2020 (Nunez, 2019). Each year, around 3.5-7 billion trees are cut down for cattle ranching, agricultural reasons, and logging (Rainforest Action Network, 2017; Nunez, 2019). Deforestation has various devastating long-term effects, including loss of habitats, increasing greenhouse gasses, and the displacement of indigenous people living in those forests. 80% of land animals and plants live in forests, so a large number of living organisms are dislocated and endangered due to changing habitats (Zhang, 2017). Trees convert carbon dioxide to oxygen, though since many trees are being cut down, carbon dioxide is being converted to oxygen at a slower rate–leading to higher concentrations of carbon dioxide in the air (Nunez 2019). As more deforestation occurs, indigenous communities that live in those forests are losing the resources that they need to live, such as food and building materials (Pachamama Alliance, n.d.). The environmental and human rights complications of deforestation have made it imperative to greatly reduce the number of trees that are cut down to produce wood and paper products. 

Due to the aforementioned issues, there have been renewed efforts to recycle paper to reduce deforestation. To create paper, wood is first cut into wood chips that are placed into pulp digesters that break apart the cellulose fibers and other parts of the wood. Extra wood components are then removed so the cellulose fibers can be separated, cleaned, and eventually turned into paper (Idaho Forests, n.d.). Because paper is created from trees, it is very biodegradable (Indumathi et al., 2022). However, paper isn’t recycled as much as it could be, even though paper and paperboard (cardboard) are the most common municipal solid waste in the United States. In 2018, 67.4 million tons of paper and cardboard were created, but only 68% of paper and cardboard was recycled (United States Environmental Protection Agency, n.d.). While this recycling rate is the highest compared to previous years (just 15% in 1970, 42% in 2000, and 66% in 2017), 25.5% of paper and paperboard is still being thrown into landfills, which equals approximately 17220 tons of paper and paperboard being wasted annually (United States Environmental Protection Agency, n.d.). Current recycling efforts haven’t been able to address the high demand for paper products, which has prompted scientists to research ways to optimize current methods and seek more effective alternatives. 

          The Froth Flotation Deinking Process (FFDP) is the most common method of paper recycling used to separate paper from inks, toners, and wastes (Indumathi et al., 2022). Waste paper is first exposed to heat and flotation reagents, such as sodium peroxide, sodium silicate, sodium hydroxide, or hypochlorite, to repulp the paper into individual fibers and remove inks, toners, and wastes (Pornpaitoonsakul, 1979). Once the fibers and inks are separated, pressurized nonreactive gas is added to the bottom of the flotation device to create air bubbles. The flotation reagents make wastes removed from fibers more hydrophobic. Figure 1 shows that, due to their increased hydrophobicity, these wastes (referred to as “pigment” in the figure) are attracted to air bubbles that lift it up to the surface, while denser fibers sink deeper into the flotation device. A frothing agent (surfactants, frothers, and dispersants are commonly used) is added to the upper portion of the flotation device so that a froth layer containing various wastes is formed on the surface (Deng & Zhu. 1999). At the end of the FFDP, the froth layer, and the wastes accumulated, are removed separately from the fibers (Deng & Zhu, 1999). Using various chemicals makes this deinking process highly effective, but it also creates a large amount of chemical wastewater as a byproduct.

The FFDP is criticized by scientists due to its heavy usage of chemicals, which manifests itself as hazardous pollutants in the resulting effluent, wastewater that is formed after the FFDP. One way to measure effluent quality is to determine the chemical oxygen demand (COD) of the effluent, which is the amount of oxygen needed to oxidize organic water contaminants into inorganic products (Millipore Sigma, n.d.). The COD level of unpolluted water is around 20 mg/L or less, but the resulting effluent from paper mills using recycled paper fiber ranges from 1500 to 1900 mg/L (Jain & Singh, 2003; Thompson et al., 2001). If exposed to the environment, marine life will face the brunt of effluent pollution. According to Thompson et al.’s review article, fish exposed to Kraft process (turning wood into wood chips) effluent in the 1980s and 1990s had liver problems, delayed sexual maturity, and changes in reproduction (2001). If bodies of water are exposed to pollutants, it can stimulate plant and algae growth, which can result in reduced oxygen levels and kill other organisms that need the oxygen (Denchak, 2023). However, the FFDP’s heavy usage of chemicals isn’t the only reason why it’s bad for the environment: some contaminants found in paper pulp can make it harder to recycle paper.

In the recycling industry, the presence of hydrophobic contaminants in recycled paper pulp can hinder paper creation processes. Hydrophobic contaminants are toxic non-polar molecules that have long environmental half-lives (Kanu & Anyanwu, 2005, p. 212). Scientists report that hydrophobic contaminants are dangerous for all organisms, since exposure can lead to certain types of human cancers and neurological diseases(Kanu & Anyanwu, 2005, p. 214 & 216). Hydrophobic contaminants are often waxes and plastics that can be found in paper used to create address labels, stamps, and envelopes (U.S. Department of Energy, 2016). These contaminants in paper pulp effluent form deposits on paper-making machines, which slows down paper-making and reduces paper quality (U.S. Department of Energy, 2016). Effectively removing hydrophobic contaminants from paper pulp can possibly increase paper quality, reduce the amount of paper that is rejected from recycling due to contamination, and allow for more paper deinking cycles (U.S. Department of Energy, 2016). However, the FFDP isn’t the most effective at removing contaminants in certain situations. 

Inks and toners are difficult to remove using the FFDP especially when a printer uses thermosetting toners. Thermosetting toners physically bind ink to paper fibers under high heat, making it hard to separate inks from fibers (Pathak et al., 2011). The FFDP requires ink and other contaminants to be hydrophobic. However, if the inks are trapped between paper fibers, they won’t gravitate towards the air bubbles and will sink to the bottom trapped within the paper fibers (Tsatsis et al., 2017). Because of the polluted effluent and limited deinking associated with the FFDP, scientists have been looking into alternative recycling methods that are more environmentally safe and cheaper, but are still effective at removing inks and toners from paper. One exciting advancement in paper recycling has been the optimization of deinking enzymes, which reduce and potentially eliminate harmful chemicals in favor of safer biological alternatives.

