Need to explain apparent ‘Hard-to-biodegrade’ cellulose remainders in CO2-based test results

Abstract

The extent of cellulose fiber biodegradation, according to many published studies, tends to reach a plateau value well below 100%. This editorial proposes that the apparent residue of not-biodegraded cellulose may be due to simplifying assumptions in a commonly used assay to quantify cellulose biodegradation. Some such tests are based on the production of CO₂. The evolved CO₂ is removed from the air by an alkaline trap, which triggers a quantified addition of O₂ gas. However, N₂ and NH₃ gases are evolved during biodegradation. The theoretical amount of nitrogen might explain a shift of up to 0.63% in the extent of biodegradation. Another possibility is that the evolution of nitrogen-based gases starves the biological system of nitrogen, thereby terminating biodegradation in the test container. This editorial asks: “Is biodegradation of cellulose usually more complete in comparison to the results from standard tests?” If yes, that would match the fact that cellulose does not build up endlessly in the environment. These findings have direct implications for natural fibers such as cotton, which might be systematically underdetermined in standard biodegradation comparisons against synthetic fibers.

Full Article

A Need to Explain Apparent ‘Hard-to-biodegrade’ Cellulose Remainders in CO₂-based Test Results

Martin A. Hubbe  ,^a,* Soojin Kwon  ,^b Jesse S. Daystar  ,^c Joel J. Pawlak  ,^a and

Richard A. Venditti  ^a

The extent of cellulose fiber biodegradation, according to many published studies, tends to reach a plateau value well below 100%. This editorial proposes that the apparent residue of not-biodegraded cellulose may be due to simplifying assumptions in a commonly used assay to quantify cellulose biodegradation. Some such tests are based on the production of CO₂. The evolved CO₂ is removed from the air by an alkaline trap, which triggers a quantified addition of O₂ gas. However, N₂ and NH₃ gases are evolved during biodegradation. The theoretical amount of nitrogen might explain a shift of up to 0.63% in the extent of biodegradation. Another possibility is that the evolution of nitrogen-based gases starves the biological system of nitrogen, thereby terminating biodegradation in the test container. This editorial asks: “Is biodegradation of cellulose usually more complete in comparison to the results from standard tests?” If yes, that would match the fact that cellulose does not build up endlessly in the environment. These findings have direct implications for natural fibers such as cotton, which might be systematically underdetermined in standard biodegradation comparisons against synthetic fibers.

DOI: 10.15376/biores.21.3.5706-5709

Keywords: Percent biodegradation; Cellulase enzymes; Hydrolysis; Denitrification; Nitrogen; Ammonia

Contact information: a: Department of Forest Biomaterials, College of Natural Resources, North Carolina State University, Campus Box 8005, Raleigh, NC, 27606; b: Gyeongsang National University, Department. of Environmental Materials Science, College of Ag. & Life Sci., South Korea; c: Cotton Incorporated, 6300 Weston Parkway, Cary, NC 27516, US; Corresponding author: hubbe@ncsu.edu

An Apparent Remainder of ‘Hard-to-biodegrade’ Cellulose

The generation of CO₂ in the course of cellulose biodegradation has been used to track and quantify the process (ISO 14851). One starts by weighing a specimen of known composition and calculating the amount of carbon dioxide that would theoretically be released following total hydrolysis of the polysaccharide content, which is assumed to be catalyzed by enzymes. A recent review article considered such studies (Hubbe et al. 2025). A remarkable finding of such studies is an apparent persistence of a remainder of about 10 to 40% of the cellulose at the end of the test period, based on the weighing and the calculations. The cellulose biodegradation appears to gradually slow down and then almost stop at about 60 to 90%, in many cases (Kwon et al. 2021; Smith et al. 2024).

There are practical reasons to expect biodegradation to continue steadily or even to accelerate in the later stages under lab conditions. First, whatever fungi or bacteria had been providing the enzymes to promote hydrolysis of the cellulose would have had time to build up, partly in response to the presence of the cellulose itself. The remaining cellulose can be expected to be substantially broken down and to become increasingly accessible to enzymatic attack, having already lost much of its substance. The remaining cellulose is expected to have a somewhat porous structure, due to the preceding enzyme action.

