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Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars
Bioresource Technology 100 (2009) 5700–5706
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Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars Pascale Champagne a,*, Caijian Li b a b
Department of Civil Engineering, Queen’s University, Kingston, ON, Canada K7L 3N6 Department of Civil and Environmental Engineering, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
a r t i c l e
i n f o
Article history: Received 14 April 2008 Received in revised form 28 January 2009 Accepted 2 June 2009 Available online 14 July 2009 Keywords: Biosolids Enzymatic hydrolysis Sugar recovery Biomass Bio-ethanol
a b s t r a c t This study examined the hydrolysis of lignocellulose extracted from municipal wastewater treatment process residuals for the purpose of investigating low-cost feedstocks for ethanol production, while providing an alternative solid waste management strategy. Primary and thickened waste activated sludges and anaerobically digested biosolids underwent various pre-treatments to enhance subsequent enzymatic hydrolysis. Half of the pre-treated samples were dried and grinded, while the other half were used as is (wet). The wet primary sludge yielded the highest reducing sugar conversions. When wet primary sludge without pre-treatment was hydrolyzed at 40 °C and an enzyme loading of 800 U/g substrate, 31.1 ± 2.7% was converted to reducing sugars in 24 h. This increased to 54.2 ± 4.0% when HCl and KOH pre-treatments were applied. FTIR analyses were used to examine differences in the sludge compositions. These indicated that the cellulose content in the primary sludge was higher than that of the thickened waste activated sludge and biosolids, which was consistent with the higher reducing sugar yields observed in the primary sludge. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the highly unstable global energy market, including fluctuations in oil and natural gas prices, has led Canada to assess future fuel developments and explore alternatives to fossil fuels and petroleum-based products. Municipal wastewaters sludges and biosolids contain large quantities of cellulose, lignocellulose, polysaccharides, proteins and other organic materials. Municipal biosolids and sludges are a waste biomass resulting from water and wastewater treatment processes. In 2001, a Canadian biosolids production rate of 0.063 kg dry biomass/person/day was estimated to generate biosolids at a rate of 387,166 tons/yr (Wood and Layzell, 2003). Based on a bio-product feasibility study conducted using municipal sludges/biosolids and livestock manures produced in Canada, an estimated 6.22 Mt/yr of sugar could be produced from this waste biomass for subsequent fermentation to bio-ethanol or other chemical bio-product precursors (Champagne, 2007). The conversion of this waste biomass to higher-value products has been recognized as an attractive alternative waste management solution (Industry Canada, 2004). However, the complex physical and chemical composition of municipal biosolids and sludges make them difficult to employ as feedstocks compared to other organic residues. Hence, to date, there have been limited ef* Corresponding author. Tel.: +1 613 533 3053; fax: +1 613 533 2128. E-mail address: [email protected] (P. Champagne). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.051
forts to convert this waste biomass to higher-value chemicals and energy on a commercial scale. As a result, waste biomass are an abundant but under-utilized biomass resource for producing biobased chemicals and bio-fuels. The main objectives of this study were to investigate the feasibility of using sludges and biosolids as low-cost feedstocks for the production of simple reducing sugars as an environmentally-friendly alternative to the disposal of solid waste. The investigation focused on the enzymatic hydrolysis of primary sludge, activated sludge and digested sludge under different pre-treatment conditions and assess the efficiency of the pretreatment applications in the production of simple reducing sugars for further use as building block chemicals. 2. Background Theoretically, all biomass materials are possible feedstocks for the production of higher-value chemical products. They differ in appearance and properties, however, they all have a relatively similar composition, which consists mainly of cellulose, hemicellulose and lignin. The two carbohydrate polymers, cellulose and hemicellulose, form the main structure of biomass materials, whereas lignin acts as a joining material and binds the fibers together. Although extraneous compounds are present in small quantities, they play an important role in structural stabilization, making cellulosic materials resistant to decomposition, decay and insect attack. Celluloses from various biomass sources are generally the
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same at the molecular level, however, they differ in their crystalline structures and in their binding to other components. Cellulose is a high molecular weight linear polymer. Each cellulose molecule is an unbranched chain consisting of 1000–1 million D-glucose units, linked together with b-1,4 glycosidic bonds. To utilize cellulose as a feedstock for the production of energy and higher chemicals, it is essential to break it down to glucose. The cellulose present in lignocellulosic materials is composed of crystalline and amorphous components. The amorphous component is usually more active than the crystalline component, thus, increasing the amorphous content enhances the rate of hydrolysis. The presence of lignin forms a physical barrier to enzymes. Hence, the separation of lignin from the cellulose and hemicellulose, reduction of cellulose crystallinity and swelling of cellulose-containing fiber to increase the accessible surface area to the cellulosic material, are essential prior to enzymatic hydrolysis to improve cellulose susceptibility to enzymes (McMillan, 1994). According to Sun and Cheng (2002) pre-treatment processes generally aim to achieve the following: (1) improve sugar yield from subsequent acid or enzymatic hydrolysis; (2) minimize the degradation or loss of carbohydrates; (3) minimize the formation of inhibitory byproducts for hydrolysis and fermentation processes; and (4) cost-effectiveness. There are three main categories of pre-treatment processes: physical, chemical and biological pretreatments. Depending on the biomass material, one or a combination of these methods may be used to improve the accessibility of enzyme to cellulose molecule. The effect of physical pre-treatment is to subdivide lignocellusic material into fine particles which are more susceptible to acid or enzymatic hydrolysis, and to reduce the degree of cellulose crystallinity. Chemical pre-treatments use a solvent to degrade the lignin, hemicellulose and/or cellulose crystalline structure. Alkaline hydrolysis pre-treatment cause structure swelling which increases the internal surface area, and decreases the degree of polymerization. It acts primarily through the saponification of intermolecular ester bonds crosslinking xylan hemicellulose and other components, which results in the separation of the structural linkages between the lignins and carbohydrates, and disrupt the lignin structure (Fan et al., 1987; Prasad et al., 2007). Acids primarily act as catalysts for cellulose hydrolysis rather than as a pre-treatment reagent, where the acid accelerates the rate of solubilization relative to structural degradation, resulting in higher conversion yields (Lloyd and Wyman, 2005; Prasad et al., 2007). The effect of pre-treatment on cellulose hydrolysis has been widely studied. Dilute acid pre-treatment has been demonstrated to be reliable in increasing the cellulose pore volume and enzyme accessibility (Esteghlalian et al., 2001). Schell et al. (2003) studied the hydrolysis of corn stovers with dilute sulfuric acid pre-treatment. The substrate was pre-treated with 0.5–1.4% (w/w) sulfuric acid at 165–195 °C for 3–12 min. Cellulose (80–87%) conversion was obtained during simultaneous saccharification and fermentation process. The disadvantages of acid pre-treatment are the costs associate with facility construction, as well as the handling and disposal of neutralization chemicals (Ward and Singh, 2002). A number of other pre-treatment processes, such as ammonia and alkaline peroxide treatment, have been shown to be very effective under certain conditions (Saha et al., 1998). For a certain biomass material, the preferred pre-treatment should meet the following criteria: achieve high mono sugar yields from enzymatic hydrolysis, minimize the degradation of carbohydrates and the formation of inhibitory byproducts, and be cost-effective (Negro et al., 2003). The enzymatic hydrolysis of cellulose is carried out by cellulase enzymes. There are a number of cellulase enzymes that are capable of degrading cellulose, of which the most intensively studied is the enzyme complex derived from filamentous fungus Trichoderma Reesei. Enzyme complexes generally consist of three components:
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endo-b-glucanase (EG) which attach to regions of low crystallinity in the cellulose fiber, generating free chain ends; exo-b-glucanase or cellobio-hydrolase (CBH) which degrade cellulose molecules further by removing cellobiose units from the free chain; and, bglucosidase which hydrolyze cellobiose to produce glucose (Prasad et al., 2007). Each component is essential to synergistically hydrolyze cellulose efficiently and completely. The enzymatic hydrolysis of cellulose takes place in three stages: (1) the adsorption of the cellulase enzyme complex onto the surface of the cellulose structure; (2) the degradation of cellulose into mono sugars; and (3) the desorption of cellulase. The rate and extent of cellulose hydrolysis by cellulase enzymes is influenced by many substrate and enzyme factors, as well as operational conditions. Because of the heterogeneous nature of the process, the overall reaction rate can be affected by mass transfer resistance; including the surface film resistance around cellulose particles, the bulk phase resistance, and the resistance through the capillary pores of the cellulose particles. Consequently, the reaction conditions such as the size of cellulose particles, the substrate concentration, enzyme concentration, pH of the buffer, and agitation intensity will influence the hydrolysis rate. At low cellulose concentrations, increases in cellulose concentrations generally lead to increases in glucose yield and reaction rates, while at high cellulose concentrations, substrate inhibition can result leading to a decrease in the rate of hydrolysis (Cheung and Anderson, 1997). Hang and Woodams (1998) have shown that at pH 5.0, the sugar yields from corn husks were higher than those at pH 4.0 and 7.0. They also found that at 40 °C, 50 °C and 60 °C, the total sugar yields were slightly different. However, significantly more glucose and xylose were converted at higher temperature. In addition, very high substrate concentrations can slow down the hydrolysis due to the inhibition from the end product. Finally, the high viscosity of slurry may also limit the mass transfer within the reaction mixture (Ingession et al., 2001). Moreover, excessive mixing speeds (>200 rpm) decrease the extent of hydrolysis because the enzyme activity is lowered. The lignin content appears as a barrier to the enzymes, but it is not always undesirable. Kaya et al. (1998) found that the addition of dissolved lignin improved the enzymatic reaction, because it was thought to bind to the enzymes in solution, then the bound enzymes were sustained and hydrolyzed the adjacent cellulose easily. 3. Methods Municipal wastewaters, sludges and biosolids include fecal materials, scraps of toilet paper, and food residues, and as such, a reasonable cellulose content is expected from these organic residuals. The various pre-treatments applied to the primary sludge, thickened waste activated sludge and anaerobically digested biosolids used as lignocellulosic feedstocks in this experimental investigation are illustrated in Fig. 1. All experiments were conducted in triplicate, and corresponding experimental means and standard deviations were reported. 3.1. Feedstock material collection and preparation Primary sludge, thickened waste activated sludge and biosolids from the anaerobic digestion process were collected from Robert O. Pickard Environmental Centre (ROPEC), a secondary wastewater treatment plant that treats dilute municipal wastewater and discharges to the Ottawa River in Ottawa (Canada). The average characteristics of these feedstocks are provided in Table 1. On average, 545,000 m3/d of wastewater is processed with average primary effluent characteristics of 110 mg cBOD5/L, 200 mg COD/L, 150 mg SS/L. ROPEC has preliminary and primary treatment
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The primary sludge and thickened waste activated sludge samples were transported in 15 L sealed plastic buckets and used directly in the experiments, while the biosolids were collected and stored refrigerated at 4 °C in sealed plastic bag. Because of the high water content in each of the feedstocks, the samples were first centrifuged to remove the liquid. For each feedstock, 2 L was divided into four 750 mL Nalgene bottles and centrifuged for 10 min at 4700 rpm. The supernatant was discarded, and the solid fraction was washed with distilled water and its pH adjusted to 7.0 using 1.0 N HCl. The mixture was then centrifuged once more to remove the excess liquid and the solid fraction was stored at 4 °C for use in subsequent pre-treatment and enzymatic hydrolysis testing.
