Protein recovery from sludge: A review

Journal Pre-proof Protein recovery from sludge: a review

Keke Xiao, Yan Zhou PII:

S0959-6526(19)34243-X

DOI:

https://doi.org/10.1016/j.jclepro.2019.119373

Reference:

JCLP 119373

To appear in:

Journal of Cleaner Production

Received Date:

10 July 2019

Accepted Date:

18 November 2019

Please cite this article as: Keke Xiao, Yan Zhou, Protein recovery from sludge: a review, Journal of Cleaner Production (2019), https://doi.org/10.1016/j.jclepro.2019.119373

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Protein recovery from sludge: a review Keke Xiaoa,b,c,d, Yan Zhoud,e* a

School of Environmental Science & Engineering, Huazhong University of Science and

Technology, Wuhan, Hubei, 430074, P.R. China b

DVGW-Research Center at the Engler-Bunte-Institut, Water Chemistry and Water

Technology, Karlsruhe Institute of Technology, Engler-Bunte-Ring 9, 76131 Karlsruhe, Germany c

Karlsruhe Institute of Technology, Engler-Bunte-Institut, Water Chemistry and Water

Technology, Engler-Bunte-Ring 9, 76131 Karlsruhe, Germany dAdvanced

Environmental Biotechnology Centre, Nanyang Environment and Water

Research Institute, Nanyang Technological University, 1 Cleantech Loop, Singapore 637141, Singapore e

School of Civil and Environmental Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798, Singapore * Corresponding Author. Tel.: +65 67906103. E-mail address: [email protected] (Y. Zhou)

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Abstract Protein recovery from sludge is a simple and well-studied waste-to-resource approach to convert the “wasted” sludge to useful “product”. This article reviews different pretreatment methods to accelerate the release/solubilization of protein from sludge, including physical, thermal, chemical, biological options, and their combinations. Based upon the papers reviewed, protein release/solubilization from sludge was found to be the determining step prior to recovery. The alkaline pretreatment at pH 12 was the most effective method for protein solubilization when compared with ultrasonic (1 W mL−1) and thermal (80 oC) pretreatments, if only considering the released protein concentration. Moreover, pretreatments can change protein properties (e.g., molecular weight, conformation, type, etc.), and the details were summarized. The recovered protein can be alternatives for poultry feedstuff, wood adhesive and fireextinguishing foam, and its separation from sludge liquor can considerably reduce the nitrogen load to the wastewater treatment plant and the environment. Finally, the integration of circular economy into protein recovery from sludge was proposed, and the related system boundaries for life cycle assessment of this technology were defined. Future research should focus on promoting its full-scale implementation by tackling with the technical, economic, social, and environmental challenges.

Keywords Sewage sludge; Sludge solubilisation; Pretreatment; Protein recovery; Protein separation and purification; Circular economy.

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Abbreviation Ca(OH)2

Calcium hydroxide

CaO2

Calcium peroxide

CE

Circular economy

COD

Chemical oxygen demand

Da

Dalton

EDTA

Ethylenediamine tetraacetic acid

EPS

Extracellular polymeric substances

FeO42-

Ferrate

FNA

Free nitrous acid

K2FeO4

Iron potassium oxide

KOH

Potassium hydroxide

LCA

Life cycle assessment

MW

Microwave

NaCl

Sodium chloride

NaOH

Sodium hydroxide

N.A.

The information is unavailable

nZVI

Nano zero valent iron 3

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•OH

Hydroxyl radicals

O3

Ozone

P

Phosphorus

SDS

Sodium dodecyl sulfate

SO4- •

Sulfate radicals

SRT

Solid retention time

3D-EEM

Three-dimension excitation emission matrix

TS

Total solids

TKN

Total Kjeldahl nitrogen

VFAs

Volatile fatty acids

VS

Volatile solids

VSS

Volatile suspended solids

WAS

Waste activated sludge

WWTPs

Wastewater treatment plants

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1. Introduction Huge amounts of waste activated sludge have been produced each year and the volume continues to increase due to the rapid urbanization. About 400-600 million ton wet sludge (water content of 80%) can be produced from wastewater treatment plants (WWTPs) in China each year, and this number is estimated to be up to 600-800 million ton (water content of 80%) at the year of 2020 (Leblanc et al., 2009). Nevertheless, over 80% of sewage sludge is improperly disposed and has become a source of secondary pollution to the environment (Feng et al., 2015). The high cost for sludge treatment and disposal can apportion up to 65% of the total operation cost of the wastewater treatment plant (Appels et al., 2008), making sludge treatment difficult. Many methods have been proposed for sludge treatment, e.g., agricultural application as fertilizer, landfill, incineration, anaerobic digestion, and pyrolysis (Praspaliauskas and Pedišius, 2017). As sewage sludge contains essential nutrients, e.g., 55% carbon, 15% nitrogen, and 3% phosphorus, it can improve the quality of unproductive soils after proper treatment (Thomsen et al., 2017). However, the use of sludge as agricultural fertilizer creates many environmental problems due to the presence of heavy metals (Zn, Pb, Cu, Cr, Ni, and Cd) and other toxic substances as well as pathogenic microorganisms, hence inciting negative public opinions (Houillon and Jolliet, 2005). Sludge landfill has been limited due to the lack of available sites and the generation of contaminated leachate and nidorous odor (Coste and Ayphassorho, 2001). Incineration of dried sludge has been widely applied as it can significantly reduce sludge volume and remove pathogens. The energy during incineration can also be recovered in terms of heat and electricity (Fytili and Zabaniotou, 2008). However, as is known, incineration can result in the accumulation of heavy metals in bottom ashes and the generation of dioxins and other toxicity (Gómez-Pacheco et al., 2012). For sludge pyrolysis, the strict requirement for oxygen-deficient 5

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environment and the generation of NOx precursors and tar hindered the wide application of sludge pyrolysis (Xiao et al., 2019). For anaerobic digestion, hydrolysis remains as the ratelimiting step. Although various pretreatment methods have been used to accelerate sludge hydrolysis, the increased solubilization by pretreatment does not always result in enhanced biogas production due to the formation of refractory compounds (Zhen et al., 2017). Besides the low hydrolysis efficiency, feed fluctuations (e.g., low quality of feeding sludge), low biogas production, and difficulty in biogas purification also limited the full-scale application of anaerobic digestion for sludge treatment. Therefore, the development of cost-effective and environmental-friendly technologies for sludge treatment is needed. It has been realized that sludge is typically consisted of dissolved organic matters such as nucleic acids, humic acids, proteins, and polysaccharides (Jung et al., 2002). Recovering useful biomaterials from excess sludge has received great attention recently. For example, Lee et al. (2014) reviewed VFAs production from different types of waste, the influential factors, and the post-application. Mulchandani and Westerhoff (2016) studied metals and nutrients (e.g., phosphorus and nitrogen) recovery from sewage sludge. Protein accounted for about 50% of the dry weight of bacterial cells (Shier and Purwono, 1994). The recovered protein from sludge can be used as fertilizers, adhesives or animal food (Adebayo et al., 2004), and it can be a new renewable source for both energy and resources recovery (Suáreziglesias et al., 2017), providing a different perspective in sludge management. The steps for protein recovery from sludge typically are shown in Fig. 1, starting from screening, then pretreatments for protein solubilization, subsequently exposing to filtration, and protein can be precipitated from protein solution. Finally, drying of protein precipitation and hence protein product (crude protein) can be separated and recovered (Chishti et al., 1992). 6