Recently, researchers have been exploring alternative paper recycling methods based on the chemical makeup of wood. Wood, which is used to create paper, is comprised of lignocellulose, which is made up of the polysaccharides cellulose, hemi-cellulose, and lignin (Nakarmi et al., 2022; Chen, 2015). Cellulose are glucose molecules linked by β-1-4 glycosidic bonds, hemi-cellulose is comprised of pentose or hexose sugars (sugars with 5 or 6 carbon atoms), and lignin is formed with three phenolic compounds: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Nature, n.d.; Westin, n.d.). As shown in Figure 2, these three lignocellulosic components form structures called microfibrils, with cellulose making up the majority of the microfibril and hemi-cellulose and lignin enclosing the cellulose in a cylindrical shape. The microfibrils then assemble themselves into macrofibrils, which are the structural basis for cell walls in plant cells (Nature, n.d.). When creating paper from wood, some lignin is removed through chemical pulping to allow the separation of cellulose and hemicellulose fibers required to create paper (Holtzapple et al., 2003). To break apart cellulose and hemi-cellulose fibers during the recycling process, the FFDP targets utilizes acid-hydrolysis to split apart the hydrogen bonds that link cellulose and hemi-cellulose (Ricardo Soccol et al., 2011; Zoghlami & Paes, 2019). Though, as previously established, the usage of chemicals in the FFDP leads to the production of toxic effluent, which is bad for the environment. Due to paper’s chemical makeup of lignocellulosic materials, researchers have instead been exploring the use of lignocellulose-degrading enzymes to deink waste paper. 

Enzymes are made up of amino acids, and act as catalysts for specific biochemical reactions. Without enzymes, some chemical reactions would either not occur or occur at a very slow rate (About Enzymes, n.d.). All organisms are dependent on enzyme catalyzed reactions to carry out bodily functions, so enzymes are necessary to sustain life (About Enzymes, n.d.; Cooper, 2000). Enzymes function at low temperatures and moderate pH because they form in living organisms, which have evolved to function in generally mild conditions (Sánchez López de Nava & Raja, 2023; Cooper, 2000). Lignocellulose-degrading enzymes, which mainly consist of cellulases, hemi-cellulases, and ligninases, use enzymatic hydrolysis and depolymerization to weaken cellulose and hemicellulose hydrogen bonds, making it easier to separate fibers and remove paper contaminants. Enzymatic hydrolysis can also lead to the removal, also known as “peeling,” of fibrils, aiding in contaminant removal (Chandra & Madakka, 2019; Basu, 2018; Tsatsis et al., 2017). A benefit in using enzymatic hydrolysis for deinking paper is the considerable reduction of the chemical waste that is produced. 

Studies have found that enzymatic deinking leads to lower COD levels in resulting effluent. COD levels can be found through conducting a laboratory assay where effluent samples are incubated with a strong chemical oxidant at usually 150 °C for 2 hours (Proteus, n.d.). Singh et al. conducted an experiment where they compared the different properties of paper and effluent after enzymatic deinking (with the enzymes xylanase and pectinase) and chemical deinking (with the chemicals NaOH, Na2SiO3, MgSO4, EDTA, and H2O2) (2012). The researchers found that the effluent produced from enzymatic deinking had COD levels of 1056 mg/L, while effluent produced from chemical deinking reached COD levels of over three times higher than enzymatic deinking at 3750 mg/L (Singh et al., 2012). Other studies have confirmed these results: Lee et al. conducted a similar experiment, which compared the chemical properties of the resulting effluent after enzymatic deinking (mixture of the enzymes xylanase and cellulase extracted from a local Aspergillus niger fungus isolate) and chemical deinking (using NaOH and sodium silicate) and found that enzymatic deinking led to a 33.8% reduction in the effluent’s COD levels (2011). Although these improvements in COD levels are great, there have been mixed results regarding the brightness of paper after chemical deinking compared to enzymatic deinking. 

Currently, it is unclear if chemical deinking or enzymatic deinking is more effective at removing inks and toners, which is often measured by the brightness of paper pulp. Brightness is measured using a spectrophotometer set to a wavelength of 457 nm. In Singh et al.’s experimentation, they found that chemical deinking led to an average paper pulp brightness of 83.68%, while enzymatic deinking was only 78.80% (2012). In contrast, researchers Lee et al. found comparable paper pulp brightness between enzymatic and chemical deinking, the average paper brightness being 83.6% and 83.1%, respectively (2011). However, Lee et al. did not chemically deink with hydrogen peroxide, a very well-known bleaching agent that is often used in chemical deinking (Bajpai, 2018). Thus, there is a possibility that if Lee et al. used hydrogen peroxide as a bleaching agent, the brightness could’ve been significantly higher than the enzymatic deinking. Due to these mixed results, paper and pulp industries are more likely to rely on chemical means to deink paper  as it’s more consistent. Therefore, more research is needed to discover, isolate, and optimize naturally occurring enzymes from bacteria and fungi that have equal or greater deinking efficiency to chemical deinking methods. 

Enzymatic deinking has been documented to have a lesser environmental impact than chemical deinking, but it isn’t yet known which specific strains of bacteria and fungi produce enzymes that are the most effective at deinking waste paper. However, through extensive field research, enzymes from various organisms should be extracted and the catalytic activity of enzymes should be examined (LibreTexts Chemistry, n.d.). Specifically, researchers should use Lineweaver-Burk Plots to identify which bacteria and fungi produce lignocellulose-degrading enzymes with high Vmax (maximum rate of reaction under substrate saturation) values for more effective deinking (Effect of Substrate, 2011). Enzymes isolated from bacteria and fungi should be optimized by finding the conditions needed for optimal enzyme activity, including pH, temperature, treatment time, enzyme dosage, and enzyme strain. Currently, researchers are particularly interested in isolating and optimizing xylanases that are extracted from fungi. 

Xylanase is one of the main classes of enzymes that is currently being analyzed by researchers. While xylanase can be produced from bacteria, algae, and insects, fungi produces xylanase at a much quicker rate than any other sources, and the xylanase produced generally has higher enzymatic activity (Polizeli et al., 2005). Xylanase is extensively used in the animal feed and baking industry (Collins et al., 2005). However, it is now being pursued for its applications in the pulp and paper industries. Xylan is one of the main components of hemicellulose, which is present in paper (Polizeli et al., 2005). Xylanase catalyzes the hydrolysis of xylan to xylose, which in turn hydrolyzes hemicellulose thus weakening the hydrogen bonds between cellulose and hemicellulose in paper.  This selective degradation allows ink to be released and paper more easily recycled (Polizeli et al., 2005; Kumar et al., 2014, p. 1; Bajpai, 2009, p. 1; Tsatsis et al., 2017).  Therefore, xylanases from filamentous fungi have great potential as possible deinking agents. 

Knowing this information, Kumar et al. conducted research on optimizing xylanase extracted from the filamentous fungus, Trichoderma viride VKF3. In their scientific article, Kumar et al. focused on optimizing this specific type of xylanase for deinking purposes by testing various paper pulp concentrations and enzyme doses to see which combination is most effective (2014). After deinking the waste paper, the researchers analyzed the contents of the resulting effluent (ie. the pH, salinity, sodium, calcium, and potassium levels) to determine the toxicity and environmental effects of the effluent (Kumar et al., 2014, p. 59). Overall, the researchers found that the chemical properties of the effluent after xylanase treatments were in compliance with India’s environmental codes for industrial effluent (Kumar et al., 2017, p. 57). However, the main focus of my replication of Kumar et al.’s scientific article will be to measure the amount of hydrophobic contaminants in the effluent after enzymatic deinking using UV-Vis spectrometry.