Lignocellulosic feedstock
Solid/liquid separation
Solid fraction
EH
Dry
KOH treatment
HCl treatment
EH EH
Dry
EH
EH
Dry
3.2. Lignocellulosic feedstock pre-treatment
KOH treatment
EH EH
Dry
EH
Fig. 1. Schematic representation of pre-treatments tested for the primary sludge, thickened waste activated sludge and biosolids feedstocks prior to enzymatic hydrolysis (EH).
followed by a conventional aerobic activated sludge unit operated at an average sludge retention time of 5 days. The waste activated sludge was characterized as young sludge based on the 5-day solids retention time of the activated sludge unit and supported by the relatively high volatile solids (VS)/total solids (TS) ratio of approximately 0.70. Ferric chloride is added to the waste activated sludge for phosphorous removal prior to waste activated sludge thickening. The thickened waste activated sludge and primary sludge are blended in a 58:42 v/v ratio and undergo mesophilic anaerobic sludge digestion to produce a stabilized biosolids product for disposal. The anaerobically digested sludges employed in this study was the final product of mesophilic sludge digesters, dewatered to 30% TS (w/w) at ROPEC. Table 1 Robert O. Picard Environmental Centre (Ottawa, Canada) average primary effluent and primary and thickened waste activated sludge characteristics. Characteristics
Primary sludge
Thickened waste activated sludge
pH TS (% w/w) VS (% w/w) VS/TS (%) TCOD (mg/L) SCOD (mg/L) SCOD/TCOD (%) NH3–N (mg/L) Alkalinity (mg/L as CaCO3) TVFA (mg/L) Ash (% TS) VS (% TS) Protein (% TS) Cellulose (% TS) Fats and grease (% TS)
6.9 4.16 0.33 7.9 52,614 1289 2.4 145 220 914 27.1 72.9 31.7 29.3 22.6
6.49 5.4 3.77 70 41,667 2,357 5.7 536 919 913 21.3 78.7 18.2 13.8 17.5
Flow (m3/d) CBOD5 (mg/L) COD (mg/L) SS (mg/L) SP (mg/L) TN (mg/L)
Primary effluent 545,000 110 200 150 2 20
Note: TS, VS, SS: total, volatile, suspended solids; SCOD, TCOD, COD: soluble, total chemical oxygen demand; CBOD5: carbonaceous 5-day biochemical oxygen demand; SP: soluble phosphorous; TN, NH3–N: total, ammonia nitrogen; TVFA: total volatile fatty acids (sum of acetic, propionic, and butyric acids).
Alkaline (KOH) pre-treatment was used in some trials to delignify the lignocellulosic material. It was assumed that the metal content in the feedstocks could potentially impact enzyme activity. Therefore, a HCl pre-treatment was applied in some trials to reduce the metal content. Procedures for organic residual pre-treatments were elaborated in previous studies (Champagne et al., 2005; Li and Champagne, 2005; Levy et al., 2003a,b; Henderson et al., 2003), where an alkaline pre-treatment of the organic residual feedstocks with 0.5 N KOH at 70 °C was found adequate for organic waste residual delignification to isolate the solid fraction which consists mainly of cellulose and pre-treatment with 1.0 N HCl at 20 °C resulted in significant metal removals. Enzymatic hydrolysis was conducted using the products derived from the four pre-treatment trials: untreated feedstocks, HCl pre-treated feedstocks, KOH pre-treated feedstocks and HCl followed by KOH pre-treated feedstocks. Enzymatic hydrolysis was applied to both wet and dried (oven dried at 70 °C for 48 h) products of each of the pre-treatment trials. The untreated and pre-treated feedstock products formed large agglomerates during the drying process. As such, the dry products were grinded using a 20 mesh prior to enzymatic hydrolysis. Each experiment was conducted in triplicate. 3.2.1. KOH pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 0.5 N KOH solution in a 500 mL beaker for 1 h at 70 °C. The solution was then centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 1.0 N HCl to a pH of 7.0. The neutralized mixture was centrifuged again and the solid fraction was stored refrigerated at 4 °C in plastic bottles. 3.2.2. HCl pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 1.0 N HCl solution in a 500 mL beaker and stirred using a magnetic stirring bar on a hot plate at 20 °C for 24 h. The mixture was then centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 0.5 N KOH. The neutralized sample was centrifuged again, and the solid fraction was kept refrigerated at 4 °C in sealed plastic bottles. 3.2.3. HCl followed by KOH pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 1.0 N HCl solution in a 500 mL beaker and stirred using a magnetic stirring bar on a hot plate at 20 °C for 24 h. The mixture was centrifuged for 10 min at 4700 rpm and the solid fraction retained. The solid fraction was then treated with 250 mL of 0.5 N KOH solution in a 500 mL beaker for 1 h at 70 °C. The solution was centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 1.0 N HCl to a pH of 7.0. The neutralized
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mixture was centrifuged again and the solid fraction was stored refrigerated at 4 °C in plastic bottles. 3.3. Enzymatic hydrolysis Cellulolytic enzymes can be produced by a number of bacteria and fungi, which can use cellulose as a primary carbon source. Of all the celluloytic fungi, T. reesei has been the most extensively studied, This research was conducted using commercially available cellulase from T. reesei (Sigma–Aldrich) with a manufacturer labeled activity of 8.3 U/mg (1 U enzyme liberating 1 lmol of glucose in 1 h at 40 °C per mg of cellulase addition). The equivalent weight of 50 mg (dry mass) of untreated or pre-treated lignocellulosic primary or activated sludge feedstock or 150 mg of digested sludge feedstock and 4.8 mg of enzyme, corresponding to an enzyme loading of 800 activity units/g substrate, were added to 2.5 mL of sodium citrate buffer (pH 4.8) in a 10 mL test tube. The reactants were then placed in a 40 °C water bath and shaken at 160 rpm for a 24-h period. The tubes were then removed from the water bath and filtered using glass filters. The filtrate was transferred into a 50 mL capped plastic bottle for reducing sugar measurement. 3.4. Reducing sugar analysis The reducing sugar concentrations were analyzed using a dinitrosalicylic acid (DNS) assay (Miller, 1959): 3 mL 1% DNS solution was added to 3 mL of sample. The mixture was then boiled for 5 min and 1 mL of 40% (w/v) potassium sodium tartrate was added to stabilize the color. After cooling, the absorbance was read at 575 nm. The percentage reducing sugar yield (RSY) was calculated as follows:
RSY ð%Þ ¼
ðReducing Sugar Concentration; mg=mLÞð50 mLÞ 100% ðSubstrate added; mgÞ
3.5. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was used to assess differences in the general functional groups of the primary sludge, thickened waste activated sludge and biosolids feedstocks. Three hundred milligrams of dry KBr was milled with a mortar and pestle, once fine enough the powder was placed in a mould and compressed into a pellet with a force 10 tons applied for 1 min. The pellet was then placed in a holder and scanned using a Bomem Michelson 120 FTIR analyzer and the background spectra were generated using Bomem Grams 386 software at Carleton University Advanced Physical Chemistry Laboratory. To obtain the spectra for each of the lignocellulosic feedstocks, centrifuged feedstock samples were first dried and grinded, then for each of the feedstocks, 5 mg of sample was mixed with 300 mg KBr and milled into a powder using a mortar and pestle. The powdered mixture was
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placed in a mould and compressed into a pellet with a force 10 tons applied for 1 min. Each sample was scanned five times between the wavelengths 4500 and 650 cm1. The generated spectra were compared to published FTIR spectra for cellulose (Cao and Tan, 2002) and lignin (Ghosh, 1998) to identify the major functional groups.