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For the shift of one technology from the laboratory-scale to the full-scale, the concept of circular economy (CE) must be taken into consideration. CE is one of the most effective transition methods for “ sludge-to-protein” towards a sustainable future. Incorporation of CE into protein recovery from sludge may help to prevent sludge production, reduce raw materials for protein production, and promote the reuse and recycle of the recovered protein products from full-chain processes of production, distribution, and consumption (Pradel et al., 2019). Unfortunately, most of current technologies narrowly focused on improving the treatment performance, ignoring the environmental, social, and economic impacts, thus resulting in unsustainable development of technologies for protein recovery from sludge. In fact, the research trend for sludge management is more and more being focused on life cycle assessment (LCA) of energy and nutrient recovery. Therefore, a closed cycle sludge management and strategy for protein recovery along with environmentally compatible sludge disposal need to be proposed. Yoshida et al. (2018) reviewed 44 LCA papers on sludge management, and found that 70% of the studied papers have considered sludge status as “waste-to-product” rather than “waste”. Pradel et al. (2019) used LCA from a “product” perspective rather than a “waste” perspective to evaluate the environmental impacts of phosphorus (P) recovery from sludge, and found that sludge-based P was both environmental-unfriendly and economical-unfavorable. Li et al. (2017) used LCA to compare the economic and environmental impacts of five different anaerobic digestion processes, and their results showed that energy output was the determining factor affecting the assessment results. Currently, limited studies have reviewed or discussed CEoriented protein recovery from sludge. Therefore, there is a need to evaluate the economic, social, and environmental effects of protein recovery from sludge and investigate the system boundaries for full-scale application.

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The efficiency of protein recovery is dependent on sludge type, the nature and severity of pretreatment method, and the choice of protein separation and purification method. The greatest challenge is yet to choose the proper sludge substrate and the optimum pretreatment conditions. The present study firstly attempts to systemically summarize and analyze literatures on protein recovery from sludge. The novelty of this review lies in its critical discussion on pretreatment technologies for protein solubilization from sludge, including the principal mechanism, efficiency in solubilizing protein and possible benefits/drawbacks. In addition, for the first time, the integration of CE into protein recovery from sludge was proposed based on current research trends. The underlying challenges were also discussed so as to evaluate its feasibility in full-scale application. 2. Methodology Current studies focused on the development of related technologies for protein recovery from sludge. The subject is still at its infancy due to the limitations of pretreatment technologies, political standards, and post-application. A comprehensive review of different pretreatment methods for protein solubilization from sludge flocs in other research areas like anaerobic digestion and sludge dewatering was also included in this study. The advantages and disadvantages of each pretreatment method were critically discussed. The methods for protein separation and purification were systematically reviewed. Finally, the challenges for the scale-up of protein recovery from sludge were discussed in terms of market, policy, and technology limitations. A life cycle assessment of protein recovery from sludge was proposed. Further investigation and future research attention were identified in promoting protein recovery from sludge. This article may pave the way to nutrients (e.g., protein) recovery from sludge and shed lights on technologies development for protein recovery and application. 8

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3. Sludge characterization Sludge is a solid or semi-solid residue and is often considered as a hazardous waste. Based on its origin, sludge can be divided into primary and secondary sludge, with the typical characteristics listed in Table 1. As can be seen in Table 1, the contents of phosphorus, iron, silica, pH, organic acids, energy contents, and alkalinity were different for the primary and secondary sludge. To be exemplified, the contents of iron and silica were higher in the primary sludge than those in the secondary sludge. However, an opposite trend was observed for the contents of protein and nitrogen, as they were higher in the secondary sludge than those in the primary sludge. These differences may be because that the primary sludge is mainly inorganic in nature as it is generated during primary sludge treatment processes, e.g., sedimentation, filtration, coagulation, and floatation (Markis et al., 2014). In contrast, the secondary sludge is biological in nature and it is mainly consisted of bacterial components like protein, polysaccharide, lipids, etc., as it is generated during secondary wastewater treatment processes like activated sludge treatment (Liu et al., 2019). Most studies recovered protein from sludge using secondary sludge as source rather than the primary sludge (García et al., 2017; Hwang et al., 2008; Xiao et al., 2017a), possibly due to its higher protein content, higher nitrogen content, and lower inorganic solids, grease and fats contents for easier isolation, enrichment and purification. 4. Methods for protein solubilization from sludge For protein recovery, the disruption of both extracellular polymeric substances (EPS) and intracellular materials (cytoplasm) to release soluble protein into the bulk liquid is a prerequisite (Gonze et al., 2003). In this section, physical, thermal, chemical, biological and several

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combinations of different pretreatment methods are briefly reviewed, and the main mechanisms are summarized in Fig. 2. 4.1 Physical methods 4.1.1 Ultrasonic pretreatment Ultrasonic pretreatment allows disruption of cell structure and floc matrix. Two main mechanisms for sludge disintegration and protein solubilization are associated with ultrasonic pretreatment. At a low ultrasonic frequency, the phenomenon of cavitation predominates, while at a high ultrasonic frequency, the chemical reactions with the formation of • OH and HO2• radicals may occur (Pilli et al., 2011) (Fig. 2a). The sonication time, type of ultrasonic heads, ultrasonic power, and the related frequency transmitted to heads can all affect the efficiency of ultrasonic disintegration (Bien et al., 2004). The operation conditions and treatment performance are summarized in Table 2. Neumann et al. (2017) reported that a specific energy input of 500 kJ kg-1 TS was not sufficient to generate significant sludge disruption or cause protein solubilization. The specific energy input threshold for sludge disintegration and protein solubilization was determined to be 1000-3000 kJ kg-1 TS (Bougrier et al., 2005). With increased specific energy input, the concentration of released soluble protein increased. For example, Appels et al. (2012) reported that the concentration of soluble protein can be increased to 450 mg L-1 when the specific energy input was applied at 8500 kJ kg-1 dry solids if compared with the raw sludge. Feng et al. (2009) reported that the concentration of soluble protein increased by 462 mg L-1 while the specific energy input increased up to 26000 kJ kg-1 dry solids. Considering the energy consumption, the applied specific energy is usually in the range of 1,000 to 16,000 kJ kg-1 TS for protein solubilization (Salsabil et al., 2009). The increased protein solubilization can be

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attributed to the increased enzyme activity in sewage sludge induced by ultrasonication (Neumann et al., 2017), thus promoting the shifts of extracellular proteins from inner pellet and tightly-bound EPS layers of sludge flocs to outer layers of soluble and loosely-bound EPS (Yu et al., 2008). 4.1.2 Microwave Compared to the conventional heating, microwave (MW) can heat up sludge more rapidly, increase reaction kinetics, own more operation feasibility (e.g., instant on/off control), selectively activate or suppress reaction pathways, and improve energy efficiency (Pino-Jelcic et al., 2006). The thermal effect during MW irradiation is produced with the rotation of dipole molecules in an oscillating electromagnetic filed (Fig. 2b). Typically for MW pretreatment, the microwave energy was set between 1 mm and 1 m, and the oscillation frequency was controlled in range of 0.3-300 GHz (Eskicioglu et al., 2007). Microorganism cells were disrupted and the related intracellular compounds were released due to the aforementioned rapid and energy-efficient heat generated during MW irradiation (Tang et al., 2010). Release of protein from sludge floc was observed under above thermal effects (Ahn et al., 2009; Eskicioglu et al., 2006; Yu et al., 2010). Eskicioglu et al. (2006) reported that the concentration of soluble protein increased from 50 to 100 mg L-1 when microwave pretreatment was applied with a ramping rate of 1.2°C min−1 till 96°C. The concentration of protein was higher when comparing microwave pretreatment with the traditional heating for the same sludge at temperature of 96°C. However, thermal pretreatment can also have negative effects, e.g., Maillard reactions, wherein carbohydrates react with amino acids to form high-molecular-weight melanoidins which were then difficult to be degraded. Typically, these reactions are observed to

