Enhancement of the enzymatic hydrolysis of fines from recycled paper mill waste rejects
© Min et al. 2015
Received: 27 April 2015
Accepted: 21 September 2015
Published: 5 October 2015
A significant fraction of short fibers commonly called “reject fines” is produced while recycling wastepaper at paper mills producing linerboard. These fines are usually rejected into the solid waste stream that further requires land filling and poses environmental problems. The major component of these rejects is cellulose that can be a potential source of fermentable sugars for biofuels, bioplastics or other products. Therefore, a feasible process for converting these reject fines into sugars can profit the paper mills by producing value for their waste products while simultaneously mitigating their adverse environmental impact by avoided solid waste. Additionally, the sugar feedstocks can be used to reduce fossil carbon contributing to the sustainability of the industry.
Enzymatic conversion of rejects fines from paper mills was achieved using commercial cellulases from Trichoderma reesei. The presence of mineral particles along with the cellulosic fines was found to have potent inhibitory effects on enzyme hydrolysis. The mineral particles are kaolin and calcium carbonate and originate from the fillers used in the wastepaper. The adsorption of the cellulase onto these mineral components was measured and quantified by the slope of the adsorption isotherm. The application of a nonionic surfactant Tween-80, decreased the adsorption of cellulase and this improved the hydrolysis yield of sugars.
Enzymatic hydrolysis of rejects from recycled paper mills is feasible and provides a source of sugars for biofuels and bioplastics. However, the presence of mineral particles can be detrimental to this bioconversion. Calcium carbonate which occurs as a filler in waste paper shows high adsorption affinity to the cellulase enzymes and thus reduces the available enzyme for cellulolysis. This can be remedied by the application of surfactants which preferentially occlude to the mineral surfaces and thus increase enzyme availability in solution. The non-ionic surfactant, Tween-80, shows the best hydrolysis enhancement at a dosage of 3 % based on the dry weight of the biomass.
KeywordsEnzymatic hydrolysis Calcium carbonate Recycled paper Surfactants Rejects Fillers Cellulase adsorption
Rising oil prices, unstable supply and the demand for sustainable environmental friendly energy sources have increased the interest in research and development of biomass resources for fuels such as bio-ethanol. Carbohydrates are a natural resource commonly available as lignocellulosic biomass that can be hydrolyzed into sugars to be further converted via fermentative or thermochemical processes into useful products (Zhang 2008; Arora et al. 2015). Among the important products that can be derived are ethanol (cellulosic), butanol and similar advanced fuels, platform chemicals such as acetone, furfural, levulinic acid, gamma valerolactone and bioplastics such as polyhydroxy butyrates or valerates (Zhang 2008; Singh et al. 2008; Galbe and Zacchi 2002; Bhuwal et al. 2014). These products are a substitute for fossil fuels or starch-based carbohydrates, thus providing an alternate sustainable resource. The plastics are biodegradable and thus are beneficial to the environment in comparison to petrochemicals and their derivatives (Kale et al. 2007). Cellulosic biomass is a promising material for bio-energy that avoids the usage of corn and other food grains and thus avoids the necessity of competing with edible sugars. One good source of cellulosic biomass is the waste rejects from recycled linerboard mills which manufacture packaging paper from old corrugated containerboard (OCC).
Repeated recycling of pulp decreases the length of fibers which become shorter and stiffer, losing their ability to bond within the paper sheet. At a certain stage, their net contribution to the sheet becomes negative and they need to be rejected. These short fibers known as fines are recovered from the wastewater stream and typically sent to landfills. The solid residue can also be applied for land use or animal bedding (Scott and Smith 1995; Monte et al. 2009; He et al. 2009; Likon and Saarela 2012). However, the fines can be a very useful resource for sugar production because they are predominantly composed of cellulose which could be converted into glucose and other monomeric sugars. Currently, some paper companies pay $ 25–$ 80/(wet) ton for disposal of the fines (Scott and Smith 1995; Villanueva and Wenzel 2007; Laurijssen et al. 2010). Besides their cost advantage, the supply of fines from paper mills is fairly homogeneous and thus there is minimal influence of seasonal- or weather-related supply challenges compared to other agricultural biomass (Villanueva and Wenzel 2007; Laurijssen et al. 2010).