4. Results and discussions 4.1. Reducing sugar yields from non-pre-treated feedstocks The percent conversions to reducing sugar based on the dry mass of lignocellulosic material yielded for each of the untreated and pre-treated lignocellulosic feedstocks are presented in Table 2. The cellulose content of the primary sludge is mainly from undigested waste paper, which has a high cellulose content and comparatively low lignin content. Table 2 indicates that for the primary sludge feedstock, 31.1 ± 2.7% and 25.0 ± 0.8% of the wet and dry substrate, respectively, was converted to reducing sugars with no pre-treatment. The percent reducing yields for thickened waste activated sludge also showed significant differences between wet (12.9 ± 2.7%) and dry (5.6 ± 1.2%) substrates, suggesting that the cellulose fibers in the activated sludge might also become less readily accessible to enzymes as a results of the drying and grinding processes. While this appears to be contrary to findings reported in other studies, the results could be attributed to the fact that in sludges, a large fraction of the cellulosic or lignocellulosic material is paper-based and has generally been processed and delignified. Hence, mechanical treatments such as drying and grinding would not necessarily enhance enzymatic hydrolysis, and could in fact shrink or collapse the existing fibers making them less readily accessible to the enzymes. For the enzymatic hydrolysis conducted on the untreated biosolids, initial substrate loadings were increased from 50 mg (dry mass) to 150 mg (dry mass) to obtain measurable reducing sugar yields. It was found that for the biosolids feedstock approximately 1% or less of the initial feedstock was converted to reducing sugars in the untreated trials. This suggested that the usable cellulose in the sludge was likely consumed or affected first during the activated sludge and then the anaerobic digestion processes. 4.2. Reducing sugar yields from KOH pre-treated feedstocks Besides waste paper, other sources of cellulose in wastewater might include plant residues such as grass, vegetables and wood. For these materials, alkaline treatment may be useful to extract the lignin and, thus, improve enzymatic hydrolysis. Therefore KOH pre-treatments were applied to the primary sludge, activated sludge and digested sludge feedstocks. The enzymatic hydrolysis was conducted under the same conditions as in the untreated feedstock trials and the conversion percentages of the KOH pre-treated materials are presented in Table 2.
Table 2 Reducing sugar yield from enzymatic hydrolysis of pre-treated municipal wastewater treatment process residuals. Pre-treatment application
No pre-treatment (wet) No pre-treatment (dry) KOH pre-treatment (wet) KOH pre-treatment (dry) HCl pre-treatment (wet) HCl pre-treatment (dry) HCl/KOH pre-treatments (wet) HCl/KOH pre-treatments (dry)
Reducing sugar conversion (% of dry mass) Primary sludge
Thickened waste activated sludge
Biosolids
31.1 ± 2.7 25.0 ± 0.8 35.4 ± 1.2 29.4 ± 1.7 42.6 ± 1.2 31.7 ± 1.2 54.2 ± 4.0 37.0 ± 1.0
12.9 ± 2.7 5.6 ± 1.2 11.3 ± 3.3 5.5 ± 0.6 – – – –
1.1 ± 0.1 0.5 ± 0.2 1.0 ± 0.3 0.9 ± 0.5 – – – –
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It was found that the KOH pre-treatment applied to the primary sludge increased the reducing sugar conversion for the wet substrate from 31.1 ± 2.7% to 35.4 ± 1.2% and from 25.0 ± 0.8% to 29.4 ± 1.7% for the dried substrate. The results only showed a slight increase in reducing sugar yield which would suggest that most of the cellulose content in the primary sludge is primarily paper based and that alkaline delignification pre-treatment would not be necessary and would not be expected enhance enzymatic hydrolysis significantly. It was also noted that the dried substrate (29.4 ± 1.7%) yielded less reducing sugars than the wet substrate (35.4 ± 1.2%). For the thickened waste activated sludge, the conversion was 11.3 ± 3.3% for wet substrate and 5.5 ± 0.6%. This can probably be attributed to the biodegradation of the more readily degradable organic materials by microorganisms in the activated sludge process. Enzymatic hydrolysis was also conducted on the KOH pre-treated biosolids feedstock. Once again, substrate loadings of 150 mg instead of 50 mg were employed in the enzymatic hydrolysis and all other pre-treatment and experimental conditions for KOH treatment and enzymatic hydrolysis were the same as with the primary sludge and thickened waste activated sludge feedstocks. Less than 1% of the biosolids feedstock material was converted to reducing sugars in the KOH treatment trials suggesting that the usable cellulose in the digested sludge was likely consumed first during the activated sludge and then the anaerobic digestion processes. From the untreated and KOH pre-treated trials on the primary sludge, thickened waste activated sludge and biosolids feedstocks, it was found that in all trials, the primary sludge feedstock provided significantly higher reducing sugar yields than the activated sludge and digested sludge feedstocks. It was also generally found that drying and grinding did not enhance the reducing sugar yield from enzymatic hydrolysis. The results would suggest that the cellulose content in the primary sludge, which has not been biologically processed along the wastewater treatment train as extensively as the thickened waste activated sludge or anaerobic digested biosolids, was more readily hydrolysable. Such a difference could have resulted because: (1) the cellulose may have been partially degraded in the aerobic and anaerobic biological treatment processes; (2) the aerobic and anaerobic biological treatment processes may have affected the physical structure of the cellulose fibers, rendering them less accessible to cellulase enzymes; (3) the chemicals added during the wastewater treatment process may have affected the chemical and/or physical structure of the cellulose; and (4) the chemicals or the microorganisms in the wastewater treatment process might suppress or compete with reducing sugar conversion enzyme activity. Thus, in order to be viable as a feedstock, primary sludge would be recommended for enzymatic hydrolysis to yield substantial reducing sugars and, subsequently, bio-ethanol. In light of these results, subsequent HCl pre-treatment and HCl/KOH pre-treatment trials were only conducted using the primary sludge feedstock. 4.3. Reducing sugar yields from HCl pre-treated primary sludge feedstock The metals contained in primary sludge, in particular heavy metals such as copper, zinc and nickel, may have adverse effects on the enzyme activity. In order to reduce the impact of metals on reducing sugar yield from enzymatic hydrolysis, primary sludge was subjected to HCl pre-treatment, where the metals would be dissolved and separated from the solid fraction. In addition, a KOH pre-treatment was applied to half of the HCl pre-treated primary sludge feedstock samples prior to enzymatic hydrolysis. The percent reducing sugar conversions are presented in Table 2. Enzymatic hydrolysis was not conducted on the HCl pre-treated activated sludge and biosolid feedstocks because of the low reducing
sugar yields obtained in the untreated and the KOH pre-treated experiments compared to the primary sludge feedstock. Table 2 shows that when treated with HCl, the percent conversion of primary sludge feedstock to reducing sugars was 42.6 ± 1.2% for wet substrate, which was approximately 11.5% higher than for the untreated primary sludge feesdstock. This indicated that through the HCl pre-treatment, the enzyme inhibition due to metals could potentially be reduced resulting in higher reducing sugar yield from the enzymatic hydrolysis. The dried substrate yielded a 34.2 ± 1.2% reducing sugar conversion, which was approximately 9.2% higher than that of the dried untreated primary sludge feedstock. When both HCl and KOH pre-treatments were applied to the primary sludge feedstock, the enzymatic reducing sugar conversion reached 54.2 ± 4.0% and 37.0 ± 1.0% for wet and dried substrates, respectively (Table 2). Such results indicated that acid and alkaline pre-treatments could significantly enhance the reducing sugar conversion from enzymatic hydrolysis. Moreover, it showed that considerable cellulose is contained in the primary sludge on a dry mass basis and that further research might be worthwhile to explore the potential energy (e.g., bio-ethanol) or value-added product (e.g., chemical precursors such as sugars) recovery from this organic residual feedstock. In the experiment, it was shown that if the non-cellulose content in primary sludge could be removed prior to the enzymatic hydrolysis, the reducing sugar yield would likely increase. Hence, to better isolate the cellulosic content from the non-cellulosic constituents in the primary sludge feedstock KOH, HCl, and HCl followed by KOH pre-treatments were applied. The resulting percent reducing sugar yields are summarized in Table 2. 4.4. FTIR Spectra of primary sludge, activated sludge and biosolids feedstocks Fourier transform infrared spectroscopy (FTIR) is widely used to identify the functional groups compare in complex organic mixtures and the similarities between substances. In this study, the generated spectra were only employed to identify possible differences between the municipal wastewater treatment process residues in comparison with published spectra, and these represented a qualitative analysis of the feedstocks as opposed to a quantitative analysis. The enzymatic hydrolysis indicated that there were considerable differences in the reducing sugar yield from primary sludge, thickened waste activated sludge and biosolids, which might suggest a variation in the cellulose content of these feedstocks. To further examine the functional group compositions and their differences, FTIR was performed on the primary sludge, thickened waste activated sludge and biosolids feedstocks pre-treated with KOH and the resulting spectra were compared with published literature cellulose (Cao and Tan, 2002) and lignin (Ghosh, 1998) spectra. Their corresponding spectra are shown in Fig. 2 and the interpretation of functional groups is presented in Table 3. The KOH pre-treated primary sludge, thickened waste activated sludge and biosolids feedstocks exhibited similar spectra. The main differences observed which could be associated with the lignocellulosic composition of the pre-treated feedstocks was the presence of peaks between 1600 and 1000 cm1 which were not apparent on the KOH pre-treated activated sludge and digested sludge spectra. This region is associated to the carbonyl groups including ketones, esters and aldehydes which are generally cellulose related groups. The KOH pre-treated thickened waste activated sludge and biosolids had similar spectra, with the exception that the former had a peak at 1250 cm1, which can be assigned to aliphatic compounds, which would indicated the presence of carbohydrates in the thickened waste activated sludge.