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occur at 100°C or higher temperatures, especially in sludge wherein the concentrations of amino acids are high (Rodríguez-Abalde et al., 2011). Besides the thermal effect, MW irradiation can also induce “athermal effect” (Hong et al., 2004). It can result in breakdown of hydrogen bonds and lead to changes in the secondary and tertiary structures of the proteins of microorganisms (Tyagi and Lo, 2013). Yu et al. (2010) reported that the concentration of soluble protein increased as the microwave energy and contact time increased. The highest soluble protein concentration of 2400 mg L-1 was achieved at the microwave energy of 900 W and the contact time of 140 s. Appels et al. (2013) reported the concentration of soluble protein increased from 600 to 1600 mg L-1 when the microwave irradiation was set at 800 W for 3.5 min. The increased concentration of soluble protein after microwave irradiation may be related with the disruption of the cell wall and/or cell membrane by irradiation (Johnghwa et al., 2009). High microwave energy and long contact time may both disintegrate sludge flocs, destroy cells, and release intracellular materials into the aqueous phase (Eskicioglu et al., 2007). 4.1.3 Electrical method Electrical pretreatment has been applied to effectively disrupt the structures of sludge flocs or other less biodegradable organic matters (Yang, 2009). The electric pulse power can generate shockwave, intense ultraviolet radiation, radicals and strong electric field, thus destructing the cell wall of sludge and releasing the intracellular protein (Šunka, 2001) (Fig. 2c). The effect of electrolysis voltage on releasing protein into the aqueous phase is more significant than the electrolysis time. Yu et al. (2014) used electrochemical technique, namely electro-oxidation method, to convert the organic substances of macromolecules into the smaller ones. The

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technique can generate either •OH or indirect oxidation where a mediator is electrochemically used for the oxidation process (Comninellis, 1994). Electrochemical pretreatment method can stimulate the rate of protein solubilization (Yu et al., 2014). Zhen et al. (2014) reported that the principle of electrolysis pretreatment was dependent on the ohmic heating, electrophoresis, and electro-osmosis, thus resulting in particle disintegration and microbial cell lysis. Their results showed that the concentration of soluble protein increased from 200 to 300 mg L-1 with electrolysis was applied at 20 V for 40 min on raw WAS. 4.1.4 Other physical methods Besides the aforementioned ultrasonic, microwave, and electrical methods, other physical pretreatment methods like high pressure jet and smash, pulse power technique, and deflaker technology were also applied for protein solubilization (Table 2). These methods typically required very simple facilities for application and additional apparatus to the pipeline of sludge transportation can be therefore avoided. Kampas et al. (2007) used a deflaker technology to pretreat thickened surplus activated sludge, and their results indicated that protein concentration increased by approximately 27-fold at the treatment time of 5 min and the specific energy input of 2500 kJ kg−1 total solids (TS). This value can be further increased if continuously increasing the treatment time till more than 10 min and the specific energy input of more than 9000 kJ kg−1 TS. Choi et al. (1997) applied high pressure jet and smash on sludge sample through firstly removing sands in sludge, and then exposing sludge to smash-flat after passing through pressure gauge. After smashing sludge for 1-5 times under 5-50 bar, the protein concentration can be increased from 63-85 mg L−1 to 108-391 mg L−1. Choi et al. (2006) used an electric pulse-power technique to pretreat sludge samples at a voltage of 19 kV, a frequency of 110 Hz, and a flow

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rate of 800 mL min−1. With the generation of shockwave, intense ultraviolet radiation, electric field, and radicals, an increase of protein concentration from 13 to 68 mg L−1 can be observed.

4.2 Thermal method The mechanism of thermal pretreatment for protein solubilization from sludge is to expose sludge to elevated temperature for long enough to promote chemical reaction and solubilize larger biomolecules for both aerobic and anaerobic sludge (Fig. 2d) (Harris and Mccabe, 2015). As shown in Table 3, different types of thermal pretreatment methods such as thermal and hyper-thermal pretreatments can be used to disintegrate sludge flocs and release protein (Shi et al., 2015; Xue et al., 2015). Xue et al. (2015) reported the effects of both low/high temperature thermal pretreatments of sewage sludge. Their results showed that the concentration of soluble protein was improved under all conditions for a wide range of temperature applied. As part of the study, they particularly investigated the effects of thermal pretreatment on high solid sludge. The highest soluble protein for high solid sludge was 30,000 mg L-1 while pretreated at 180°C pretreated for 180 min. A high temperature pretreatment (130-190 oC) is usually accompanied with high pressure (6-8 bar), with heating by injection of live stream from a boiler (Shana et al., 2013). Although higher protein solubilization can be achieved, huge amounts of energy consumption, strict operational control, and high costs are often needed. High temperature can increase the quantity of accessible substrates and favor protein solubilization (Li et al., 2014). However, temperature exceeded 150°C could induce the production of recalcitrant soluble organics or toxic-inhibitory intermediates (Wilson and Novak, 2009). Lu et al. (2018) pretreated sludge samples at 172 oC, and they found that non-biodegradable steroid-like compounds and

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aromatics, e.g., benzenoids, flavonoids, pyridines, and their derivatives can also appear at this high temperature. Nowadays, the low-temperature thermal pretreatment with temperature in range of 50-90°C has gained popularity, e.g., protein solubilization at 50°C for 48 h (Nges and Liu, 2009), or at 70 C for 9 h (Ferrer et al., 2008). Yan et al. (2013) investigated the effects of mild thermal pretreatment (50-120 ̊C) on the solubilization of sludge and protein release. The results indicated that the concentration of soluble protein showed an increasing trend as the temperature increased, with the maximum released protein of 3,000 mg L-1 was achieved at 120 ̊C. García et al. (2017) proposed a new method to recover protein from sludge by hydrothermal and a maximum protein concentration of 7.2 g L-1 can be obtained after a hydrothermal pretreatment for 87 min. Besides the improved protein solubilization, the thermal pretreatment also showed improved pathogen destruction and sludge dewaterability, and removal of micropollutants, e.g., steroidal hormones (Hamid and Eskicioglu, 2013; Yu et al., 2009).

4.3 Chemical methods 4.3.1 Acid, alkaline and salt pretreatments Alkaline pretreatment is one of the most widely used chemical pretreatment methods for protein recovery from sludge (Weemaes and Verstraete, 1998) (Fig. 2e). Alkaline compounds such as sodium hydroxide (NaOH) (Chishti et al., 1992; Li et al., 2013; Liu et al., 2012; Rani et al., 2012; Sun et al., 2014; Xu et al., 2014; Yuan et al., 2006), calcium hydroxide (Ca(OH)2) (Czechowski and Marcinkowski, 2006), and potassium hydroxide (KOH) are typically used. Alkali can induce solubilization of membrane proteins, saponification of the membrane lipids, and damage of the microbial cell (Mendonca et al., 1994). Ca(OH)2 has been reported to be less 15

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effective than NaOH in protein solubilization as the bivalent cation (Ca2+) can connect cell with EPS and result in re-flocculation of dissolved organic polymers (Li et al., 2008). But it demonstrated superiority in terms of low cost, easy storage, slow solubility for long reaction time, good sludge dewaterability, and low phosphorus content in the liquid phase (Li et al., 2014). KOH has also been reported to solubilize less protein than NaOH, and the cost of KOH to obtain the similar protein content was higher than NaOH (Rani et al., 2012). However, a significant disadvantage of alkaline pretreatment is related with the irrecoverable salts, and the high residual salt concentrations posed a big challenge for the post-treatment of the remaining sludge after protein solubilization (Zheng et al., 2009). Protein recovered with alkaline pretreatment may be also not suitable for application in food industry, e.g., racemization of amino acid residues (Linder et al., 1995). Acid pretreatment has also been used to solubilize sludge for protein release (Fig. 2e). For example, Devlin et al. (2011) reported that the concentration of soluble protein increased from 50 to 450 mg L-1 with acid pretreatment at pH of 1. However, acid pretreatment was usually reported to be less effective than alkaline pretreatment for hydrolyzing biomass cells in WAS (Liu et al., 2008). In recent years, numerous researchers reported HNO2 as an efficient pretreatment method for sludge disintegration, and the true role of HNO2 in sludge disintegration was due to the effects of free nitrous acid (FNA) as it can be extraordinarily biocidal to microbes (Ma et al. 2015; Zhao et al.2015). Compared with the common acid like HCl, the use of HNO2 was more environmental-friendly as it can be reduced into N2 in the denitrification step and was more cost-effective as it can be generated in-situ by adding nitrite in a sludge reject water system (Pijuan et al. 2012). Ma et al. (2015) reported that the concentration of soluble protein increased from 4.3 to 20 mg g-1 volatile suspended solids (VSS) while treating sludge with 2.04 mg HNO216