A number of different processes including incineration, gasification and pyrolysis may be used for treating this waste fines stream (Monte et al. 2009). However, direct hydrolysis of the cellulose into sugars can be particularly attractive due to the simplicity of the process and ready use of the sugar solution after concentration (Wang et al. 2012). These sugars can be used as a feedstock for conversions into biofuels and bioplastics such as polyhydroxy alkanoates or into platform chemicals such as succinic acid, lactic acid, levulinic acid and furfurals (Zhang 2008; Bhuwal et al. 2014; Graf and Koehler 2000; Lark et al. 1997; Kádár et al. 2004) Of the varieties of paper mill fines rejects, those from recycled pulp mills using old corrugated cartons are particularly important. Some modern OCC mills find that rejecting ‘inactive’ fines into the waste stream can be more profitable than using them in the manufactured product, particularly recycled linerboard. The reject stream thus contains higher cellulosic fines contents and typically lower minerals than deinked pulp rejects in the waste streams of fine papers or tissue mills.
The present study focuses on the enzymatic hydrolysis of OCC fines rejects from a recycled linerboard mill. The objective was to identify a method for and optimize the saccharification of this waste stream to yield fermentable sugars. The effect of enzyme activity (characterized by their FPUs, in terms of the filter paper units of activity of the enzyme, per g of cellulose substrate), and impact of hydrolysis temperature, pH, pulp type and filler composition were also studied. Furthermore, methods of enhancing the enzyme activity and sugar yields by binding the minerals using different surfactants (ionic and nonionic) were studied.
The fines were procured from a recycled linerboard-manufacturing mill in New York State. Unbleached softwood kraft pulp (USKP), unbleached hardwood kraft pulp (UHKP) and mixtures of fiber and fillers were used for hydrolysis. For comparison purposes, samples of old corrugated cartons were also slushed and used. Pulps were ground and screened through a 200 mesh screen (accepts were less than 75 μm in size) using a Wiley mill. The fines were not ground given their small particle size. A sample of microcrystalline cellulose (Avicel) was also used as a reference cellulosic source for hydrolysis.
Solid content and ash content were computed according to the National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure (LAP, NREL/TP-510-42627, NREL/TP-510-42622). Enzyme activity was determined by NREL LAP (NREL/TP-510-42628). Particle size and Zeta potential were quantified by a particle size analyzer (90 Plus/BI-MAS, Brookhaven Instruments Co.)
The hydrolysis of fines was carried in a medium with a solid to liquid ratio of 1:20 with a cellulase dosage of 5–50 FPU using 20 mL sodium acetate buffer (pH 5). Commercial grade cellulases from Trichoderma reesei ATCC 26921 and Aspergillus sp. (CAS No. 9012-54-8) were obtained from Sigma-Aldrich. The hydrolysis flask was placed in a shaking incubator (Reciprocal Shaking Bath 51221080, Precision Scientific Co., Denver CO) and hydrolyzed at 50 °C up to 72 h at 120 rpm. Samples were withdrawn to analyze for sugar concentration at different time intervals up to 72 h. The hydrolyzed material was then filtered through 0.1 μm filters and the filtrate was analyzed for composition using HPLC (Yasarla and Ramarao 2012; Alves et al. 2010). The hydrolysis yield was calculated gravimetrically with reducing sugars based on the substrate loading.
Effect of fillers
To determine the effect of filler on hydrolysis yield, synthetic reject mixtures were generated in the laboratory using unbleached softwood kraft pulp (USKP) mixed with various proportions of calcium carbonate (in two commonly used forms: ground, GCC, and precipitated, PCC) and kaolin. The filler content was varied to understand the influence of each on hydrolysis yield. The total filler content in this mixture was adjusted to yield comparable filler content levels in the original fines (the proportions of calcium carbonate and kaolin were adjusted to a total of 30 % (w/w) and the ratio of fillers was varied between 0 and 30 %) (Lavrykov and Ramarao 2012; Singh et al. 2009).
Effect of surfactants
Since fillers provide adsorption surfaces for the cellulase enzymes (they are nonproductive in terms of sugar production), inactivation by shielding their surfaces with a suitable surfactant to prevent enzyme adsorption was investigated. Anionic (sodium dodecyl sulfate, Amresco®), cationic (1-hexadecyl trimethyl ammonium bromide, Alfa Aesar®) and a nonionic surfactant (Tween-80, Amresco®) were chosen for this purpose.