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Transmittance
Biosolids Activated Sludge Primary Sludge
4500
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber, cm-1 Fig. 2. FTIR spectra of KOH pre-treated primary sludge, thickened waste activate sludge and biosolids.
Table 3 Functional group assignments for the spectra of primary sludge, thickened waste activated sludge and biosolids. Observed peaks (cm1)
Functional group assignment
Comment
3400–3350 2915
Aliphatic primary amine NH stretching Methylene C–H asymmetric/symmetric stretching Methylene C–H asymmetric/symmetric stretching Primary amides in solid state a-Amino carboxylates R2NCH2COO Secondary amides Carboxylic acids, or aliphatic nitro compounds Aryl aldehydes, or organic sulphates Alkyl ketones CH deformation of CH3 or CH2 bending Ester C–O–C stretching vibration Ester asymmetric vibration Organic siloxane or silicone Aromatic CH out of plane bending
PAB PAB
2850 1650 1590 1540 1450–1430 1370 1320 1230 1170 1120 1060–1040 900–880
PAB PAB P PAB P PAB PAB PA P P AB PAB
Note: P – observed in the spectrum primary sludge; A – observed in the spectrum of thickened waste activated sludge; B – observed in the spectrum of biosolids.
In summary, the examination of the FTIR spectra would indicate that the cellulose content in the KOH pre-treated primary sludge was more prominent than in the KOH pre-treated thickened waste activated sludge and biosolids, which would be consistent with the higher reducing sugar yields generally observed in the primary sludge compared to the thickened waste activated sludge and biosolids feedstocks. 5. Conclusions Biosolids and sludges contain large quantities of lignocellulose, polysaccharides, proteins, and other biological materials, and the ability to convert these materials into value-added products has been recognized as an attractive technology (Industry Canada, 2004). Wet and dried primary sludge, thickened waste activated sludge and anaerobically digested sludge (biosolids) feedstocks were enzymatically hydrolyzed for 24 h at 40 °C with an enzyme loading of 800 U/g of substrate after various feedstock pre-treatments. It was found that the primary sludge feedstocks yielded sig-
nificantly higher reducing sugars than the thickened waste activated sludge and biosolids, which would suggest that the cellulosic material readily available for reducing sugar conversion in the primary sludge is consumed in activated sludge process and further degraded in the anaerobic digestion process in a wastewater treatment facility. For each feedstock, the dried substrate yielded less reducing sugars than the corresponding wet substrate, indicating that the drying process might affect subsequent enzyme access to the cellulosic fiber during enzymatic hydrolysis. The alkaline (KOH) delignification pre-treatment had little effect on the reducing sugar yield from enzymatic hydrolysis, while the acidic (HCl) pre-treatment was more effective, removing metals from the cellulosic substrate. The highest reducing sugar conversion was achieved for primary sludge when a combination of HCl followed by KOH pre-treatment was performed. FTIR analysis of the primary sludge, thickened waste activated sludge and biosolids spectra appeared to exhibit more peaks associated with cellulosic material fractions in the primary sludge when compared to the thickened waste activated sludge and biosolids spectra. Based on bio-product feasibility studies conducted on livestock manures and municipal biosolids and sludges, an estimated 6.22 Mt/yr of sugar could be produced from this waste biomass in Canada (Champagne, 2007; Wood and Layzell, 2003; Cheung and Anderson, 1997). Despite the large potential that residual and waste biomass can offer to meet Canada’s future energy and bio-products needs, there are significant hurdles that must be overcome before the large-scale use of residual and waste biomass as an energy resource becomes economically and technologically viable. Further research, as presented in this study, is critical to investigate its application beyond the laboratory-scale and to develop the necessary biotechnologies. To date, the most significant challenge of this biotechnology development is the cost of hydrolysis. This barrier may be overcome through the development of cost-effective separation, pre-treatment and conversion methodologies. The long-term benefits of this research will be to introduce innovative and sustainable solid waste management strategies; contribute to the mitigation in greenhouse gases through sustained carbon and nutrient recycling; reduce the potential for water, air and soil contamination associated with landspreading of organic wastes; broaden the source of raw materials for the emerging bio-products industry and reduce the burden on non-renewable resources such as petroleum.