In UV-Vis spectrometry, the amount of a chemical compound dissolved in solution can be determined from electronic transitions that occur when conjugated systems are exposed to certain wavelengths of UV light (Soderberg, n.d.). A spectrophotometer is a machine that measures the intensity of light absorbed, which can be used to calculate the concentration of a certain chemical substance in the solution (Vo, n.d.). The electrons of a molecule at ground state start at the Highest Occupied Molecular Orbital (HOMO). After the end of an electronic transition, the electron will have jumped to the Lowest Unoccupied Molecular Orbital (LUMO). When a molecule is exposed to light with a wavelength equal in energy to the HOMO-LUMO energy gap, the wavelength of the light will be absorbed and the electron will jump to the LUMO (Soderberg, n.d.). Thus, the difference in energy between the HOMO and the LUMO determines the wavelength that a conjugated system needs to absorb for the electron transition (Soderberg, n.d.). To find hydrophobic contaminants in treated effluent, a UV wavelength of 465 nm was used, as the conjugated systems in hydrophobic contaminants uniquely utilize this wavelength of light for their HOMO-LUMO transition  (Kumar et al., 2017, p. 59). 

UV-Vis spectroscopy is a method that has been used by researchers to analyze the inks and toners removed from paper pulp. Specifically, researchers look for characteristic “peaks” on a UV spectra that indicates certain compounds have been released. For example, Singh et al. used a spectrophotometer set to 260 nm and 280 nm, to measure the peak in absorbance corresponding to lignin-related contents that were released into the effluent (Singh et al., 2019). On the other hand, Xu et al., who studied the deinking efficiency of enzymes in tandem with acid hydrolysis, measured UV spectra for different enzymatic and chemical deinking combinations. These researchers compared the UV spectra to each other by analyzing different signals at 205 nm and 280 nm, which corresponds to the release of lignin-related contents (2009). Kumar et al. used a similar approach leveraging spectroscopy to determine the amount of hydrophobic compounds released into effluent after varying xylanase doses and paper pulp consistencies (2014). After reviewing the literature, UV-Vis spectroscopy is an effective method utilized by researchers to affirm that certain contaminants have been released into the effluent after deinking paper.

First, I attempted to replicate Kumar et al.’s experiment on the effects of recycling paper pulp with varying xylanase doses and measuring the amount of hydrophobic contaminants released into the effluent. Kumar et al. hypothesized that a higher xylanase dose should lead to more hydrophobic contaminants being released because more xylanase is available to break apart the paper fibers, which releases more ink and hydrophobic contaminants. For my replicate study, I kept the pulp consistency at 5% for all experiments and analyzed the difference between the hydrophobic contaminants released into effluent after a no enzyme treatment and a 5% xylanase treatment. With these parameters, Kumar et al. found that paper pulp treated with no enzyme had an absorbance at 465 nm of around 0.04, while effluent that resulted from paper pulp treated with 5% xylanase had an absorbance of around 0.12 (Kumar et al., 2017, p. 57). These results indicate that enzymatic treatment trials should lead to a higher absorbance than no enzyme trials. This replicate study helped validate the methods that I used using for future experimentation. In future experimentation, I analyzed wastepaper deinking of cellulase and cutinase in synergy. 

Cellulase is one of the most studied enzymes in the field of wastepaper deinking. Cellulases are enzymes that catalyze cellulolysis, the degradation of cellulose. Cellulase breaks down cellulose polysaccharides by hydrolyzing β-1,4-linkages that breaks down the cellulose into glucose monosaccharides (Lakhundi et al., 2015; Ophardt, n.d.). Cellulases are produced by a wide variety of bacteria and fungi, and are applicable in the paper and pulp, textile, laundry, feed, brewing, and agriculture industry (Kuhad et al., 2011). Cellulase is especially important in the paper and pulp industry, as paper is mostly made of cellulose. Past research has shown that cellulase improves paper brightness and strength properties (Singh et al., 2016). 

Cutinase, on the other hand, is a very understudied enzyme in the wastepaper deinking field. Cutinase catalyzes the hydrolysis of ester bonds between cutin and is capable of degrading the plant cell wall (Chen et al., 2008). Cutinase is produced from fungi and bacteria (Chen et al., 2008). Cutinase is often used in the food, cosmetics, pesticide, textiles, and plastic recycling industries (Pio & Macedo, 2009; Furukawa et al., 2019). While cutinase is understudied in the paper and pulp industry, researchers Hong et al. argued that cutinase should be able to degrade the carbon-oxygen bonds (also known as ester bonds) that links the pigments and resins in the ink solvent together, releasing the pigments off of the paper’s surface (2017). The researchers were using brightness as a measure of how well cutinase deinked paper and found that paper treated by the cutinases resulted in a brightness at least 4% higher than the paper treated by the chemical deinking method (Hong et al., 2017). This research indicates that cutinase has potential as a deinking agent.  

Given the consistent deinking ability of cellulase and the potential of cutinase, I sought to answer the following experimental question: can cellulase and cutinase be used in synergy to increase removal of inks and toners from paper pulp? I hypothesized that cellulase and cutinase can be used in synergy because the cellulase would degrade the cellulose in the paper fibers and the cutinase would break down the ester bonds connecting the pigments and resin inside of the ink particles. I conducted one negative control where paper pulp was treated with just distilled water, an experimental arm where paper pulp was treated with a 37.5% concentration of cellulase, another experimental arm where paper pulp was treated with a 37.5% concentration of cutinase, and a final experimental arm where paper pulp was treated with 18.75% concentration of cellulase and cutinase each. 

Large-scale enzymatic deinking of waste paper can greatly assist in alleviating the deforestation problem. Even though there are efforts to increase recycling initiatives, there were still approximately 17220 million tons of waste paper discarded into landfills (United States Environmental Protection Agency, n.d.). Moreover, current recycling methods can be incredibly harmful to ocean ecosystems if they are exposed to the resulting effluent, due to the high COD levels present (Thompson et al., 2001; Denchak, 2023). Instead of promoting current recycling practices that have detrimental effects on the environment, resources should be put into future studies that seek greener alternatives that leverate enzymes to sustainably deink waste paper. 