Enzyme adsorption test
Results and discussion
Hydrolysis of fines, pulps and avicel
Characteristics of fines rejects from OCC paper mill
Fines (rejected fines containing fillers and contaminants)
% (by mass)
Others (plastics, synthetic fibers, other organics)
We also conducted control experiments with a sample of an unbleached kraft hardwood pulp (UKHP: sugar maple) in the never dried state and another sample of an unbleached kraft softwood pulp (UKSP: spruce) obtained as dried laps and reslushed in water. The hydrolysis was conducted with a dosage of 50 FPU of cellulase at 50 ℃ for 72 h and the yields were measured after 72 h. The yields of the Avicel, UKHP, UKSP and the fines sample were 92 (4.5), 92 (1.3), 58 (1.8) and 44 (1.7), respectively. The yields were determined from an assay of the sugars and the numbers in the parentheses represent the standard deviations determined from hydrolysis runs in triplicates. Nearly all the cellulose in Avicel was readily converted into glucose. Similarly, the conversion of the sample of the unbleached Kraft hardwood pulp (UKHP) was extremely high. Unbleached kraft softwood pulp (UKSP) shows lower yields primarily because this pulp was dried and reslushed before enzymatic hydrolysis. The process of drying causes the pulps to hornify, i.e., limits the accessibility of cellulose by reducing the cell wall porosity. Upon reslushing, therefore, a dried pulp fiber will not rehydrate to the same extent as virgin fibers and the cellulases are blocked from entering the crystalline structure (Cao et al. 1999). The maximum hydrolysis yield was around 44 % of OD (oven dry mass basis) fines and was found at 50 FPU of the enzyme. The maximum hydrolysis yield was the same at 100 FPU but is not shown here.
Hydrolysis with surfactants
Some solutions had to be considered to improve the enzyme efficiency for fine hydrolysis process because of the cost contribution of enzyme to sugar conversion from lignocellulosics (Klein-Marcuschamer et al. 2012). The enzyme dosage of 50 FPU for the maximum hydrolysis yield is too high to produce sugars from fines economically since other researchers reported that enzyme less than 10 FPU is sufficient to reach the highest conversion yields of pretreated corn stover, bleached hardwood and softwood pulp, or copy papers (Chen et al. 2012; Roche et al. 2009). We conducted an analysis of fine constitutions and properties to determine the presence of any inhibitory components that may cause inactivation or otherwise hinder the hydrolysis of cellulose in these fines. Table 1 shows an analysis of the fines in terms of their mineral and lignin compositions as well as other characteristic features. It is interesting to note that the amount of ash, representing minerals as fillers in the original pulp fines, was a significant fraction, consisting 41 % (g/g) of the total. We also determined that calcium carbonate (CaCO3) composed around half of this ash. Lignin was also present in the fines at 4 %. The particle size of fines was very small and the pH was close to neutral (6.5–7), but the magnitude of the zeta potential was small (−9 mV).
The presence of significant mineral particles can reduce the yield of sugar by interference with the action of the enzymes. Most often, this can take the form of simple competitive adsorption of the enzymes reducing the net activity in solution. The impact of mineral fillers was demonstrated in the present study by mixing kaolin or calcium carbonate filler with samples of unbleached hardwood kraft pulps and subjecting them to hydrolysis. The hydrolysis yield was measured for several enzyme dosages. The results indicate that calcium carbonate particles have a dramatic impact, reducing hydrolysis yields as compared to kaolin, which was minimally active.
Effect of surfactant (3 % dosage per 1 g of fines) on the enzymatic hydrolysis yield
Hydrolysis yield (%)
Impact of nonionic surfactant on mineral fillers
Effect of fillers and nonionic surfactant on the hydrolysis yield
Additives in USKP (g/g of OD fine)
Enzyme dosage (FPU/g of OD fine)
Besides providing surfaces for competitive and nonproductive, i.e., non-hydrolyzing sites for enzyme adsorption, the CaCO3 could perform as an inhibitor in other important ways. For example, the presence of CaCO3 alters the pH from the optimal value for hydrolysis and Ca2+ ions could bind to the enzymes and neutralize their charges, thus changing their conformation and size in solution.