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References Cao, Y., Tan, H., 2002. The properties of enzyme-hydrolyzed cellulose in aqueous sodium hydroxide. Carbohydr. Res. 337, 1453–1457. Champagne, P., 2007. The feasibility of producing bio-ethanol from agricultural waste residues: a Canadian perspective. Resour. Conserv. Recyc. 50, 211–230. Champagne, P., Levy, T., Tudoret, M.J., 2005. Recovery of value-added products from hog manure: a feasibility study. Int. J. Solid Waste Technol. Manage. 31, 147– 157. Cheung, S.W., Anderson, B.C., 1997. Laboratory investigation of ethanol production from municipal primary wastewater solids. Bioresour. Technol. 59, 81–96. Esteghlalian, A., Srivastava, V., Gilkes, N., Gregg, D., Saddler, J., 2001. Cellulase action and synergism. Am. Chem. Soc. Symp. Ser. 769, 100–111. Fan, L.T., Gharpuray, M.M., Lee, Y.H., 1987. Cellulose Hydrolysis. Springer-Verlag, Berlin. Ghosh, I., 1998. Blends of Biodegradable Thermoplastics with Lignin Esters. Master’s thesis, Virginia Polytechnic Institute and State University. Hang, Y.D., Woodams, E.E., 1998. Enzymatic production of soluble sugars from corn husk. Lebensm-Wiss. U.-Technol. 32, 208–210. Henderson, B., Champagne, P., Tudoret, M.J., Dinel, H., 2003. The chemical separation of cellulose from lignin in sugarcane bagasse. In: Proceedings of the Eighth Specialty Conference on Environment and Sustainable Engineering. 31st Annual CSCE Congress, June 4–7, Moncton, NB. Industry Canada, BioProducts Canada, 2004. Innovation Roadmap on Bio-based Feedstocks, Fuels and Industrial Products. ISBN: 0-662-36411-2. Ingesson, H., Zacchi, G., Yang, B., Esteghlalian, A.R., Saddler, J.N., 2001. The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose. J. Biotechnol. 88, 177–182. Kaya, F., Heitmann, J.A., Joyce, T.W., 1998. Effect of dissolved lignin and related compounds on the enzymatic hydrolysis of cellulose model compound. Cellul. Chem. Technol. 23, 203–213. Levy, T., Champagne, P., Tudoret, M.J., Dinel, H., 2003a. Bio-chemical integrated recycling of hog manure. In: Proceedings of the Eighth Specialty Conference on
Environment and Sustainable Engineering. 31st Annual CSCE Congress, June 4– 7, Moncton, NB. Levy, T., Champagne, P., Tudoret, M.J., Dinel, H., 2003b. Feasibility study on the recovery of commodity chemicals and agri-products from hog manure. In: Proceedings of the 18th International Conference on Solid Waste Technology and Management, March 23–26, Philadelphia, PA. Li, C., Champagne, P., 2005. Feasibility of using waste materials as feedstocks for ethanol production. Int. J. Solid Waste Technol. Manage. 31, 93–101. Lloyd, T.A., Wyman, C.E., 2005. Combined sugar yields for dilute sulfuric acid on xylan, lignin, and total mass removal from corn stovers. Bioresour. Technol. 96, 1967–1977. McMillan, J.D., 1994. Pre-treatment of lignocellulose biomass in conversion of hemicellulose hydrolysates to ethanol. In: Himmel, M.E., Baker, J.O., Overend, R.P. (Eds.), American Chemical Society Symposium. Washington, DC, pp. 292– 324. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 13, 420–428. Negro, M., Manzanares, P., Ballesteros, I., Oliva, J., 2003. Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass. Appl. Biochem. Biotechnol., 87–100. Prasad, S., Singh, A., Joshi, H.C., 2007. Ethanol as an alternative fuel from agricultural, industrial and urban residues. Resour. Conserv. Recyc. 50, 1–39. Saha, B., Dien, B., Bothast, R., 1998. Fuel ethanol production from corn fiber: current status and technical prospects. Appl. Biochem. Biotechnol. 70, 115–125. Schell, D.J., Farmer, J., Newman, M., McMillan, J.D., 2003. Dilute sulfuric acid pretreatment of corn stover in a pilot-scale reactor: investigation of yields, kinetics, and solids enzymatic digestibilities. Appl. Biochem. Biotechnol. 105, 69–86. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic material for ethanol production: a review. Bioresour. Technol. 83, 1–11. Ward, O., Singh, A., 2002. Bioethanol technology: developments and perspectives – VI cellulase production. Adv. Appl. Microbiol. 51, 60–63. Wood, S., Layzell, D.B., 2003. A Canadian Biomass Inventory: Feedstocks for a Biobased Economy”.. Industry Canada Contract #5006125. BIOCAP Foundation.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars Pascale Champagne a,*, Caijian Li b a b
Department of Civil Engineering, Queen’s University, Kingston, ON, Canada K7L 3N6 Department of Civil and Environmental Engineering, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
a r t i c l e
i n f o
Article history: Received 14 April 2008 Received in revised form 28 January 2009 Accepted 2 June 2009 Available online 14 July 2009 Keywords: Biosolids Enzymatic hydrolysis Sugar recovery Biomass Bio-ethanol
a b s t r a c t This study examined the hydrolysis of lignocellulose extracted from municipal wastewater treatment process residuals for the purpose of investigating low-cost feedstocks for ethanol production, while providing an alternative solid waste management strategy. Primary and thickened waste activated sludges and anaerobically digested biosolids underwent various pre-treatments to enhance subsequent enzymatic hydrolysis. Half of the pre-treated samples were dried and grinded, while the other half were used as is (wet). The wet primary sludge yielded the highest reducing sugar conversions. When wet primary sludge without pre-treatment was hydrolyzed at 40 °C and an enzyme loading of 800 U/g substrate, 31.1 ± 2.7% was converted to reducing sugars in 24 h. This increased to 54.2 ± 4.0% when HCl and KOH pre-treatments were applied. FTIR analyses were used to examine differences in the sludge compositions. These indicated that the cellulose content in the primary sludge was higher than that of the thickened waste activated sludge and biosolids, which was consistent with the higher reducing sugar yields observed in the primary sludge. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, the highly unstable global energy market, including fluctuations in oil and natural gas prices, has led Canada to assess future fuel developments and explore alternatives to fossil fuels and petroleum-based products. Municipal wastewaters sludges and biosolids contain large quantities of cellulose, lignocellulose, polysaccharides, proteins and other organic materials. Municipal biosolids and sludges are a waste biomass resulting from water and wastewater treatment processes. In 2001, a Canadian biosolids production rate of 0.063 kg dry biomass/person/day was estimated to generate biosolids at a rate of 387,166 tons/yr (Wood and Layzell, 2003). Based on a bio-product feasibility study conducted using municipal sludges/biosolids and livestock manures produced in Canada, an estimated 6.22 Mt/yr of sugar could be produced from this waste biomass for subsequent fermentation to bio-ethanol or other chemical bio-product precursors (Champagne, 2007). The conversion of this waste biomass to higher-value products has been recognized as an attractive alternative waste management solution (Industry Canada, 2004). However, the complex physical and chemical composition of municipal biosolids and sludges make them difficult to employ as feedstocks compared to other organic residues. Hence, to date, there have been limited ef* Corresponding author. Tel.: +1 613 533 3053; fax: +1 613 533 2128. E-mail address: [email protected] (P. Champagne). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.051
forts to convert this waste biomass to higher-value chemicals and energy on a commercial scale. As a result, waste biomass are an abundant but under-utilized biomass resource for producing biobased chemicals and bio-fuels. The main objectives of this study were to investigate the feasibility of using sludges and biosolids as low-cost feedstocks for the production of simple reducing sugars as an environmentally-friendly alternative to the disposal of solid waste. The investigation focused on the enzymatic hydrolysis of primary sludge, activated sludge and digested sludge under different pre-treatment conditions and assess the efficiency of the pretreatment applications in the production of simple reducing sugars for further use as building block chemicals. 2. Background Theoretically, all biomass materials are possible feedstocks for the production of higher-value chemical products. They differ in appearance and properties, however, they all have a relatively similar composition, which consists mainly of cellulose, hemicellulose and lignin. The two carbohydrate polymers, cellulose and hemicellulose, form the main structure of biomass materials, whereas lignin acts as a joining material and binds the fibers together. Although extraneous compounds are present in small quantities, they play an important role in structural stabilization, making cellulosic materials resistant to decomposition, decay and insect attack. Celluloses from various biomass sources are generally the