Overview

The replicate study, based on Nathan et al.’s research, investigates the release of hydrophobic compounds into effluent after deinking newspaper using xylanase (2014). Nathan et al. were able to test the efficacy (through analyzing the physio-chemical properties and release of hydrophobic and phenolic compounds into the effluent) of commercial xylanase and xylanase extracted from the fungus Trichoderma viride VKF3 in deinking newspaper. However, due to time and budget constraints, this study could only find the hydrophobic compounds released into the effluent by baking-grade xylanase sold by enzymes.bio (CAS Number: 9025-57-4). This senior study will branch away from deinking paper pulp using xylanase and study the deinking capacity of cellulase and cutinase-hydrolase enzymes alone and in synergy with each other. It is hypothesized that the combination of both enzymes will increase the release of ink into the effluent, measured using UV Vis Spectrophotometry because cellulase would break down cellulose in the paper fibers while the cutinase would degrade the ester bonds that connect the pigments and resins inside of ink particles themselves (Structure of Lignocellulose, n.d.; Hong et al., 2017).. 

Safety Protocol

Personal protective equipment (lab coat, nitrile gloves, safety goggles) should be worn during experimentation to prevent contamination of paper pulp samples and protect against irritation that may occur through the exposure of xylanase (MSDS), cellulase (MSDS), and cutinase (MSDS). Heat gloves should be worn alongside personal protective equipment when working with the Bio-Rad Temperature Controlled Water Bath (Model 1660504EDU) and any materials heated inside of the water bath. 

Replicate Study

The replicate study analyzed the deinking capacity of xylanase by treating newspaper pulp with a 5% xylanase dose and a 10% xylanase dose. All arms of the experiment contained nine experimental replicates. The negative control measured the release of hydrophobic contaminants after no xylanase treatment. This data was compared to data collected in Experimental Arms #1 and #2 (5% and 10% xylanase respectively) to see if xylanase was effective at breaking apart paper fibers and releasing more hydrophobic contaminants than distilled water alone. The absorbance of the resulting effluent was collected at 465 nm to find the amount of hydrophobic compounds released from the paper pulp due to the xylanase treatment. 

Paper Pulp Creation

First, approximately 40 pages of newspaper were shredded using a Bonsaii paper shredder (Model #Bonsaii18CC) and further cut into smaller (around 1 cm) segments using a pair of scissors. The paper was placed into a 13 ⅝ in. x 8 ¼ in. x 4 ⅞ in. Sterilite 6 quart plastic storage bin. The storage bin was filled halfway with warm tap water and soaked for 10 hours. The paper was blended to a slurry the following day using a Hamilton Beach Blender (Model #58148A) at setting “3” until the paper was blended into a uniform gray mush with no visible paper segments. Mondal et al., who tested xylanase and pectinase from Aspergillus niger SKN1 to deink mixed office paper, used a home grinder to blend their paper mush, so a Hamilton Beach Blender was deemed suitable for this application (2022). Afterward, the macerated paper mush was placed into a Hamilton Beach Food Dehydrator (Model #32100), where it was evenly spread across the sheets, and dried at 50°C for 6 hours. This same temperature and drying time was used by Soni et al., who optimized cellulase from Aspergillus fumigatus for deinking wastepaper (2010). If the paper pulp was still damp after drying, the paper pulp was dried in the dehydrator at the same temperature for 4 more hours. The paper pulp should be placed at least an inch away from the middle of the dehydrator because, in past experimentation, some paper pulp near the middle was discolored and burned. After drying, the paper pulp will be clumped together (due to the way it was placed into the dehydrator), so it should be placed back into the Hamilton Beach Blender and blended at setting “3” to separate the dried paper fibers from each other. The resulting paper pulp should be soft and individual fibers should be visible. Once blended, the paper pulp should be stored in 16-ounce plastic containers when not in use. 

Sample Creation and Enzyme Dosage

In the replicate study, the paper pulp samples were at a 5% (w/v) pulp concentration: 100 mL of distilled water, measured using a 100 mL graduated cylinder, and 5 grams of paper pulp, measured using a Flinn Scientific Electronic Scale (Model #OB2090). The distilled water and paper pulp were placed into a 250 mL flask. The paper pulp was very light and didn’t sink to the bottom of the flask, so a laboratory spatula was used to push it down so it was completely submerged. During the experimental arms, 5 grams (if conducting Experimental Arm #1) or 10 grams (if conducting Experimental Arm #2) of xylanase were added to each paper pulp sample. The xylanase was weighed using a 5.51" x 5.51" x 0.87" weighing boat placed on top of a Flinn Scientific Electronic Scale (Model #OB2090). Since baking-grade xylanase (enzymes.bio; CAS Number: 9025-57-4) is very powdery and lightweight, a scoopula was used to pick up small amounts which were added directly to the paper pulp sample. Xylanase was scraped off the weighing boat with a scoopula as needed. The sample was stirred with a glass stirring rod for thirty seconds to ensure the xylanase was adequately mixed with the paper pulp. Once stirred, the flasks containing samples of the paper pulp mixtures were covered with aluminum foil.

When first conducting these experiments, the time that paper pulp was submerged in distilled water was not accounted for. However, the difference in time was a confounding variable that led to varying hydrophobic contaminant concentrations in the effluent. Paper pulp samples submerged in water for a longer time had higher absorbance values (higher hydrophobic contaminant concentrations) in the effluent, and the reverse was true as well. Therefore, all paper pulp samples were submerged in water overnight for 16-24 hours to eliminate time as a confounding variable and ensure more consistent absorbance values. Incubation would occur the following day.   

Incubation and Boiling Water Bath

Right before incubation, fill half of the Bio-Rad Temperature Controlled Water Bath (Item #1660504EDU) with water and set the temperature to 100°C, as the paper samples will need to be heat inactivated in boiling water to deactivate the xylanase (LibreTexts, n.d.). In the replicate study, the paper pulp samples inside of the 250 mL flasks were covered with aluminum foil and placed into OHAUS 250 mL Flask Clamps (Item#217274) screwed into a Flinn Scientific Shaking Incubator (Model #17002945EDU).  The incubation ran for one hour, which is the optimal length of time documented by Nathan et al. and Indumathi et al.’s research (2014; 2021). The temperature should be set to 60°C because the published optimal temperature of baking-grade xylanase is between 50°C to 65°C (Xylanase Enzyme, n.d.). Tsasis et al. used a container equipped with a stirrer to keep the paper pulp samples mixed during enzymatic treatment (2017). While this container wasn’t available, a shaking incubator set to 200 RPM (rotations per minute) was used instead to ensure consistent mixing. 

After incubation, put on heat gloves and place the flasks containing the paper pulp samples into the water bath for 15 minutes. The 250 mL flasks were weighed down with 150 mL beakers placed on top of them to ensure they didn’t move during the boiling water bath. Researchers Kumar and Dutt, who compared the physical properties of mixed office paper after enzymatic deinking and chemical deinking, did a 15-minute boiling water bath to deactivate their enzymes, so a 15-minute water bath was deemed suitable (2021). After 15 minutes, the paper pulp samples were removed from the water bath and cooled to room temperature on a countertop for five minutes. 