Initial Attempts to Explain the “Hard to Break Down” Cellulose

Table 1 contains several initial explanations that were considered by the authors to account for the reported results. Each explanation is paired with some reasons that the authors were not satisfied with those explanations:

Table 1. Initially Considered Explanations for the Apparent Remainder of Unbiodegraded Cellulose

Proposed Explanation Based on N₂ Evolution

Biodegradation cannot occur in the absence of proteins, which are a necessary component of both biological cells and enzymes. The biodegradation of proteins can be expected to result in the evolution of a combination of nitrogen gas (Jiang et al. 2026), the oxides of nitrogen (N₂O and NO) (Jiang et al. 2026), and ammonia (Kappaun et al. 2018), each of which can be released to the gas phase. The test protocol ISO 14851-2019 that is often used to quantify biodegradation calls for the addition of ammonium chloride as part of the test medium. As a first step, a “Solution A” is prepared that contains 0.5 g of NH₄Cl per liter, along with some other electrolytes. Each specimen is tested with the addition of 10 mL of Solution A per liter. In the work of Kwon et al. (2021), each specimen suspension was 400 mL. This implies 2 mg of NH₄Cl per specimen. If 100% of the NH₄Cl ended up as N₂, that would amount to about 18.7 μmol of nitrogen gas. Kwon et al. (2021) added 80 mg of cellulose per test (with 400 mL of suspension). If all of that cellulose were biodegraded with the production of carbon dioxide gas, one can calculate that there would be 2960 μmol of CO₂ gas. The ratio of theoretical N₂ gas volume to CO₂ gas volume implies a maximum error (lower biodegradation than actual) by about 0.63%.

Proposed Explanation Based on NH₃ from Degradation of Proteins

Biodegradation of protein results in some production of NH₃ by a urease pathway (Kappaun et al. 2018). Biodegradation of proteins also gives rise to CO₂ (Pols et al. 2020). The ratio between release of NH₃ and CO₂ will depend on details of protein molecular structure, reaction pathways, and reaction kinetics. Arginine, a building block of proteins, is known to break down to urea, which then yields NH₃ (Wu 1995), with an expected ratio of NH₃ to CO₂ of 2:1 (Pols et al. 2020). To estimate the theoretical maximum amount of NH₃ gas, it will again be assumed that 100% of the NH₃ content will come from the NH₄Cl. The calculation is the same as when considering N₂ evolution, above, except for a factor of two. Each mole of N atoms can result in one mole of NH₃ but only half a mole of N₂. Thus, the theoretical maximum NH₃ production can account for about 1.26%, as an estimate for the erroneous lack of biodegradability of cellulose during a standard test.

Proposed Explanation Based on Active Nitrogen Depletion

The action of enzymes, upon which biodegradation occurs, can be regarded as a catalytic process. A relatively small amount of proteinaceous material can theoretically break down a much larger amount of cellulose by operating multiple times. However, as was just shown, there exists a mechanism by which the system might become starved of the active nitrogen that is required for all biological processes. The biochemical pathways by which active nitrogen can be lost can be complex and hard to predict (Jiang et al. 2026).

Proposed Explanation Based on Solubility of CO₂ in Water

At room temperature, the amount of CO₂ gas that will dissolve in water from the atmosphere is about 1.5 g/L. Assuming the same 400 mL of suspension, that would imply 0.6 g, which is equivalent to 13600 μmol. Although this value is about 4.6 times that of the CO₂ expected from the biodegradation of cellulose, there are several uncertainties to consider. The standard test employs an alkaline trap to remove CO₂ from the air space. Assuming complete equilibration, all of the CO₂ should eventually pass into the alkaline trap. However, it is not certain how much time would be required for such exchange. Hypothetically, the equilibration might be slowed down by a monolayer at the water surface, e.g. fatty acids from some cellulosic materials.

Proposed Explanations Based on Dynamics of Microbiology

Pfeiffer and Bonhoeffer (2004) showed that when microbes are cultured under isolated conditions, analogously to the conditions of ISO 14851, there can be a shift of dominance in the microbial community. A second microbe may employ different enzymes that act on degradation products from the first. As a result of such changes, the production of cellulase might stop, or the cellulase already produced might become denatured. As microorganisms degrade biomass, they consume materials and grow in numbers. This additional biomass is not necessarily accounted for in biodegradation tests. The carbon content of bacteria has been reported to be ~50% by weight (Fagerbakke et al. 1996). Thus, a simple mass balance shows that a significant portion of the cellulose (e.g. 10% to 50%) could be converted into biomass carbon.

Summation

Many questions remain. Is there evidence of depletion of active nitrogen when conducting standard tests of biodegradation? Do conditions allow suitably fast equilibration of CO₂ among the aqueous, gas, and alkaline trap phases? Does N₂ or NH₃ build up in the air space, thus displacing some of the intended flow of oxygen during the test? Are microbial population changes large enough to explain plateau effects? At least some of these questions could be addressed by future experimentation. The authors hope that his editorial helps to draw more research attention to these issues. Among the explanations considered, the hypothesis based on denitrification and starvation of the protein-based enzyme system can explain not only the plateauing of biodegradation levels and the magnitude of the effects, but also the fact that such plateaus can be at widely different levels of apparent degradation. For commercially significant natural fibers such as cotton, resolving these measurement questions would confirm what field observation already suggests: that cellulose biodegrades more completely than standard tests indicate, providing a meaningful contrast to synthetic fibers that persist in the environment.

References Cited

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