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same at the molecular level, however, they differ in their crystalline structures and in their binding to other components. Cellulose is a high molecular weight linear polymer. Each cellulose molecule is an unbranched chain consisting of 1000–1 million D-glucose units, linked together with b-1,4 glycosidic bonds. To utilize cellulose as a feedstock for the production of energy and higher chemicals, it is essential to break it down to glucose. The cellulose present in lignocellulosic materials is composed of crystalline and amorphous components. The amorphous component is usually more active than the crystalline component, thus, increasing the amorphous content enhances the rate of hydrolysis. The presence of lignin forms a physical barrier to enzymes. Hence, the separation of lignin from the cellulose and hemicellulose, reduction of cellulose crystallinity and swelling of cellulose-containing fiber to increase the accessible surface area to the cellulosic material, are essential prior to enzymatic hydrolysis to improve cellulose susceptibility to enzymes (McMillan, 1994). According to Sun and Cheng (2002) pre-treatment processes generally aim to achieve the following: (1) improve sugar yield from subsequent acid or enzymatic hydrolysis; (2) minimize the degradation or loss of carbohydrates; (3) minimize the formation of inhibitory byproducts for hydrolysis and fermentation processes; and (4) cost-effectiveness. There are three main categories of pre-treatment processes: physical, chemical and biological pretreatments. Depending on the biomass material, one or a combination of these methods may be used to improve the accessibility of enzyme to cellulose molecule. The effect of physical pre-treatment is to subdivide lignocellusic material into fine particles which are more susceptible to acid or enzymatic hydrolysis, and to reduce the degree of cellulose crystallinity. Chemical pre-treatments use a solvent to degrade the lignin, hemicellulose and/or cellulose crystalline structure. Alkaline hydrolysis pre-treatment cause structure swelling which increases the internal surface area, and decreases the degree of polymerization. It acts primarily through the saponification of intermolecular ester bonds crosslinking xylan hemicellulose and other components, which results in the separation of the structural linkages between the lignins and carbohydrates, and disrupt the lignin structure (Fan et al., 1987; Prasad et al., 2007). Acids primarily act as catalysts for cellulose hydrolysis rather than as a pre-treatment reagent, where the acid accelerates the rate of solubilization relative to structural degradation, resulting in higher conversion yields (Lloyd and Wyman, 2005; Prasad et al., 2007). The effect of pre-treatment on cellulose hydrolysis has been widely studied. Dilute acid pre-treatment has been demonstrated to be reliable in increasing the cellulose pore volume and enzyme accessibility (Esteghlalian et al., 2001). Schell et al. (2003) studied the hydrolysis of corn stovers with dilute sulfuric acid pre-treatment. The substrate was pre-treated with 0.5–1.4% (w/w) sulfuric acid at 165–195 °C for 3–12 min. Cellulose (80–87%) conversion was obtained during simultaneous saccharification and fermentation process. The disadvantages of acid pre-treatment are the costs associate with facility construction, as well as the handling and disposal of neutralization chemicals (Ward and Singh, 2002). A number of other pre-treatment processes, such as ammonia and alkaline peroxide treatment, have been shown to be very effective under certain conditions (Saha et al., 1998). For a certain biomass material, the preferred pre-treatment should meet the following criteria: achieve high mono sugar yields from enzymatic hydrolysis, minimize the degradation of carbohydrates and the formation of inhibitory byproducts, and be cost-effective (Negro et al., 2003). The enzymatic hydrolysis of cellulose is carried out by cellulase enzymes. There are a number of cellulase enzymes that are capable of degrading cellulose, of which the most intensively studied is the enzyme complex derived from filamentous fungus Trichoderma Reesei. Enzyme complexes generally consist of three components:
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endo-b-glucanase (EG) which attach to regions of low crystallinity in the cellulose fiber, generating free chain ends; exo-b-glucanase or cellobio-hydrolase (CBH) which degrade cellulose molecules further by removing cellobiose units from the free chain; and, bglucosidase which hydrolyze cellobiose to produce glucose (Prasad et al., 2007). Each component is essential to synergistically hydrolyze cellulose efficiently and completely. The enzymatic hydrolysis of cellulose takes place in three stages: (1) the adsorption of the cellulase enzyme complex onto the surface of the cellulose structure; (2) the degradation of cellulose into mono sugars; and (3) the desorption of cellulase. The rate and extent of cellulose hydrolysis by cellulase enzymes is influenced by many substrate and enzyme factors, as well as operational conditions. Because of the heterogeneous nature of the process, the overall reaction rate can be affected by mass transfer resistance; including the surface film resistance around cellulose particles, the bulk phase resistance, and the resistance through the capillary pores of the cellulose particles. Consequently, the reaction conditions such as the size of cellulose particles, the substrate concentration, enzyme concentration, pH of the buffer, and agitation intensity will influence the hydrolysis rate. At low cellulose concentrations, increases in cellulose concentrations generally lead to increases in glucose yield and reaction rates, while at high cellulose concentrations, substrate inhibition can result leading to a decrease in the rate of hydrolysis (Cheung and Anderson, 1997). Hang and Woodams (1998) have shown that at pH 5.0, the sugar yields from corn husks were higher than those at pH 4.0 and 7.0. They also found that at 40 °C, 50 °C and 60 °C, the total sugar yields were slightly different. However, significantly more glucose and xylose were converted at higher temperature. In addition, very high substrate concentrations can slow down the hydrolysis due to the inhibition from the end product. Finally, the high viscosity of slurry may also limit the mass transfer within the reaction mixture (Ingession et al., 2001). Moreover, excessive mixing speeds (>200 rpm) decrease the extent of hydrolysis because the enzyme activity is lowered. The lignin content appears as a barrier to the enzymes, but it is not always undesirable. Kaya et al. (1998) found that the addition of dissolved lignin improved the enzymatic reaction, because it was thought to bind to the enzymes in solution, then the bound enzymes were sustained and hydrolyzed the adjacent cellulose easily. 3. Methods Municipal wastewaters, sludges and biosolids include fecal materials, scraps of toilet paper, and food residues, and as such, a reasonable cellulose content is expected from these organic residuals. The various pre-treatments applied to the primary sludge, thickened waste activated sludge and anaerobically digested biosolids used as lignocellulosic feedstocks in this experimental investigation are illustrated in Fig. 1. All experiments were conducted in triplicate, and corresponding experimental means and standard deviations were reported. 3.1. Feedstock material collection and preparation Primary sludge, thickened waste activated sludge and biosolids from the anaerobic digestion process were collected from Robert O. Pickard Environmental Centre (ROPEC), a secondary wastewater treatment plant that treats dilute municipal wastewater and discharges to the Ottawa River in Ottawa (Canada). The average characteristics of these feedstocks are provided in Table 1. On average, 545,000 m3/d of wastewater is processed with average primary effluent characteristics of 110 mg cBOD5/L, 200 mg COD/L, 150 mg SS/L. ROPEC has preliminary and primary treatment
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The primary sludge and thickened waste activated sludge samples were transported in 15 L sealed plastic buckets and used directly in the experiments, while the biosolids were collected and stored refrigerated at 4 °C in sealed plastic bag. Because of the high water content in each of the feedstocks, the samples were first centrifuged to remove the liquid. For each feedstock, 2 L was divided into four 750 mL Nalgene bottles and centrifuged for 10 min at 4700 rpm. The supernatant was discarded, and the solid fraction was washed with distilled water and its pH adjusted to 7.0 using 1.0 N HCl. The mixture was then centrifuged once more to remove the excess liquid and the solid fraction was stored at 4 °C for use in subsequent pre-treatment and enzymatic hydrolysis testing.
Lignocellulosic feedstock
Solid/liquid separation
Solid fraction
EH
Dry
KOH treatment
HCl treatment
EH EH
Dry
EH
EH
Dry
3.2. Lignocellulosic feedstock pre-treatment
KOH treatment
EH EH
Dry
EH
Fig. 1. Schematic representation of pre-treatments tested for the primary sludge, thickened waste activated sludge and biosolids feedstocks prior to enzymatic hydrolysis (EH).
followed by a conventional aerobic activated sludge unit operated at an average sludge retention time of 5 days. The waste activated sludge was characterized as young sludge based on the 5-day solids retention time of the activated sludge unit and supported by the relatively high volatile solids (VS)/total solids (TS) ratio of approximately 0.70. Ferric chloride is added to the waste activated sludge for phosphorous removal prior to waste activated sludge thickening. The thickened waste activated sludge and primary sludge are blended in a 58:42 v/v ratio and undergo mesophilic anaerobic sludge digestion to produce a stabilized biosolids product for disposal. The anaerobically digested sludges employed in this study was the final product of mesophilic sludge digesters, dewatered to 30% TS (w/w) at ROPEC. Table 1 Robert O. Picard Environmental Centre (Ottawa, Canada) average primary effluent and primary and thickened waste activated sludge characteristics. Characteristics
Primary sludge
Thickened waste activated sludge
pH TS (% w/w) VS (% w/w) VS/TS (%) TCOD (mg/L) SCOD (mg/L) SCOD/TCOD (%) NH3–N (mg/L) Alkalinity (mg/L as CaCO3) TVFA (mg/L) Ash (% TS) VS (% TS) Protein (% TS) Cellulose (% TS) Fats and grease (% TS)
6.9 4.16 0.33 7.9 52,614 1289 2.4 145 220 914 27.1 72.9 31.7 29.3 22.6
6.49 5.4 3.77 70 41,667 2,357 5.7 536 919 913 21.3 78.7 18.2 13.8 17.5
Flow (m3/d) CBOD5 (mg/L) COD (mg/L) SS (mg/L) SP (mg/L) TN (mg/L)
Primary effluent 545,000 110 200 150 2 20
Note: TS, VS, SS: total, volatile, suspended solids; SCOD, TCOD, COD: soluble, total chemical oxygen demand; CBOD5: carbonaceous 5-day biochemical oxygen demand; SP: soluble phosphorous; TN, NH3–N: total, ammonia nitrogen; TVFA: total volatile fatty acids (sum of acetic, propionic, and butyric acids).