Filtration 

Vacuum filtration apparati were set up by placing a 9 cm Büchner funnel and a rubber filter adapter onto a 500 mL Büchner flask. A 9 cm diameter filter paper was placed into a Büchner funnel and wetted with distilled water so that the filter paper adhered to the funnel. A vacuum filtration apparatus was made for each paper pulp sample and labeled using marking tape to match the corresponding label on the paper pulp sample. The paper pulp samples were poured into their respective filtration apparati. The VIVOHOME Rotary Vane Vacuum Pump (Model #‎VH409-RE) was attached to a filtration apparatus by twisting the rubber tubing onto the handle of the Buchner flask. The VIVOHOME Vacuum Pump needs to be filled with a minimum of 220 mL of oil and a max of 310 mL of oil to function (VIVOHOME Air Vacuum, n.d.). The vacuum pump was turned on for 10 seconds as that was a sufficient amount of time to filter out effluent from the paper pulp samples. Leaving the vacuum pump on for longer increased the chance that effluent was sucked into the pump. After all of the effluent was filtered from the paper pulp, the vacuum pump was turned off and the rubber tubing was carefully twisted off of the handle to prevent it from breaking. This process was repeated for all of the remaining paper pulp samples. 

12 mL conical centrifuge test tubes were labeled with the same name given to the paper pulp and blank samples using washing tape and a sharpie. A DLAB P-1000 Micropipette (PIPET-DL1000) with 1000 μL United Micropipet Tips was set to 1000 μL and 1 mL of filtered effluent was transferred from the vacuum flask to its corresponding test tube ten times for a total of 10 mL of effluent. The effluent is often cloudy after being treated with xylanase, so all of the test tubes were centrifuged (Premiere XC-1000 Centrifuge; Model#XC-1000) for 5 minutes at 4000 RPM. This allows any insoluble compounds that pass through the vacuum filtration apparatus to accumulate at the bottom of the test tube leaving clear supernatant at the top. 

A 0.45 μm syringe filter was placed onto the end of a 5 mL disposable syringe, and a fresh 12 mL test tube (labeled with the same name given to the paper samples) was placed under the syringe in a test tube rack. Using a 3 mL disposable micropipette, the supernatant from one of the effluent samples was placed into the syringe. The supernatant was filtered through the syringe filter by pressing on the plunger of the syringe. If the syringe filter got clogged, the syringe was flipped upside down and the syringe filter was replaced with a new one. Syringe filtration removes any xylanase that may have remained in the supernatant after centrifugation. This is beneficial because insoluble products (including xylanase) was  large confounding variable during experimentation. All of the filtered effluent samples were then stored in a 4°C refrigerator until data collection. 

Data Collection

The Spectronic 200 Spectrophotometer (Model#840-281700) was set to 465 nm, as Nathan et al. and Indumathi et al. used this wavelength to see how much light was absorbed by the effluent, which corresponds to the concentration of hydrophobic contaminants in the resulting effluent (2014; 2021; Vo, n.d.). Singh et al. and Xu et al. used spectrophotometry to quantify lignin-related contents released into effluent but used a higher energy range of 205 nm to 280 nm (2012; 2009). Unfortunately, the spectrophotometer available can’t emit wavelengths below 340 nm. Therefore, only data on hydrophobic contaminants at 465 nm were collected. A 3 mL disposable micropipette was used to dispense distilled water into a cuvette and the distilled water sample was placed in the spectrophotometer as a blank. The spectrophotometer was zeroed by pressing the ‘0.00’ button, which calibrated the spectrophotometer. Once calibrated, disposable micropipettes were used to dispense effluent from the paper samples into separate cuvettes. The cuvettes were then placed into the spectrophotometer and the absorbance of each paper pulp samples’ effluent was recorded in a data table. 

Data Analysis 

All effluent absorbance values were inputted into a Google Sheet that performed standard deviation and mean value calculations. A two-tailed independent t-test was also conducted on Google Sheet to determine if there was a statistical difference between the no wastewater treatment and the enzymatic treatments. There was also a two-tailed independent t-test conducted between the 5% xylanase treated samples and the 10% xylanase treated samples to see if the difference in enzyme dosage made a difference in deinking efficiency. If the P-value is below 0.05, this indicates that the data is statistically different and there is a significant difference in the release of hydrophobic compounds between the negative and experimental arms being compared. 

Senior Study

The senior study will analyze the deinking capacity of cellulase and cutinase by treating used copy paper pulp with cellulase (enzymes.bio; Model#SC60), cutinase (enzymes.bio; CAS number: 9014-01-1), and both enzymes to see if there is a synergistic effect. All arms of the experiment contained nine experimental replicates. The negative control recorded data on the release of inks and toners after no enzymatic treatment. This data was compared to data collected in the experimental arms to see if the enzymes were effective at breaking apart paper fibers and releasing inks and toners. In each of the three experimental arms, used copy paper with blue ballpoint pen ink was treated with: 3 mL of cellulase, 3 mL of cutinase, and 1.5 mL of both enzymes in the final experimental arm. A full scan of the absorbance wavelengths (400 nm to 900 nm) was conducted on the effluent to detect diagnostic absorbance peaks that can be used to analyze the amount of inks released from the paper pulp.

Paper Pulp Creation

Most of the paper pulp creation procedure from the replicate study remained the same. However, the source of used paper was changed for the senior study. Lignocellulosic peaks are observed between 205-280 nm (Xu et al., 2009), but the spectrophotometer available (Spectronic 200 Spectrophotometer; Model#840-281700) does not emit wavelengths lower than 340 nm. Therefore, it was difficult to find information on the lignin-related contents that were released into the effluent. Singh et al. experimented with different chemical and enzymatic treatments using copy paper with blue ballpoint pen ink, and found that after the release of blue ballpoint pen ink, there were unique absorbance peaks at 575 nm (2012). Furthermore, Kumar and Sharma found that most blue ballpoint pen ink has absorbance peaks between 580-590 nm due to the presence of the dye crystal violet (2017). Additionally, Lee et al. found that enzymatic deinking may not be effective for removing inks and toners from newspaper waste as newspaper is commonly printed with flexographic ink, which is strongly attached to paper fibers and difficult to separate (2013). Therefore, for the senior study, BIC Round Stic Xtra Comfort Blue Ballpoint pens (Item#GSMG144E-BLU) were used to create 44 uniform lines on both sides of 5 pages of Quill Brand 8.5" x 11" Multipurpose Copy Paper (Model#720700). After making the marks on the copy paper, the soaking, blending, and drying process was the exact same as in the replicate study. Refer to Paper Pulp Creation in the Replication Study section for more information. 