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N L-1 for 24 h. Zhao et al. (2015) reported that the soluble protein concentration after pretreatment with 1.54 mg FNA L−1 can be as high as 1100 mg chemical oxygen demand (COD) L−1 after a 10 h treatment, which was even higher than the sludge sample pretreated at pH 10 (500 mg COD L−1) after the same treatment period. Chishti et al. (1992) investigated the effects of sodium chloride (NaCl) on the solubilization of sludge protein, and the results indicated that the concentration of soluble protein was increased from 970 to 1230 mg L-1 with NaCl concentration was increased from 0.5 to 1 mol L-1, while this only constituted nearly 12% of the total available protein in the primary sludge. If continuously increasing NaCl concentration, the concentration of soluble protein showed unobservable improvement. Only a very small amount of sludge protein was solubilized with NaCl pretreatment (Sridhar and Pillai, 1973). This may be related with the relatively lower indigenous protein contents in the primary sludge than WAS (Table 1). 4.3.2 Oxidants During the oxidation pretreatment, the reactive species, e.g., hydroxyl radicals (•OH), sulfate radicals (SO4-•) or ferrate (FeO42-, VI), are generated to promote sludge solubilization through destructing bacterial cell membranes, discharging biomass particulates, and transforming them into soluble composition (Fig. 2e). Ozone (Chu et al., 2008; Zhang et al., 2016), calcium peroxide (CaO2) (Chen et al., 2016), potassium ferrate (Wu et al., 2015; Ye et al., 2012; Zhang et al., 2012), hydrogen peroxide (H2O2) (Neyens and Baeyens, 2003), oxone (Xiao et al., 2017b), wet oxidation (Urrea et al., 2016), potassium permanganate (Wu et al., 2014), and potassium persulfate (Zhen et al., 2012) have been reported to be effective for protein solubilization from sludge (Table 4). Many researchers have reported that sludge ozonation was the most effective 17

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technique for protein solubilization as it produces less toxic by-products and is more environmental-friendly (Chu et al., 2008). Moreover, it can significantly inactivate total coliforms/Escherichia coli (E. coli) in sludge samples, thus easing the subsequent sludge hygienization for biosolids reuse in agriculture (Yu et al., 2019). Compared to other methods, cell lysis is typically accompanied with protein solubilization due to the strong oxidation effect (Tokumura et al., 2009). Moreover, it is noteworthy that higher oxidant dosage may not necessarily contribute to improve protein solubilization (Chu et al., 2009), as the oxidizing radicals may react with the solubilized protein and mineralize soluble protein (Xiao et al., 2017b). 4.3.3 Surfactants Surfactants have been widely investigated to improve protein solubilization from sludge (Fig. 2e). Sludge is known to be mainly composed of microbiologically produced protein and carbohydrate, and these biopolymers were usually absorbed onto sludge surface (Liu and Fang, 2002). The addition of surfactant could change the microbial cell structure through causing a perceptible increase in the aqueous solubility and accelerating the rate of non-aqueous phase substrate dissolution into the aqueous phase, thus making cell materials detach cell surface and dissolve the biopolymers in aqueous solution (Chen et al., 1999; Chen et al., 2004; Chen et al., 2001). The surfactants are typically added in form of sodium dodecyl sulfate (SDS) (Jiang et al., 2007). The anionic SDS is widely used in disintegrating sludge flocs as it has excellent dispersive, wetting, and emulsifying characteristics and is more economically beneficial compared to other synthetic surfactants (Ying, 2006). Through disruption and denaturation of functional groups in sludge samples, the native conformation and function of sludge flocs can be destabilized, and the biopolymers immobilized in matrix are liquefied and then dissolved into the liquid part. Kavitha et al. (2016) reported the effects of SDS on WAS can be divided into dual 18

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phases, namely floc disruption phase (EPS release), and disintegration phase (cell lysis). In the disruption phase, EPS (responsible for bio-flocculation) located in the exterior region and exocellular protein are released. In the disintegration phase, release of enormous amounts of intracellular DNA would happen when increase of SDS dosage caused cleavage of sludge biomass. 4.4 Biological methods Biological stabilization (enzymatic or microbial pretreatment) has been regarded as an attractive method for solubilizing sludge compared to chemical or physical methods as it is environmental-friendly, neither causing pollution nor requiring special equipment (Parmar et al., 2001) (Fig. 2f). Biological pretreatment can be achieved either by adding enzyme or specific microbe which can secrete certain enzymes. The commercial enzymes are very expensive, which makes the direct enzymatic method unfeasible, and therefore bio-augmentation with enzymeproducing strains isolated form the sewage sludge are considered (Yu et al., 2013). Extracellular enzymes (e.g., peroxidases, oxidoreductases, lipases, amylases, glucosidases, proteases, etc.) can be divided into ectoenzymes (cell-bound enzyme, associated with the microbial cell surface) and exoenzymes (cell-free enzyme, dispersed in the bulk liquid) (Burgess and Pletschke, 2008). The enzyme activity depends on the diffusion rate of enzyme surface active sites into the sludge matrix particles (Cadoret et al., 2002) as well as operational factors, such as substrate types and concentration, incubation time, system configuration, temperature and pH (Linder et al., 1995). The addition of lipases and the related strains, e.g., Penicillium restrictum, for removal of oil and grease (Cammarota and Freire, 2006; Masse et al., 2001) and addition of cellulose, hemicellulose, and β-glucosidase for lignocellulosic biomass pretreatment

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have been widely applied (Akao et al., 1992; Jørgensen et al., 2007; Rintala and Ahring, 1994; Romano et al., 2009; Sangave and Pandit, 2006; Sonakya et al., 2001). However, the application of enzyme pretreatment for sewage sludge is still at its infancy, and contradictory results have been reported. Sesay et al. (2006) used enzymes to extract biopolymers, e.g., protein, from activated sludge. Their results indicated that proteinase (2.8 U mg-1) from Aspergillus sp. can increase concentration of soluble protein from 0 to 1 mg g-1 VSS after 6 h incubation. However, protein can be easily degraded and converted into polypeptides, two peptides, and amino acids after enzyme hydrolysis (Ji and Brune, 2005). Ayol (2005) reported the added enzymes (e.g., protease and lipase) resulted in decreased protein concentration of the biosolids samples. Kavitha et al. (2013) added two bacterial strains (Bacillus jerish 03 and Bacillus jerish 04) into WAS sample, and the concentration of protein in sludge samples increased from 50 to 200 mg L-1.

4.5 Combined methods Table 5 summarizes the main combined pretreatments on protein solubilization from sludge. Alkaline pretreatment becomes especially effective when combined with thermal pretreatment (Neyens et al., 2004). Combined thermo-chemical method has been consistently reported to improve protein solubilization. For example, Cho et al. (2013) reported that the released protein after combined thermo-alkaline pretreatment was 2.4 times higher than that of the non-pretreated sludge and 2.1 times higher than that with the alkali pretreatment alone. Liu et al. (2008) reported that the combined thermal-alkaline can improve the concentration of crude protein by 60.2-61.6% with 7.9 g L-1, which was significantly higher than any individual method.