Alkaline (KOH) pre-treatment was used in some trials to delignify the lignocellulosic material. It was assumed that the metal content in the feedstocks could potentially impact enzyme activity. Therefore, a HCl pre-treatment was applied in some trials to reduce the metal content. Procedures for organic residual pre-treatments were elaborated in previous studies (Champagne et al., 2005; Li and Champagne, 2005; Levy et al., 2003a,b; Henderson et al., 2003), where an alkaline pre-treatment of the organic residual feedstocks with 0.5 N KOH at 70 °C was found adequate for organic waste residual delignification to isolate the solid fraction which consists mainly of cellulose and pre-treatment with 1.0 N HCl at 20 °C resulted in significant metal removals. Enzymatic hydrolysis was conducted using the products derived from the four pre-treatment trials: untreated feedstocks, HCl pre-treated feedstocks, KOH pre-treated feedstocks and HCl followed by KOH pre-treated feedstocks. Enzymatic hydrolysis was applied to both wet and dried (oven dried at 70 °C for 48 h) products of each of the pre-treatment trials. The untreated and pre-treated feedstock products formed large agglomerates during the drying process. As such, the dry products were grinded using a 20 mesh prior to enzymatic hydrolysis. Each experiment was conducted in triplicate. 3.2.1. KOH pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 0.5 N KOH solution in a 500 mL beaker for 1 h at 70 °C. The solution was then centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 1.0 N HCl to a pH of 7.0. The neutralized mixture was centrifuged again and the solid fraction was stored refrigerated at 4 °C in plastic bottles. 3.2.2. HCl pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 1.0 N HCl solution in a 500 mL beaker and stirred using a magnetic stirring bar on a hot plate at 20 °C for 24 h. The mixture was then centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 0.5 N KOH. The neutralized sample was centrifuged again, and the solid fraction was kept refrigerated at 4 °C in sealed plastic bottles. 3.2.3. HCl followed by KOH pre-treatment of feedstock For each of the centrifuged feedstocks, 10 g (dry weight) of the feedstock was treated with 250 mL of 1.0 N HCl solution in a 500 mL beaker and stirred using a magnetic stirring bar on a hot plate at 20 °C for 24 h. The mixture was centrifuged for 10 min at 4700 rpm and the solid fraction retained. The solid fraction was then treated with 250 mL of 0.5 N KOH solution in a 500 mL beaker for 1 h at 70 °C. The solution was centrifuged for 10 min at 4700 rpm and the solid fraction was washed with distilled water and neutralized with 1.0 N HCl to a pH of 7.0. The neutralized
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mixture was centrifuged again and the solid fraction was stored refrigerated at 4 °C in plastic bottles. 3.3. Enzymatic hydrolysis Cellulolytic enzymes can be produced by a number of bacteria and fungi, which can use cellulose as a primary carbon source. Of all the celluloytic fungi, T. reesei has been the most extensively studied, This research was conducted using commercially available cellulase from T. reesei (Sigma–Aldrich) with a manufacturer labeled activity of 8.3 U/mg (1 U enzyme liberating 1 lmol of glucose in 1 h at 40 °C per mg of cellulase addition). The equivalent weight of 50 mg (dry mass) of untreated or pre-treated lignocellulosic primary or activated sludge feedstock or 150 mg of digested sludge feedstock and 4.8 mg of enzyme, corresponding to an enzyme loading of 800 activity units/g substrate, were added to 2.5 mL of sodium citrate buffer (pH 4.8) in a 10 mL test tube. The reactants were then placed in a 40 °C water bath and shaken at 160 rpm for a 24-h period. The tubes were then removed from the water bath and filtered using glass filters. The filtrate was transferred into a 50 mL capped plastic bottle for reducing sugar measurement. 3.4. Reducing sugar analysis The reducing sugar concentrations were analyzed using a dinitrosalicylic acid (DNS) assay (Miller, 1959): 3 mL 1% DNS solution was added to 3 mL of sample. The mixture was then boiled for 5 min and 1 mL of 40% (w/v) potassium sodium tartrate was added to stabilize the color. After cooling, the absorbance was read at 575 nm. The percentage reducing sugar yield (RSY) was calculated as follows:
RSY ð%Þ ¼
ðReducing Sugar Concentration; mg=mLÞð50 mLÞ 100% ðSubstrate added; mgÞ
3.5. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FTIR) was used to assess differences in the general functional groups of the primary sludge, thickened waste activated sludge and biosolids feedstocks. Three hundred milligrams of dry KBr was milled with a mortar and pestle, once fine enough the powder was placed in a mould and compressed into a pellet with a force 10 tons applied for 1 min. The pellet was then placed in a holder and scanned using a Bomem Michelson 120 FTIR analyzer and the background spectra were generated using Bomem Grams 386 software at Carleton University Advanced Physical Chemistry Laboratory. To obtain the spectra for each of the lignocellulosic feedstocks, centrifuged feedstock samples were first dried and grinded, then for each of the feedstocks, 5 mg of sample was mixed with 300 mg KBr and milled into a powder using a mortar and pestle. The powdered mixture was
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placed in a mould and compressed into a pellet with a force 10 tons applied for 1 min. Each sample was scanned five times between the wavelengths 4500 and 650 cm1. The generated spectra were compared to published FTIR spectra for cellulose (Cao and Tan, 2002) and lignin (Ghosh, 1998) to identify the major functional groups.
4. Results and discussions 4.1. Reducing sugar yields from non-pre-treated feedstocks The percent conversions to reducing sugar based on the dry mass of lignocellulosic material yielded for each of the untreated and pre-treated lignocellulosic feedstocks are presented in Table 2. The cellulose content of the primary sludge is mainly from undigested waste paper, which has a high cellulose content and comparatively low lignin content. Table 2 indicates that for the primary sludge feedstock, 31.1 ± 2.7% and 25.0 ± 0.8% of the wet and dry substrate, respectively, was converted to reducing sugars with no pre-treatment. The percent reducing yields for thickened waste activated sludge also showed significant differences between wet (12.9 ± 2.7%) and dry (5.6 ± 1.2%) substrates, suggesting that the cellulose fibers in the activated sludge might also become less readily accessible to enzymes as a results of the drying and grinding processes. While this appears to be contrary to findings reported in other studies, the results could be attributed to the fact that in sludges, a large fraction of the cellulosic or lignocellulosic material is paper-based and has generally been processed and delignified. Hence, mechanical treatments such as drying and grinding would not necessarily enhance enzymatic hydrolysis, and could in fact shrink or collapse the existing fibers making them less readily accessible to the enzymes. For the enzymatic hydrolysis conducted on the untreated biosolids, initial substrate loadings were increased from 50 mg (dry mass) to 150 mg (dry mass) to obtain measurable reducing sugar yields. It was found that for the biosolids feedstock approximately 1% or less of the initial feedstock was converted to reducing sugars in the untreated trials. This suggested that the usable cellulose in the sludge was likely consumed or affected first during the activated sludge and then the anaerobic digestion processes. 4.2. Reducing sugar yields from KOH pre-treated feedstocks Besides waste paper, other sources of cellulose in wastewater might include plant residues such as grass, vegetables and wood. For these materials, alkaline treatment may be useful to extract the lignin and, thus, improve enzymatic hydrolysis. Therefore KOH pre-treatments were applied to the primary sludge, activated sludge and digested sludge feedstocks. The enzymatic hydrolysis was conducted under the same conditions as in the untreated feedstock trials and the conversion percentages of the KOH pre-treated materials are presented in Table 2.
Table 2 Reducing sugar yield from enzymatic hydrolysis of pre-treated municipal wastewater treatment process residuals. Pre-treatment application
No pre-treatment (wet) No pre-treatment (dry) KOH pre-treatment (wet) KOH pre-treatment (dry) HCl pre-treatment (wet) HCl pre-treatment (dry) HCl/KOH pre-treatments (wet) HCl/KOH pre-treatments (dry)
Reducing sugar conversion (% of dry mass) Primary sludge
Thickened waste activated sludge
Biosolids
31.1 ± 2.7 25.0 ± 0.8 35.4 ± 1.2 29.4 ± 1.7 42.6 ± 1.2 31.7 ± 1.2 54.2 ± 4.0 37.0 ± 1.0
12.9 ± 2.7 5.6 ± 1.2 11.3 ± 3.3 5.5 ± 0.6 – – – –
1.1 ± 0.1 0.5 ± 0.2 1.0 ± 0.3 0.9 ± 0.5 – – – –
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It was found that the KOH pre-treatment applied to the primary sludge increased the reducing sugar conversion for the wet substrate from 31.1 ± 2.7% to 35.4 ± 1.2% and from 25.0 ± 0.8% to 29.4 ± 1.7% for the dried substrate. The results only showed a slight increase in reducing sugar yield which would suggest that most of the cellulose content in the primary sludge is primarily paper based and that alkaline delignification pre-treatment would not be necessary and would not be expected enhance enzymatic hydrolysis significantly. It was also noted that the dried substrate (29.4 ± 1.7%) yielded less reducing sugars than the wet substrate (35.4 ± 1.2%). For the thickened waste activated sludge, the conversion was 11.3 ± 3.3% for wet substrate and 5.5 ± 0.6%. This can probably be attributed to the biodegradation of the more readily degradable organic materials by microorganisms in the activated sludge process. Enzymatic hydrolysis was also conducted on the KOH pre-treated biosolids feedstock. Once again, substrate loadings of 150 mg instead of 50 mg were employed in the enzymatic hydrolysis and all other pre-treatment and experimental conditions for KOH treatment and enzymatic hydrolysis were the same as with the primary sludge and thickened waste activated sludge feedstocks. Less than 1% of the biosolids feedstock material was converted to reducing sugars in the KOH treatment trials suggesting that the usable cellulose in the digested sludge was likely consumed first during the activated sludge and then the anaerobic digestion processes. From the untreated and KOH pre-treated trials on the primary sludge, thickened waste activated sludge and biosolids feedstocks, it was found that in all trials, the primary sludge feedstock provided significantly higher reducing sugar yields than the activated sludge and digested sludge feedstocks. It was also generally found that drying and grinding did not enhance the reducing sugar yield from enzymatic hydrolysis. The results would suggest that the cellulose content in the primary sludge, which has not been biologically processed along the wastewater treatment train as extensively as the thickened waste activated sludge or anaerobic digested biosolids, was more readily hydrolysable. Such a difference could have resulted because: (1) the cellulose may have been partially degraded in the aerobic and anaerobic biological treatment processes; (2) the aerobic and anaerobic biological treatment processes may have affected the physical structure of the cellulose fibers, rendering them less accessible to cellulase enzymes; (3) the chemicals added during the wastewater treatment process may have affected the chemical and/or physical structure of the cellulose; and (4) the chemicals or the microorganisms in the wastewater treatment process might suppress or compete with reducing sugar conversion enzyme activity. Thus, in order to be viable as a feedstock, primary sludge would be recommended for enzymatic hydrolysis to yield substantial reducing sugars and, subsequently, bio-ethanol. In light of these results, subsequent HCl pre-treatment and HCl/KOH pre-treatment trials were only conducted using the primary sludge feedstock. 