Sample Creation and Enzyme Dosage

Similar to the replicate study, the paper pulp samples were also at a 5% (w/v) pulp concentration. However, the paper pulp samples were much smaller in size: 5 mL of distilled water, measured using a 25 mL graduated cylinder, and 0.25 grams of paper pulp, measured using a Flinn Scientific Electronic Scale (Model #OB2090). This smaller sample size saved time during experimentation and was easier to handle than the larger samples. Instead of a 250 mL flask, the paper pulp samples were placed into 12 mL conical centrifuge test tubes. In the replicate study, first the paper pulp and distilled water were kept in the test tubes overnight and the enzyme was added to the sample afterward. However, Singh et al. mixed their liquid medium (containing other chemicals) and the enzyme solution together before adding in paper pulp to ensure that the enzyme would be properly distributed across the paper pulp (2012). Therefore, for the experimental arms of the senior study, the distilled water and enzyme solution were added first, mixed thoroughly with a glass stirring rod for thirty seconds, and the paper pulp was added afterward. For all arms of the experiment (including the negative control), incubation occurred right after sample creation, rather than the following day. 

For the senior study, I transitioned to deinking paper pulp using liquid enzyme rather than powder enzyme (like the xylanase used in the replicate study) because the powder xylanase from the replicate study didn’t fully dissolve into the distilled water during incubation, meaning that the xylanase may not have been evenly distributed or deinked the paper pulp to its fullest extent. There are four arms of experiment in the senior study: one negative control where paper pulp will only be treated with distilled water, the next experimental arm will test the deinking capacity of 3 mL of cellulase, another will test the deinking capacity of 3 mL of cutinase, and the final experimental arm will test the deinking capacity of 1.5 mL of cellulase and 1.5 mL of cutinase. First, 5 mL of distilled water was added to one test tube followed by 3 mL of enzyme depending on what experimental arm was occurring. In the experimental arms with just cellulase or cutinase, a DLAB P-1000 Micropipette (PIPET-DL1000) with 1000 μL United Micropipet Tips was set to 1000 μL and transferred 3 mL of either enzyme into its corresponding test tube three times for a total of 3 mL of enzyme per sample. For the experimental arm with both enzymes, the micropipette was set to 1000 μL to add 1 mL of cellulase and then set to 500 μL to add another 0.5 mL of cellulase (adding a total of 1.5 mL of cellulase). The same process was repeated for cutinase. 

For the experimental arms of the senior study, one “blank” sample of just enzyme solution and distilled water (no paper pulp) was created. These samples were created because the enzyme solution is colored and, when added to the paper pulp samples, increases the absorbance due to extra pigmented substances in the solution. Therefore, these “blank” enzyme samples were used to zero the spectrophotometer and subtract the absorbance contributed by the enzyme solution

Incubation and Boiling Water Bath

Due to the change in sample size and materials, the test tubes containing the samples were placed into the shaking incubator in 250 mL beakers with a paper towel. Each beaker should contain a maximum of five test tubes that are sandwiched in between the paper towel to ensure that they do not move while shaking. The optimal temperature ranges of cutinase and cellulase are 45-65℃ and 35-55℃ respectively, so all of samples were heated to 50°C (Cutinase Hydrolase, n.d.; Cellulase Enzyme, n.d.). Because the test tubes were much smaller than the erlenmeyer flasks and have less space to mix, the shaking incubator was increased to 250 RPM from 200 RPM. 

After incubation, a test tube rack was placed inside of the boiling water bath to keep the test tubes standing up. The test tubes and rack take up less volume than the 250 mL flasks, so the boiling water bath should be filled ¾ with tap water, rather than half-full like in the replicate study. The paper pulp samples were left in the boiling water bath for 15 minutes (same as the replicate study). Afterward, the test tubes were placed on the countertop to cool down for 5 minutes.

Filtration

The paper pulp samples were very small, so it would be a waste of time to do vacuum filtration to remove a small amount of effluent. It is also possible to lose effluent with a large filtration apparatus through the effluent being absorbed by the filter paper. Centrifugation was utilized instead. All paper pulp samples remained in their test tubes and were centrifuged at 4000 RPM using a Premiere XC-1000 Centrifuge (Model#XC-1000) for 5 minutes allowing the effluent to rise to the top while the paper pulp and insoluble substances accumulated at the bottom. The syringe filtration process from Filtration in the Replicate Study Section was repeated to filter out paper fibers and any insoluble compounds. The filtered effluent samples were stored in a 4°C refrigerator until data collection. 

Data Collection

In the replicate study, the effluent that resulted from the paper pulp samples was analyzed at 465 nm to measure the amount of hydrophobic contaminants released after the xylanase treatment.. Due to the new paper pulp creation method with blue ballpoint pen ink, there should be a peak at around 580-590 nm (Kumar & Sharma, 2017). Therefore, a spectrophotometric scan from 400 nm to 900 nm was conducted on each effluent sample, and the absorbance of the tallest peaks (particularly those near 580-590 nm) were collected in a data table. During experimentation, a consistent peak was found at 601 nm. Also, rather than using a distilled water blank for the experimental arms (as was done in the replicate study), the enzyme “blank” samples for each experimental arm were used to zero the spectrophotometer.  This ensures that the absorbance accurately reflects the inks and toners removed from the paper pulp (though distilled water was still used as a blank for the negative control, which had no enzyme added). 

Data Analysis 

All absorbance values of the paper pulp samples were inputted into a Google Sheet that performed standard deviation and mean value calculations. A two-tailed independent t-test was also conducted on Google Sheets to determine if there was a statistical difference between no wastewater treatment and each of the enzymatic treatments, and the experimental arms with each other. If the P-value is below 0.05, this indicates that the data is statistically different and there is a significant difference in the release of inks and toners between the negative control and the experimental arms. The negative control is expected to be significantly lower than all of the other experimental arms. The cellulase and cutinase alone experimental arms is expected to be significantly higher than the negative control, lower than the mixed enzyme experimental arm, and can go either way with each other. The mixed enzyme experimental arm is expected to be significantly higher than the other three experimental arms.