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Microwave and alkaline pretreatments are based on different sludge solubilization mechanisms, and the combination of these two methods can take good advantages of both methods, as alkaline pretreatment can weaken cell walls and result in more release of protein when combined with microwave. Doğan and Sanin (2009) observed a clear synergistic effect of MW + pH 12 on protein release when they investigated different pH pretreatments in association with MW pretreatment. Liu et al. (2008) combined ultrasonic-alkaline pretreatment method and found that the soluble protein was increased to 7.9 g L-1 when the ultrasonic pretreatment was set at 28 kHz at pH 12 after a 60 min pretreatment. The combined ultrasonic-alkaline pretreatment showed a synergistic effect on protein solubilization from sludge, with the ultrasonic method disintegrated sludge flocs and the subsequent alkaline pretreatment damaged microbial cells through solubilizing membrane proteins and saponifying membrane lipids (Mendonca et al., 1994). Sahinkaya (2015) reported the combined effects of ultrasonic-acid pretreatment and 1750 mg L-1 of soluble protein was observed compared to 1200 mg L-1 for ultrasonic pretreatment at original pH value of sludge. The optimum pretreatment condition was at the ultrasonic power density of 1 W mL-1 for 10 min and the initial sludge pH of 2.0. The combined ultrasonic-acid pretreatment can firstly dissipate sludge flocs by hydro-mechanical shear force and then the microbial cells released from the disrupted flocs were more effectively exposed to H+ ions. Assawamongkholsiri et al. (2013) reported that the combined acid-heat pretreatment with 0.5% (w/v) HCl for 6 h followed by a 60 min incubation at 110°C gave the highest concentration of soluble protein (580% increase in soluble protein) for WAS.

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Zhen et al. (2014) used combined electrolysis and alkaline pretreatment to solubilize protein from sludge flocs. The results showed that the prior electrolysis can crack the microbial cells trapped in sludge gels and release protein with the aid of alkaline. Electrolysis at 20 V applied voltage and pH of 11 can release 900 mg L-1 protein, while it was 250 mg L-1 at 20 V applied voltage alone and was 500 mg L-1 with alkaline pretreatment at pH 11 alone. Ultrasonication assisted chemical oxidation has been proved to be more efficient than chemical oxidation alone (Wang et al., 2011). Ning et al. (2015) reported the amounts of protein obtained with different pretreatments were in the sequence of raw sludge ≤ iron potassium oxide (K2FeO4) pretreatment ≤ ultrasonication pretreatment ≤ combined K2FeO4 and ultrasonication pretreatment. This can be related with a two-step mechanism: in the first step, the hydromechanical shear forces disintegrated sludge flocs, with extracellular and intracellular polymers were released into the liquid phase; in the second step, radicals generated during the oxidation process can further oxidize the soluble polymers to low molecular biodegradable substances. García et al. (2017) for the first time investigated the effects of hydrothermal (combination of thermal hydrolysis and wet oxidation) pretreatment on protein recovery from sewage sludge. Their results indicated that a maximum protein concentration of 7.7 g L-1 was achieved after 87 min reaction during a thermal pretreatment at 160°C and 40 bar with presence of oxidants. Chemical coupled with biological methods have been applied to increase the concentration of soluble protein. The application of chemical methods aims to release trapped EPS and disintegrate hard cell walls. The common chemical agents are ethylenediamine tetraacetic acid (EDTA), SDS, formaldehyde, and NaOH. For example, SDS can set free the immobilized organics in the flocs matrix and then the organics are consumed by the inoculated bacteria, thus

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enhancing the concentration of soluble protein. Kavitha et al. (2016) investigated surfactantstimulated thermophilic bacterial pretreatment on WAS disintegration. The added surfactant SDS firstly disintegrated sludge flocs, released trapped EPS in the solid phase into the liquid phase, thus providing more substances for the subsequent bacterial pretreatment. The measured concentration of soluble protein was significantly higher compared to those in the control groups with single SDS pretreatment or bacterial pretreatment alone. While in the combined EDTAbacterial pretreatment, EDTA can remove the cations (Ca2+, Mg2+, Fe2+ and Fe3+) from the flocs structure and disrupt the sludge matrix, thus releasing the enzymes (absorbed and immobilized) and other dissolved organic matters into the liquid phase (Wawrzynczyk et al., 2007). The subsequent biological pretreatment with bacterial strains can use the released substrates and enhance protein solubilization. Kavitha et al. (2013) investigated the combined EDTA and biological pretreatment with Bacillus jerish 03 and Bacillus jerish 04. These two strains can further enhance the concentration of soluble protein to 400 mg L-1 through combinatorial enzyme activity. Combination of chemo-mechanical pretreatment did not significantly cause increase of protein concentration. For example, Saha et al. (2011) used alkaline pretreatment to weaken the microbial cell wall and a high pressure homogenizer to lyse the cells of WAS drawn from pulp mill. The results indicated that such chemo-mechanical pretreatment was effective in solubilizing sugar (total sugar increased from 7 to 36%), while the related protein solubilization was not statistically significant. This may be due to the pulp mill WAS contains residual cellulose, hemicellulose, lignin and chemical components, thus facilitating sugar solubilization.

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4.6 Comparison of different pretreatment methods for protein solubilization It is challenging to compare all the methods due to the limited information on the cost estimation, sludge property variation, and operation scales etc. Xiao et al. (2017a) tried to compare the energy and economic benefits of ultrasonic, alkaline and thermal pretreatments for protein solubilization from WAS, with the results summarized in Table 6. As can be seen, tyrosine-like protein with molecular less than 20 kDa predominated in various sludge samples pretreated by ultrasonic, thermal, and alkaline methods (Table 6). If compared with the raw sludge without any pretreatment, the solubilized protein concentration of sludge samples was increased by 11, 23, and 12 times by ultrasonic (1 W mL−1), alkaline (pH 12), and thermal (80 oC) pretreatments, respectively. It is therefore deduced that alkaline pretreatment (pH 12) was more efficient than ultrasonic pretreatment (1 W mL−1) and thermal pretreatment (80 oC) to solubilize protein from sludge samples, accompanied with more release of both low molecular weight (< 20 kDa) and high molecular weight proteins (> 20 kDa). This may be possibly related with the relatively higher removal of protein-N in solid phase at pH 12. Comparing various methods for protein solubilization from sludge, the physical methods, e.g., ultrasonication, microwave and grinding, generally showed low operational costs and easy control, however, high energy demand should be noted (Zhen et al., 2017). Thermal method can effectively remove odor and pathogens while solubilizing sludge protein, however, high energy demand and capital cost are needed. Moreover, recalcitrant compounds would be formed and ammonia can be released during thermal pretreatment. Chemical method, particularly the alkaline pretreatment, is effective in disrupting sludge flocs and easy to be operated with simple device, however, chemical cost and the associated corrosion due to acid and alkaline addition need to be considered. Moreover, the risk of the liberated cellular materials mineralization 24