Equilibrium constant K ad of GCC, PCC, and Kaolin, with (B) and without 3 % of Tween-80 (A)
K ad (ml/ml)
Optimal surfactant dosage
Addition of acid buffer to maintain optimal pH
Impact of drying of fines on hydrolysis
Drying effect of materials on enzymatic hydrolysis yield
25 FPU, 72 h
Hydrolysis yield (%, g/g)
Dry effect (%)
Unbleached kraft pulp
Effect of temperature on hydrolysis
Paper mill rejected fines are a good source of biomass for sugar production given the low lignin content, negative cost, pre-processed nature (which negates requirement of a pretreatment regime) and the large surface area and porous nature of the particles compared to other naturally occurring biomass. The particle size is smaller than typical milled biomass particles (sizes for which are in the sub-millimeter ranges). Approximately, 75 % of the enzymatic hydrolysis yield based on reducing sugars was achieved. The sugar yield of rejected fines is similar to the hydrolysis yield of woody biomass which was reported as 70–90 % for lignocellulosic biomass (Galbe and Zacchi 2002; Sun and Cheng 2002).
The commercialization of “waste cellulosic fiber” based sugar requires deactivation of inhibitory potential of contaminants and ash which includes fillers, calcium carbonate being one of the most powerful inhibitors (Chen et al. 2012). Although ash removal can be attempted, it is not feasible under many industrial circumstances. The filler particles are usually bound to the fibers due to the application of different types of flotation agents in the deinking operations. It renders them difficult to remove by conventional flotation steps. Furthermore, any de-ashing steps using screening methods add to the costs of the process and can prove uneconomical.
The fines have a potential to produce sugars as a resource of biomass. The main inhibitor of enzymatic hydrolysis fines was CaCO3 which decreases the enzyme activity by enzyme adsorption and pH increase. Nonionic surfactant Tween-80 improved enzymatic hydrolysis yield of fines in addition to 50 % increase at 10 FPU and reduced enzyme dosage to obtain the maximum hydrolysis yield. A nonionic surfactant was able to significantly reduce the affinity of the fillers to the enzymes. This mechanism appears to be one of the main reasons improving enzymatic hydrolysis yield of pulp. The surfactant application was simple and an economical option to increase profitability and productivity of sugars from waste cellulosic fibers. Using proper pH buffer was also found to be a critical factor to improve sugar productivity from fines. It was found that addition of surfactants and acid mitigated the inhibitory effect of CaCO3 which has a high inhibitory potential and resulted in decrease of enzyme demanding from 50 FPU to 20 FPU for maximum sugar yield around 95 % (reducing sugars g/g of total sugar in fines). A separation processes to reduce fillers and other contaminants from fines prior to a hydrolysis process can be used to further decrease the enzyme dosage.
unbleached kraft hardwood pulp (sugar maple, Acer saccharum was chosen here)
unbleached kraft softwood pulp (spruce was chosen here)
oven dry weight
filter paper units (of cellulase concentration, represented per 1 g of substrate)
old corrugated cartons (these are extensively recycled all over the world)
ground calcium carbonate, used as filler in many white (fine) paper grades
precipitated calcium carbonate, used as filler in many paper grades such as copiers, digital printing and other fine and coated paper grades
cetyl trimethyl ammonium bromide (cationic surfactant)
sodium dodecyl sulfonate (anionic surfactant)
adsorbed enzyme on fillers or pulp fines or fiber substrates
enzyme concentration in solution
- K :
slope of the adsorption isotherm. The adsorption isotherm is assumed to pass through 0, 0
BCM conducted the majority of the experiments and analysis described in this work. BVB assisted the initial fines hydrolysis experiments with different enzymes. VSJ assisted in experimentation and data analysis. BVR initiated the work and supervised it. All authors read and approved the final manuscript.
The support of the New York State Energy Research and Development Authority under grant No. 25922 to SUNY ESF is gratefully acknowledged. The partnership of Minimill Technologies, LLC, Dewitt NY in sourcing mill rejects is also gratefully acknowledged.
Compliance with ethical guidelines
Competing interests SUNY ESF has applied for US patents for the incorporation of surfactants for hydrolysis of reject fines from paper mills. (US Patent Application Nos. 14/212,361; 61/953,152). Avatar Sustainable Technologies LLC, co-founded by the authors (BVB, BVR) has taken a limited time option for possible commercialization.
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