RESULTS

Deinking Newspaper with Powdered Xylanase

Preliminary data collection was required to optimize the experimental methods in order to decrease experimental error, save materials, and increase effectiveness. When first deinking paper pulp with powdered xylanase for the negative control, the absorbance correlated with how long the paper pulp was submerged in distilled water. The mean absorbance of the samples submerged in distilled water for 1 hour was 0.07, while the mean absorbance of the samples submerged in distilled water for 16-24 hours was 0.22. The longer the paper pulp was submerged in distilled water, the higher the absorbance. One sample submerged in distilled water for one week led to an even higher absorbance of 0.42. However, analyzing this trend further was abandoned due to timeline constraints. A two-tailed independent t-test was conducted to compare samples submerged in distilled water for 1 hour and overnight (16-24 hours). This statistical test showed a significant difference (p = 0.0029, p < .05) in absorbance with higher absorbance values being detected for longer submersion times. Therefore, when conducting experiments with powdered xylanase, all paper pulp samples were submerged in distilled water for 16-24 hours before experimentation to fit better with an SRD schedule and reduce sample variation.

After conducting preliminary experimental trials with the powdered xylanase, it seemed that the xylanase wasn’t sufficiently filtered out with the ToyBasics Lab Vacuum Filtration Pump (X002LOW46H) as the wastewater produced was very opaque and murky. Centrifugation was used to gather paper pulp at the bottom and push clarified supernatant to the top of the test tube. The supernatant was used to collect absorbance data, but the xylanase was still present in varying amounts. This caused a systematic error in absorbance because the spectrophotometer would detect the variable amounts of insoluble xylanase in the absorbance. 

Thus, a much stronger vacuum pump and syringe filters were proposed for more efficient filtration. However the stronger vacuum pulp was rather ineffective, as the paper pulp became very sticky after being mixed with the xylanase, so wastewater wasn’t filtering out of the paper pulp. As a result, centrifugation was utilized again, but the clarified supernatant went through a modified filtration step prior to data collection. To prevent paper fibers from being present in the effluent, syringe filtration was used to filter them out (the supernatant post-syringe filtration will be referred to as effluent). Syringe filters are membrane-based filters that have a small pore size to remove particulate matter (Syringe Filters, n.d.). In these experiments, the syringe filters filter out materials larger than 0.45 µm.

Preliminary results of the negative control (n = 3) and 5% xylanase treatment (n = 4) after undergoing syringe filtration indicated that the xylanase treatment produced effluent with a higher absorbance than the negative control, though the actual effluent itself looked very similar to the yellow-ish hue of the Negative Control. A two-tailed independent t-test was used to compare negative control samples (mean absorbance is 0.08 (+/- 0.032)) and the samples treated with a 5% xylanase solution (mean absorbance is 0.21 (+/- 0.055)). The statistical test displayed a significant difference between the samples (p = 0.0138, p < .05). While there was a significant difference between the two data sets, the paper pulp itself seemed to have stayed the same color and the hue of the effluent was very similar between the untreated samples and the xylanase-treated samples. This implies that no inks and toners were being removed from the paper pulp and being released into the effluent. The powdered nature of this xylanase may have inhibited its ability to deink wastepaper as the xylanase didn’t fully dissolve into the distilled water. This could have led to an uneven xylanase distribution throughout the paper pulp. Past researchers such as Singh et al. have used solubilized, liquid enzyme mediums for experimentation (2012). Therefore, liquid enzymatic mediums were used for future paper deinking experiments to potentially increase deinking, specifically the enzymes cellulase and cutinase. I hypothesized that cellulase and cutinase could be used in synergy to increase paper pulp deinking because cellulase would break down cellulose in paper while cutinase would degrade the ester bonds that connect resins and pigments inside of ink particles (Structure of Lignocellulose, n.d.; Hong et al., 2017). 

Deinking Newspaper with Liquid Cellulase and Cutinase

First, the Negative Control was conducted again so that all of the samples could be syringe-filtered. However, even after syringe filtration, the absorbance of the Negative Control samples was much higher–similar to the 5% xylanase-treated samples. A potential uneven ink distribution throughout the paper pulp batches may cause this higher mean absorbance. As seen in Figure 4, the Negative Control was conducted on two separate days, and, through a two-tailed independent t-test, the mean of the absorbance values collected from the effluent on each day were significantly different from each other (p = 0.0004, p < .05). The difference in absorbance may have to do with a potential difference in ink in each batch of paper pulp. For the most accurate results, future experimentation will have to include creating newspaper pulp samples from scratch and making sure there is a uniform spread of ink throughout the pulp. 

Experimentation on newspaper was continued because of the difficulty and time constraints that were associated with creating newspaper from scratch. The 12.5% cellulase treatment of paper pulp was conducted after the Negative Control. One major change made during the cellulase trials was measuring data from effluent using a different absorbance. Up until this point, effluent had been measured at 465 nm because past researchers have previously used this wavelength to detect ink, such as Kumar et al. and Indumathi et al. (2014; 2021). However, not all newspapers utilize the same pigments and inks, which could be a confounding variable in this procedure preventing the accurate detection of inks and toners released into the effluent. However, as seen in Figure 5, after zeroing the spectrophotometer with an enzyme blank (so that the absorbance only reflects the inks and toners released and not the enzyme itself) and doing a full absorbance scan, a small absorbance peak was found at 515 nm for every cellulase-treated sample and almost every cutinase-treated sample (in later experiments). This suggests that the inks removed from the newspaper used in this study absorb light at 515 nm. In future experimentation, a full absorbance scan was done for every sample processed to identify the absorbance peaks characteristic of the inks and toners released from the paper pulp. 

The data from the cellulase trials referenced in the prior paragraph are represented in Figure 6. When comparing the absorbance of the cellulase blank to the 12.5% cellulase-treated paper pulp samples (zeroed with distilled water), there is very little difference in absorbance, indicating that the cellulase isn’t removing many inks and toners from the newspaper pulp. Therefore, smaller-scale experiments were conducted to test the effect of different concentrations of cellulase and cutinase on paper pulp to see if higher concentrations of enzyme treatment lead to more paper pulp deinking.

An error was made when creating the paper pulp samples for the cellulase and cutinase varying concentration trials because instead of using v/v calculations for the distilled water and the liquid enzyme, w/v calculations were used instead. The paper pulp consistency was meant to stay constant at 5% but instead ranges from 2.5%-4.2%. Figures 7 and 8 show that there wasn’t a great difference in absorbance between any of the varying cellulase concentrations or cutinase concentrations. However, the absorbance of the cutinase samples was generally lower than the cellulase samples, which could potentially mean that the cellulase deinks better than the cutinase. The cellulase-treated samples also showed a more distinct peak at 515 nm than the cutinase-treated samples, though none of the samples displayed a high removal of inks and toners. Looking back at past research, researchers Xu et al., Singh et al., Kumar et al., and Indumathi et al., searched for larger lignocellulosic peaks from between 205-280 nm, and Kumar et al. and Indumathi et al. searched for smaller peaks at 465 nm (2009, 2012, 2014, 2021). Therefore, it is possible that the inks removed from the newspaper weren’t measurable by the spectrophotometer. This study relied on observing lignocellulosic peaks at higher wavelengths, as the spectrophotometer available only measures wavelengths from 340 nm to 1000 nm. Due to low absorbance values, a paper pulp switch was made. Instead of deinking newspaper, the study shifted to deinking copy paper with blue ballpoint pen as it has unique and strong absorption of visible light at higher wavelengths.  