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existed when oxidants were used. The application of biological method is typically limited due to the slow start-up, mild-treatment conditions and difficulty in up-scaling (Harris and Mccabe, 2015). Xiao et al (2017a) compared the energy and economic costs of ultrasonic, alkaline and thermal pretreatments on protein solubilization from sludge. The results indicated that the chemical/energy costs for ultrasonic (1 W mL−1), alkaline (pH 12), and thermal (80 oC) pretreatments were 7.11, 0.47, and 14.76 USD ton−1 wet sludge, respectively. Regardless of the cost for installation, electricity, and personnel, the alkaline treatment at pH 12 showed the highest economic benefit with net saving of 25.57 USD ton−1 wet sludge when compared with the conventional sludge treatment and disposal method. 4.7. Changes of protein characteristics by different pretreatment methods As discussed above, although the application can significantly improve the release of protein from the solid phase to the liquid phase, it is noteworthy that the different pretreatment methods applied may destroy/change protein molecular weight, conformation, type, ect. For example, Xiao et al. (2017c) reported that the molecular weight of protein of less than 20,000 Dalton (Da) predominated after the oxidaiton process by SO4-•. Xiao et al. (2019) found that the molecular weight distribution of protein in sludge sample pretreated by centrifugation and heating was similar, and the pretreatment of centrifugation can even result in similar type of protein compared with the raw sludge. However, the protein conformation was changed by the heating method, while the centrifugation method can result in the maximum retention of the organic secondary structure. Zhong et al. (2007) investigated the effects of pulsed electric fields on the secondary structures of two typical proteins, e.g., peroxidase and polyphenol oxidase. Their results indicated that 22.63% reduction of the α-helix fraction for peroxidase was observed at 25 kV cm-1 for 124 μs while 50.72% reduction for polyphenol oxidase was noted at 25 kV cm25

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for 52 μs. Xiao et al. (2014) reported that the alkaline pretreatment at pH of 12 can cause the

unfolding of protein through destroying both the hydrogen bonding networks and disulfide bridges of proteins. Chodankar et al. (2008) investigated the structural evolution in denaturation of bovine serum albumin at different temperatures, and their results indicated that temperature more than 60 oC can change the prolate ellipsoidal shape of bovine serum albumin to a fractal structure, and its fractal dimension incresed as temperature increased. Wang et al. (2016) found that the heat-alkaline pretreatment at pH 12 for 24 h and temperature of 120 oC for 30 min can alter the main S–S stretching pattern of protein conformation from gauche-gauche-gauche to gauche-gauche-trans, thus resulting in increased protein susceptibility to proteolytic hydrolysis (76.3% versus 47.0%). With the changes of protein characteristics by different pretreatments in mind, it may be possible to recover structure-oriented protein by choosing a specific pretreatment method. 5. Protein separation and purification After solubilization, further protein separation and purification from the complex solution are necessary to separate protein from the non-protein parts of the mixture. The principle for protein separation is to disrupt the hydration shell of proteins and precipitate proteins from the liquid phase to the solid phase (Xu et al., 2018). Acids, inorganic salts, and organic solvents have been applied to precipitate protein (Sastry and Virupaksha, 1967). The common precipitating agents have been reported as hydrochloric acid, sulphuric acid, sodium lignosulphonate (Knorr et al., 1997), trichloroacetic acid, and ammonium sulphate (Chishti et al., 1992). Chishti et al. (1992) compared the efficiency of hydrochloric acid, sodium lignosulphonate, sulphuric acid, acetic acid and ammonium sulphate to precipitate protein, and they concluded that ammonium sulphate was the most effective precipitant reagent with a maximum protein recovery of 91%. García et al. 26

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(2017) also reported that the addition of ammonium sulphate was the best separation method, which can achieve 87% and 86% of protein recovery for thermal hydrolysis and wet oxidation treated sludge samples, respectively. However, this method was not capable of removing heavy metals. In contrast, pretreatment with hydrochloric acid, sulphuric acid or trichloroacetic acid between pH 3 and 4 was found to be effective in precipitating protein, as well as removing the heavy metals, with trichloroacetic acid being the best agent (Chishti et al., 1992). Similar results have been reported by Pervaiz and Sain (2011) and Hwang et al. (2008) that the highest protein precipitation can be achieved at pH 3, with the final recovered protein can be as high as 80-92%. After precipitation, further analytical purification is necessary. Many researchers have reviewed methods for protein purification in details (Janson, 2011; Scopes, 1987). Briefly, a pH graded gel or an ion exchange column can be used for protein separation from other organic compounds basing on the different isoelectric points (Yamamoto and Ishihara, 1999). The size exclusion chromatography can also be used for protein purification basing on the different size/molecular weight of proteins compared to other compounds (Kawate and Gouaux, 2006). Opiteck et al. (1998) used the high-performance liquid chromatography to separate proteins basing on their polarity/hydrophobicity. 6. Challenges for protein recovery As previously stated, there is a demand for protein recovery from sludge either for agricultural activity/food production or as value-added products (i.e., adhesives or fire extinguisher) in the market (Fig. 3). Currently, there are a few patented technologies that have been applied in practice for protein recovery from sludge. Markham and Reid (1988) used thermal method to convert biological sludge and primary float sludge to animal protein

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supplement. Rudolf et al. (1993) used combined thermal and mechanical method (high-pressure homogenizer) to extract solid matter containing protein from sludge. However, except the aforementioned two patents, most of the work on protein recovery from sludge is still at the laboratory-scale stage. System scale-up is necessary to make the solubilized protein practically feasible for industrial applications. Besides, the pretreatment methods described in previous sections can result in partial degradation of protein as indicated by the increased concentration of ammonium (Negral et al., 2013; Xue et al., 2015), thus destructing protein structure (Fig. 3). Wang et al. (2016) also reported that thermal-alkaline pretreatment altered the protein conformation and destroyed protein disulphide bonds. However, little is known about the impact of changes on the subsequent application of recovered protein. Future work is necessary to investigate the changes of protein conformation in liquid with Raman spectroscopy and the N transformation pathway with X-ray photoelectron spectroscopy. Furthermore, the application of recovered protein remains a huge barrier to its development. Currently, the recovered protein shows potentials to be used as feed stuff (Hwang et al., 2008), wood adhesive (Pervaiz and Sain, 2011), and fire extinguishing foam (Zhu, 1994). The market price of recovered protein still needs to be compared with the existing feedstuffs, e.g., corn grain, fish mill, soybean and dried yeast, however, their comparison of market price remains less clear till now. Moreover, the application of recovered protein as feedstuff still has a long way to go. It is essential to determine whether protein extracted from sludge is safe for animals based on a long-term toxicity investigation (Hwang et al., 2008). Moreover, most of current application focuses on crude protein, with the recovered protein types are rarely known so far, thus limiting its wide application. Different protein types are composed of different amino acids, thus 28

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affecting its application. Once the types of proteins can be explored, other applications of recovered protein need to be further investigated. Moreover, the post-treatment of remaining sludge after protein recovery is also a big challenge. For example, the chemicals used for protein solubilizaiton may be present in the residuals, making it difficult for the conventional treatment, e.g., composting. Unfortunately, until yet most of previous studies applying treatments to recovery sludge have mainly focused on improving crude protein recovery with hardly considering economic and environmental issues. The dosed chemicals may become an emerging environmental pollutant due to their residual levels in the remaining sludge after protein solubilisation. The cost for protein recovery is the determining factor for its scale-up in real application. It is also noteworthy that the related environmental standards and incentive policies on protein recovery from sludge are lacking (Fig. 3), thus hindering the wide application of protein recovery from sludge. With the concept of CE in mind to alleviate the contradiction between economic development and energy shortage, LCA can be proposed to evaluate the “from cradle to grave” process of protein recovery from sludge so as to evaluate the economic benefits and technical feasibility of this technology (Fig. 4). The scenarios considered for LCA included upstream processes for sludge production, transportation of sludge in the wastewater treatment plant, manufacturing (protein solubilization, separation and purification), and the post-treatment of sludge residuals (landfill or incineration). During these processes, the waste emissions to air, water, and soil, as well as the related environmental, economic, and social impacts need to be evaluated. In the future work, in order to investigate the optimum method for protein solubilization, a systematic assessment of different pretreatment options, in terms of cost balance, technique feasibility and solubilization\recovery efficiency from sludge biomass, the 29

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concentration of residual heavy metals, is essential for the industrial application. An economic system with the CE concept operated at the micro level (sludge, WWTP, protein product, company, protein consumers), meso level (eco-industrial parks), and macro level (village, city, country and beyond) should be evaluated for sustainable development for protein recovery from sludge. The related operational/environmental standards and incentive policies should be established to promote protein recovery from sludge.