4.3. Reducing sugar yields from HCl pre-treated primary sludge feedstock The metals contained in primary sludge, in particular heavy metals such as copper, zinc and nickel, may have adverse effects on the enzyme activity. In order to reduce the impact of metals on reducing sugar yield from enzymatic hydrolysis, primary sludge was subjected to HCl pre-treatment, where the metals would be dissolved and separated from the solid fraction. In addition, a KOH pre-treatment was applied to half of the HCl pre-treated primary sludge feedstock samples prior to enzymatic hydrolysis. The percent reducing sugar conversions are presented in Table 2. Enzymatic hydrolysis was not conducted on the HCl pre-treated activated sludge and biosolid feedstocks because of the low reducing
sugar yields obtained in the untreated and the KOH pre-treated experiments compared to the primary sludge feedstock. Table 2 shows that when treated with HCl, the percent conversion of primary sludge feedstock to reducing sugars was 42.6 ± 1.2% for wet substrate, which was approximately 11.5% higher than for the untreated primary sludge feesdstock. This indicated that through the HCl pre-treatment, the enzyme inhibition due to metals could potentially be reduced resulting in higher reducing sugar yield from the enzymatic hydrolysis. The dried substrate yielded a 34.2 ± 1.2% reducing sugar conversion, which was approximately 9.2% higher than that of the dried untreated primary sludge feedstock. When both HCl and KOH pre-treatments were applied to the primary sludge feedstock, the enzymatic reducing sugar conversion reached 54.2 ± 4.0% and 37.0 ± 1.0% for wet and dried substrates, respectively (Table 2). Such results indicated that acid and alkaline pre-treatments could significantly enhance the reducing sugar conversion from enzymatic hydrolysis. Moreover, it showed that considerable cellulose is contained in the primary sludge on a dry mass basis and that further research might be worthwhile to explore the potential energy (e.g., bio-ethanol) or value-added product (e.g., chemical precursors such as sugars) recovery from this organic residual feedstock. In the experiment, it was shown that if the non-cellulose content in primary sludge could be removed prior to the enzymatic hydrolysis, the reducing sugar yield would likely increase. Hence, to better isolate the cellulosic content from the non-cellulosic constituents in the primary sludge feedstock KOH, HCl, and HCl followed by KOH pre-treatments were applied. The resulting percent reducing sugar yields are summarized in Table 2. 4.4. FTIR Spectra of primary sludge, activated sludge and biosolids feedstocks Fourier transform infrared spectroscopy (FTIR) is widely used to identify the functional groups compare in complex organic mixtures and the similarities between substances. In this study, the generated spectra were only employed to identify possible differences between the municipal wastewater treatment process residues in comparison with published spectra, and these represented a qualitative analysis of the feedstocks as opposed to a quantitative analysis. The enzymatic hydrolysis indicated that there were considerable differences in the reducing sugar yield from primary sludge, thickened waste activated sludge and biosolids, which might suggest a variation in the cellulose content of these feedstocks. To further examine the functional group compositions and their differences, FTIR was performed on the primary sludge, thickened waste activated sludge and biosolids feedstocks pre-treated with KOH and the resulting spectra were compared with published literature cellulose (Cao and Tan, 2002) and lignin (Ghosh, 1998) spectra. Their corresponding spectra are shown in Fig. 2 and the interpretation of functional groups is presented in Table 3. The KOH pre-treated primary sludge, thickened waste activated sludge and biosolids feedstocks exhibited similar spectra. The main differences observed which could be associated with the lignocellulosic composition of the pre-treated feedstocks was the presence of peaks between 1600 and 1000 cm1 which were not apparent on the KOH pre-treated activated sludge and digested sludge spectra. This region is associated to the carbonyl groups including ketones, esters and aldehydes which are generally cellulose related groups. The KOH pre-treated thickened waste activated sludge and biosolids had similar spectra, with the exception that the former had a peak at 1250 cm1, which can be assigned to aliphatic compounds, which would indicated the presence of carbohydrates in the thickened waste activated sludge.
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Transmittance
Biosolids Activated Sludge Primary Sludge
4500
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber, cm-1 Fig. 2. FTIR spectra of KOH pre-treated primary sludge, thickened waste activate sludge and biosolids.
Table 3 Functional group assignments for the spectra of primary sludge, thickened waste activated sludge and biosolids. Observed peaks (cm1)
Functional group assignment
Comment
3400–3350 2915
Aliphatic primary amine NH stretching Methylene C–H asymmetric/symmetric stretching Methylene C–H asymmetric/symmetric stretching Primary amides in solid state a-Amino carboxylates R2NCH2COO Secondary amides Carboxylic acids, or aliphatic nitro compounds Aryl aldehydes, or organic sulphates Alkyl ketones CH deformation of CH3 or CH2 bending Ester C–O–C stretching vibration Ester asymmetric vibration Organic siloxane or silicone Aromatic CH out of plane bending
PAB PAB
2850 1650 1590 1540 1450–1430 1370 1320 1230 1170 1120 1060–1040 900–880
PAB PAB P PAB P PAB PAB PA P P AB PAB
Note: P – observed in the spectrum primary sludge; A – observed in the spectrum of thickened waste activated sludge; B – observed in the spectrum of biosolids.
In summary, the examination of the FTIR spectra would indicate that the cellulose content in the KOH pre-treated primary sludge was more prominent than in the KOH pre-treated thickened waste activated sludge and biosolids, which would be consistent with the higher reducing sugar yields generally observed in the primary sludge compared to the thickened waste activated sludge and biosolids feedstocks. 5. Conclusions Biosolids and sludges contain large quantities of lignocellulose, polysaccharides, proteins, and other biological materials, and the ability to convert these materials into value-added products has been recognized as an attractive technology (Industry Canada, 2004). Wet and dried primary sludge, thickened waste activated sludge and anaerobically digested sludge (biosolids) feedstocks were enzymatically hydrolyzed for 24 h at 40 °C with an enzyme loading of 800 U/g of substrate after various feedstock pre-treatments. It was found that the primary sludge feedstocks yielded sig-
nificantly higher reducing sugars than the thickened waste activated sludge and biosolids, which would suggest that the cellulosic material readily available for reducing sugar conversion in the primary sludge is consumed in activated sludge process and further degraded in the anaerobic digestion process in a wastewater treatment facility. For each feedstock, the dried substrate yielded less reducing sugars than the corresponding wet substrate, indicating that the drying process might affect subsequent enzyme access to the cellulosic fiber during enzymatic hydrolysis. The alkaline (KOH) delignification pre-treatment had little effect on the reducing sugar yield from enzymatic hydrolysis, while the acidic (HCl) pre-treatment was more effective, removing metals from the cellulosic substrate. The highest reducing sugar conversion was achieved for primary sludge when a combination of HCl followed by KOH pre-treatment was performed. FTIR analysis of the primary sludge, thickened waste activated sludge and biosolids spectra appeared to exhibit more peaks associated with cellulosic material fractions in the primary sludge when compared to the thickened waste activated sludge and biosolids spectra. Based on bio-product feasibility studies conducted on livestock manures and municipal biosolids and sludges, an estimated 6.22 Mt/yr of sugar could be produced from this waste biomass in Canada (Champagne, 2007; Wood and Layzell, 2003; Cheung and Anderson, 1997). Despite the large potential that residual and waste biomass can offer to meet Canada’s future energy and bio-products needs, there are significant hurdles that must be overcome before the large-scale use of residual and waste biomass as an energy resource becomes economically and technologically viable. Further research, as presented in this study, is critical to investigate its application beyond the laboratory-scale and to develop the necessary biotechnologies. To date, the most significant challenge of this biotechnology development is the cost of hydrolysis. This barrier may be overcome through the development of cost-effective separation, pre-treatment and conversion methodologies. The long-term benefits of this research will be to introduce innovative and sustainable solid waste management strategies; contribute to the mitigation in greenhouse gases through sustained carbon and nutrient recycling; reduce the potential for water, air and soil contamination associated with landspreading of organic wastes; broaden the source of raw materials for the emerging bio-products industry and reduce the burden on non-renewable resources such as petroleum.
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