Deinking Blue Ballpoint Pen Copy Paper with Liquid Cellulase and Cutinase

Instead of deinking newspaper with liquid enzymes, copy paper written on with blue ballpoint pen ink was deinked instead because blue ballpoint pen ink contains crystal violet, a chemical that can be measured at 585 nm (Singh et al., 2012). Though in this study, a diagnostic peak was detected at 601 nm, potentially due to the machine’s accuracy or the variable composition of commercially available blue ball point pen ink. Figure 9 displays the data gathered after deinking paper pulp written with blue ballpoint pen. The no enzyme trials have a mean absorbance of 0.06 (+/- 0.0083), the cellulase only trials have a mean absorbance of 0.43 (+/- 0.045), the cutinase only trials have a mean absorbance of 0.03 (+/- 0.018), and the mixed enzyme trials (cellulase and cutinase) have a mean absorbance of 0.16 (+/- 0.054). Through various independent two-tailed t-tests, all of the data sets depicted in Figure 9 were compared and found to be significantly different from each other. The data refutes my hypothesis because the mixed cellulase and cutinase trials did not yield a higher absorbance than the individual cellulase or cutinase trials. Instead, the cutinase trials seemed to have negligible removal of ink (due to its low absorbance). This was corroborated by the fact that the effluent samples from the cellulase-only and mixed enzyme trials displayed a peak at 601 nm, while the cutinase-only trials didn’t show absorbance peaks at any wavelength. As indicated by its peak at 601 nm, the mixed enzyme trials seemed to remove some ink, but it did not remove as much as the cellulase-only trials. This is interesting because the enzymes that were added in all of the different experimental arms should have oversaturated the paper pulp. According to the enzyme provider, the optimal cellulase dosage was 0.05% to 0.5% and the optimal cutinase dosage was 10-2000 g per ton of paper pulp, or 0.001% to 0.2% (Enzymes.bio, n.d.), and overall, the enzyme dosage in all of these experiments were well over 0.5%. These results indicate that the cellulase is not oversaturated and could potentially deink paper pulp better at higher doses. Alternatively, if the enzymes are truly oversaturated, it could be that the cutinase and the cellulase interact with each other in a way that impedes the cellulase’s ability to deink paper pulp.  

Another interesting development in Figure 9 is that the mean absorbance of the effluent from the cutinase trials is lower than the effluent from the no enzyme trials. Hypothetically, if the cutinase didn’t activate, the absorbance of both the cutinase and no enzyme trials should be around the same. However, this may have happened because the spectrophotometers were zeroed with different solutions (the no enzyme trials had a distilled water blank while the cutinase trials used a no paper pulp sample containing the same cutinase concentration), so this may have contributed to which inks were detected.

Because it wasn’t clear why the cutinase didn’t deink the wastepaper, a final trial was completed with higher varying cutinase concentrations to see if the paper pulp would be deinked at higher concentrations. All of the concentrations were higher than the 3:5 cutinase concentration (3 mL cutinase and 5 mL distilled water; 37.5%) because this concentration showed little change in the paper pulp’s physical appearance and absorbance. As shown in Figure 10, this trial yielded very little change in absorbance in all of the concentrations tested, which supports the idea that no concentration of cutinase would lead to the deinking of paper pulp. 

DISCUSSION 

It was hypothesized that a combined cellulase and cutinase paper pulp treatment would lead to the release of more inks. The data collected in this study currently refutes this hypothesis because, while past data reporting on the efficiency of cellulase’s deinking was corroborated, the cutinase did not seem to remove any inks from the paper pulp. Amid the current climate crisis, it is essential to reduce deforestation to prevent an increase of greenhouse gasses, habitat loss, and displacement of indigenous people (Zhang, 2017). Recycling paper is extremely important to the ongoing reforestation movement and deinking methods should not cause more environmental destruction. Therefore, a transition should be made from chemical deinking, which has negative ramifications on the environment, to using enzymes, which have lower environmental pollution (Singh et al., 2012). The current largest knowledge gap is finding an enzyme that definitively deinks wastepaper to either the same or a higher degree than current chemical-based methods. This study analyzes the use of two enzymes to observe if they display a novel, synergistic effect when mixed together to deinking wastepaper. While the hypothesis was ultimately refuted and no synergistic effect was observed, it is crucial to analyze the effects of different enzyme combinations to find an equivalent deinking method to chemical-deinking. 

There are a few limitations to this study. First and foremost, only one type of commercial cutinase was used as only one type (Cutinase enzyme SJ30) was commercially available. This cutinase did not show any evidence of deinking in this study, but Hong et al. found that cutinases derived from Thermobifida fusca and Fusarium solani pisi displayed strong deinking potential by analyzing the brightness of the resulting paper pulp (2017). The researchers claim that the brightness of the paper pulp deinked by the cutinases were 4%-5% higher than the chemically deinked paper pulp in this study, and higher than the brightness of paper pulp deinked by cellulases, hemi-cellulases, and laccase (Hong et al., 2017). It is possible that the cutinase used in this study could have been defective or not have as much enzymatic activity as the cutinases analyzed by Hong et al. The second limitation of this study is that, due to time constraints, it was not possible to test different concentrations of cellulase on blue ballpoint pen ink removal. It is not clear if the mixed enzyme trials in Figure 9 displayed a lower mean absorbance than the cellulase trials because the dosage of cellulase was lower, or that the cellulase was oversaturated in the enzyme solution but the cutinase inhibited the cellulase’s deinking ability. Lastly, this study was also conducted on a very small scale, as less than 1 gram of paper pulp was tested during many of the experimental trials, so future researchers should conduct their future experiments with larger amounts of enzyme and paper pulp to better understand the issues that may arise while deinking using enzymes in paper recycling mills. 

Deforestation plays a significant role in the climate crisis, partially due to the global demand of paper. In 2018,  67,390,000 tons of paper was generated in the United States and only 68% of the paper was recycled, leaving 17,220,000 tons of paper to be landfilled (United States Environmental Protection Agency, n.d.). Thus, it is essential to increase paper recycling efforts to counteract deforestation. Future researchers should continue studying deinking paper pulp using different enzymes to find enzyme combinations that lead to higher deinking of paper pulp. This research could publicize a cheaper and more environmentally friendly method of deinking, financially and morally incentivizing the recycling industry to transition out of expensive chemical deinking methods that pollute the environment. Advancing enzymatic research on deinking paper pulp is crucial for a more sustainable future. 

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• Optimized Materials and Procedural Document

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