7. Conclusions Through the analysis of current status for protein recovery from sludge, some conclusions could be put forward as follows: (1) This study has indicated that protein recovery from sludge can be a potential technology to convert the “wasted” sludge to useful “product”, and the recovered proteins can be suitable substrates for the production of wood adhesive, fire extinguishing agent, poultry feed stuff, etc. (2) Protein solubilization can be enhanced with the use of different pretreatment methods. If only considering the concentration of released protein, alkaline pretreatment at pH 12 was the most effective one when compared with ultrasonic (1 W mL−1) and thermal (80 oC) pretreatments. However, the extra costs for chemicals, energy, electricity, personnel, and installment need to be justifiable. (3) The application of pretreatments can also significantly change protein molecular weight, conformation, type, etc. The future work can be focused on recovering a specific protein by modifying protein structure through using a specific pretreatment method.

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(4) Besides protein solubilization, a proper choice of chemicals for protein separation and purification is also necessary. The addition of ammonium sulphate has been reported as the best separation method, followed by the pH graded gel, ion exchange column, size exclusion chromatography, and high-performance liquid chromatography for protein purification. (5) The lack of operational/environmental standards and incentive policies for protein recovery from sludge may hinder the shift of protein recovery from the laboratory-scale study to the commercial market. Pilot-scale/full-scale studies are necessary to fine-tune the technologies. Moreover, a systematic life cycle assessment of protein recovery from sludge is essential to evaluate the related economic, social, technological, and environmental impacts.

Acknowledgements The authors were grateful to the funding support of National Natural Science Foundation of China (No. 51708239) and National Key Research and Development Program of China (2018YFD1100601). Dr. Keke Xiao also acknowledges the support from Alexander von Humboldt-Stiftung. The authors acknowledge the Analysis and Testing Center of Huazhong University of Science and Technology. References Adebayo, O.T., Fagbenro, O.A., Jegede, T., 2004. Evaluation of Cassia fistula meal as a replacement for soybean meal in practical diets of Oreochromis niloticus fingerlings. Aquac. Nutri. 10(2), 99–104.

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Protein recovery Filtration

Fig. 1. The process of protein recovery from sludge.

1

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(a)

Radius (microns)

Booster Probe

Microwave Waveguide

Resonant cavity Bubble forms

Unstable state

Microwaves

Transducer

Electromagnet

Ultrasonic

Power supply

Physical treatment

(b)

Anode Cathode Violent collapse

Bubble grows

Time (μs)

Thermal

Physical treatment

(c) Electrical

DC power generator

Magnetron

Applicator Athermal

Thermal treatment

(d) More negative charges

Sludge flocs

Higher release of Total EPS contents

Multimeter

Graphite electrode

Feed tank

(e)

Sludge flocs

Oxidants

Chemical treatment

Negative charges

Biological treatment

Enzymes

Sludge flocs

O

Proteins

Microbes

CH

C Structure denaturation

EPS

(f)

•OH/SO4- •

Chemicals

Heat

More dispersive colloids with higher Great erosion of molecular weight floc structure

Anode (+)

Cathode (-)

Flash mixture

Motor Ammeter

Aerobic sludge Cool Anaerobic sludge

Mixer regulation

Oxidation to components with low molecular weight

Fig.2. Schematic of (a) ultrasonic treatment (Pilli et al. 2011), (b) microwave treatment (Afolabi and Sohail 2017), (c) electrical treatment (Veluchamy et al. 2018), (d) thermal treatment (Wang et al. 2017), (e) chemical treatment, and (f) biological treatment.

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Limitations

 Agricultural activity  Lack of environmental

 Food production  Value-added products

Markets

Trade relations

standard Policy

(adhesives)

New product introductions

Industrial policy

Technology Constraints  High investment  Low efficiency  Protein denaturation

Fig.3. Challenges for protein recovery.

3

 Lack of incentive policy

Upstream supply chain Cradle to gate

Protein solubilizaiton, separation, and purification

Use and end of life

Use process

End of treatment process Transportation

Wastewater treatment Secondary tank

Transportation Manufacturing process Waste emissions

Transportation

Waste emissions

Environmental impact

Fig. 4. The proposed life cycle assessment for protein recovery from sludge.

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Waste emissions

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Table 1. Characteristics of primary and secondary sludge, adapted from Tyagi and Lo (2013). Parameters

Primary sludge

Secondary sludge

Total solids (TS, %)

5-9

0.8-12

Volatile solids (VS, %)

60-80

59-68

Nitrogen (% TS)

1.5-4

2.4-5.0

Phosphorus (% TS)

0.8-2.8

0.5-0.7

Potash (K2O % TS)

0-1

0.5-0.7

Cellulose (%TS)

8-15

7-9.7

Iron (Fe g/kg)

2-4

-

Silica (SiO2 %TS)

15-20

-

pH

5.0-8.0

6.5-8.0

Grease and fats (%TS)

7-35

5-12

Protein (%TS)

20-30

32-41

Alkalinity (mg L-1 as CaCO3)

500-1500

580-1100

Organic acids (mg L-1 as acetate)

200-2000

1100-1700

Energy content (kJ kg-1 TS)

23,000-29,000

19,000-23,000

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Table 2. Summary of main physical pretreatments on protein solubilization from sludge. Treatment

Feed

Treatment

Increased

Protein assay

method

stock

conditions

soluble

method

References

protein (mg L-1) Ultrasonic

WAS

0-8500 kJ kg-1

450

dry solids Ultrasonic

WAS

0-26000 kJ kg-1

462

dry solids

Bicinchoninic

(Appels et al.,

acid method

2012)

Coomassie

(Feng et al.,

brilliant blue G-

2009)

250 method Ultrasonic

WAS

0-71 kJ kg-1 dry

660

solids, 20 mins Ultrasonic

Microwave

WAS

WAS

42 kHz, 120 min

32

900 W for 140s

1900

Bicinchoninic

(Gonze et al.,

acid

2003)

Bradford

(Kim et al.,

method

2003)

Coomassie

(Yu et al.,

brilliant blue G-

2010)

250 method Microwave

Microwave

WAS

WAS

50

96°C

15 min, 700 W

750

2

Bradford

(Eskicioglu et

method

al., 2006)

Kjeldahl

(Ahn et al.,

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Microwave

Deflaker

WAS

WAS

800 W, 3.5 min

1000

3000 rpm, 15

2000

mins Jet and

WAS

30 bar

215

pressure Pulse-power

WAS

17 kV, 150 Hz

55

treatment

nitrogen method

2009)

Bicinchoninic

(Appels et al.,

acid method

2013)

Bradford

(Kampas et al.,

method

2007)

Bradford

(Choi et al.,

method

1997)

Bradford

(Choi et al.,

method

2006)

Coomassie

(Yuan et al.,

Brilliant Blue

2011)

method Electrolysis

WAS

20 V,15 mins

10

G-250 method Electrolysis

WAS

20 V, 15 mins

100

Lowry method

(Zhen et al., 2014)

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Table 3. Summary of main thermal pretreatment on protein solubilization from sludge. Treatme

Feed stock

nt

Treatment

Improved

Protein assay

conditions

soluble

method

method

References

protein (mg L-1)

Thermal

WAS

55°C, sludge

202

Lowry method

2014)

alkaline Thermal

Dewatered

120 ̊C; 1 h

(Li et al.,

1550

WAS

Kjeldahl

(Zhang et

nitrogen

al., 2015)

method Thermal

Mixed

7 bar pressure,

primary and secondary

N.A.

Estimated from

(Shana et

170 ̊C for 30

nitrogen value by

al., 2013)

mins

multiplying a

sludge

conversion factor of 6.25

Thermal

Dewatered

180 ̊C, 180

high

min;

27,500

Lowry

(Xue et al.,

method

2015)

Lowry

(Yan et al.,

method

2013)

solid sludge Thermal

WAS

120°C for 30

2,993

min Noted: “N.A.” denotes the information is not available.

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Table 4. Summary of main chemical pretreatment on protein solubilization from sludge. Treatment

Feed-stock

method

Treatment

Improved

Protein assay

conditions

soluble

method

References

protein concentration (mg L-1) CaO2

WAS

100 mg

-

3D-EEM

CaO2 g-1

(Chen et al., 2016)

TSS NaOH

80% primary

0.1 mol L-1

sludge, 20%

NaOH

2.2

Dairy waste

method pH 11

-

Lowry method

activated sludge NaOH

Primary sludge

(Li et al., 2013)

brilliant blue

biofilm sludge NaOH

Coomassie

(Rani et al., 2012a)

pH 12

-

Multiplying total

(Chishti et al.,

organic nitrogen

1992)

by 6.25

NaOH

Proteinaceous

pH

12,000

Lowry method

sewage sludge

NaOH

(Liu et al., 2012)

Dewatered

80 mg

activated sludge

NaOH, 4

900

5

Coomassie

(Shi et al.,

brilliant blue

2015)

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̊C, 24 h NaOH

WAS

0.92 ± 0.01

method 4,100

g NaOH g1

NaOH

WAS

VSS

Bicinchoninic

(Sun et al.,

acid protein assay

2014)

kit

pH 10

337

Bradford method

(Xu et al.,

with bovine

2014)

serum albumin as standard NaOH

WAS

pH 11

1,010

Lowry method

(Yuan et al., 2006)

Ca(OH)2

Raw primary

24% (w/w)

sludge

Ca(OH)2,

-

-

(Czechowski and Marcinkowski, 2006)

KOH SDS

WAS

0.3 g SDS

545

Soluble protein:

(Jiang et al.,

g-1 dry

Lowry-Folin

2007)

sludge

method; total protein: estimated from TKN (1.5*(TKN – inorganic nitrogen)/0.16)

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SDS

WAS

0.02 g

30

SDS g-1 SS O3

WAS

0.16 g O3

-

Soluble protein:

(Kavitha et al.,

Lowry method

2014)

Bradford

(Chu et al.,

g-1 TS O3

WAS

0.1 g O3 g1

O3

Pharmaceutical waste sludge

K2FeO4

WAS

2008) 45

TS

(Pei et al., 2016)

0.1 g O3 g1

-

207

-

TS

(Pei et al., 2016)

40 mg g-1

63

SS

Modified Lowry

(Ye et al.,

method with

2012)

casein as standard K2FeO4

WAS

1200 mg

7

Coomassie

(Zhang et al.,

L-1 sludge,

Brilliant Blue G-

2012)

pH

250

controlled at 3 K2FeO4

WAS

1000 mg

23

L-1 HCl

WAS

pH 1

400

Modified Lowry

(Wu et al.,

method

2015)

a RC-DC protein

(Devlin et al.,

assay kit (Biorad,

2011)

UK) HNO2

WAS

2.04 mg

182

HNO2-N

7

Lowry-Folin

(Ma et al.,

method

2015)

Journal Pre-proof

L-1 for 24 h HNO2

WAS

1.54 mg

667

Lowry method

FNA L-1

(Zhao et al., 2015)

for 12 h NaCl

Primary sludge

3N NaCl,

-

Multiplying total

(Chishti et al.,

incubated

organic nitrogen

1992)

for 4 h

by 6.25

Noted: “N.A.” denotes the information is not available.

8

Journal Pre-proof

Table 5. Summary of main combined pretreatment on protein solubilization from sludge. Treatment

Feed-stock

method

Treatment

Improved concentration

Protein

conditions

of soluble protein

assay

(mg L-1)

method

1,350

Lowry-

Thermal-

Mixture of 40%

pH 12，60 ̊C

alkaline

primary sludge

for 60 mins

References

(Cho et al., 2013)

Folin

and 60%

method

secondary sludge Thermal-

Dairy waste

pH 12, 60 ̊C for

alkaline

activated sludge

60 mins

Thermal-

Waste activated

0.5% (w/v)

acid

sludge

HCl, 110 ̊C for

2,000

Lowry’s

(Rani et al., 2012b)

method 8,100

Lowry’s

(Assawamongkholsiri

method

et al., 2013)

Lowry’s

(Doğan and Sanin,

method

2009)

Lowry’s

(Kavitha et al., 2016)

60 min Alkaline-

Waste activated

Microwave for

microwave

sludge

16 min,

1,250

ultrasonic power of 600 W SDS-

Waste activated

SDS of 0.03 g-1

bacterial

sludge

SS, bacterial

300

method

strain of 2 g dry cell weight L-1 EDTA-

Waste activated

0.2 g-1 SS

200 9

Lowry’s

Journal Pre-proof

bacterial

sludge

EDTA,

method

(Kavitha et al., 2013)

Lowry’s

(Liu et al., 2008)

thermophilic bacterial strain of 2 g dry cell weight L-1 Ultrasonic-

Waste activated

Ultrasonic at 28

alkaline

sludge

kHz for 60 min,

7,900

method

pH 12 Ultrasonic-

Textile dyeing

0.5936 g-1 SS

K2FeO4

sludge

K2FeO4 for 60

Brilliant

min, and US of

Blue G-250

0.72 W mL-1

method

188

Coomassie

(Ning et al., 2015)

intensity for 10 mins Ultrasonic-

Waste activated

Ultrasonic at 1

acid

sludge

W mL-1 for 10

550

Lowry’s

(Sahinkaya, 2015)

method

min; pH of 2.0

Electrolysis-

Waste activated

Electrolysis at

alkaline

sludge

20V for 40 min;

650

Lowry’s method

pH 11

10

(Zhen et al., 2014)

Table 6. Energy and economic evaluation of protein recovery from sludge by different pretreatment methods adapted from Xiao et al (2017a). Ultrasonic

Alkaline

Thermal

-1

(°C)

(W mL ) Raw

0.25

0.5

1

pH 8

pH 10

pH 12

40

60

80

56.72

232.84

478.92

615.35

315.96

343.87

1109.00

123.37

589.63

681.47

5.42

33.17

59.57

78.25

18.57

18.54

104.40

9.82

22.43

41.72

22.77

82.55

178.45

227.58

138.46

152.36

446.77

51.50

270.61

296.97

0.72

1.32

2.31

3.15

1.52

2.01

6.31

1.11

3.09

4.33

1.00

2.33

4.22

6.63

2.38

3.60

12.37

1.90

5.67

8.40

0

9.12

17.89

30.93

0

0

0

17.50

40.83

64.17

0.022

0.046

0.1

Protein concentration (mg L−1) Molecular weight (> 20 kDa) (ppm-C) Molecular weight (< 20 kDa) (ppm-C) Tyrosine-like protein (106 RU) Tryptophan-like protein (106 RU) Energy consumption (kWh ton−1 wet sludge) Chemical dosage

0

_ 11

_

−1

(g g TS) Chemical cost (USD ton−1 wet sludge) Energy cost (USD ton−1 wet sludge)

0

_

0.093

0.18

0

2.10

4.11

7.11

8.10

36.96

76.02

97.68

50.15

54.58

150

0

0

0

0

−141.90

−115.14

−78.09

−59.43

−99.94

0.47

_

_ 4.03

9.39

14.76

176.04

19.58

93.59

108.17

0

0

0

0

0

−95.60

25.57

−134.45

−65.80

−56.59

Credit from protein recovery (USD ton−1 wet sludge) Cost of sludge transport and disposal (USD ton−1 wet sludge) Net saving compared to conventional treatment (USD ton−1 wet sludge)

12