Chemical extraction methods for activated sludge extracellular polymeric substances (EPS): A review

Waste activated sludge (WAS) is formed by a diverse microorganisms, organic and inorganic compounds merged and mixed together in an extracellular polymeric substances (EPS) network. EPS is a complex high-molecular weight macromolecules in WAS that happens to be one of the common analysis to determine the efficiency of treatment. Therefore, different extraction methods has been applied in order to achieve better EPS extraction yield. This paper serves as a base to review the commonly used chemical extraction methods to extract EPS components. The mechanisms, conditions and efficiencies of each of the chemical extraction methods were discussed and compared accordingly. The possible use of different chemical extraction methods for different type of activated sludge were summarized.


Introduction
Waste activated sludge (WAS) is a by-product generated after the biological aerobic and anaerobic digestion [1]. It is formed by a diverse microorganisms, organic and inorganic compounds merged and mixed together in an extracellular polymeric substance (EPS) polymeric network [2]. Due to the restriction of the law, WAS can no longer be directly dispose of to the landfill or by incineration prior treatment as it contains viruses or/and heavy metals which possess threats to the environment. Therefore, law enforcements and restrictions has been set to prevent direct disposal of WAS for a sound and responsible waste management. Extracellular polymeric substance (EPS), one of the common component that is measured to measure the efficiency of WAS treatment.
EPS can be known as a complex high-molecular weight (HWM) macromolecules that consists of proteins, carbohydrates as the main constituents and other small amount of components such as nucleic acids, uronic acids, deoxyribonucleic acids (DNA), humic-like __________________________ substances and lipids which have cohesive and adhesive characteristics in binding the cells together [3][4][5]. EPS is a form of metabolic secretion by the microorganisms that tends to create a layer termed as "biofilm" enveloping the cell to prevent possible loss of water and protect them from extreme conditions [6][7][8][9]. By having said this, the protection layer formed outside has caused flocculation and settling of sludge to occur and thus making the cells difficult to be degraded [2,6,7,10]. EPS are normally classified into two type: (a) soluble microbial products (SMP) and (b) extractable-EPS (eEPS) [11,12]. SMP are the slim and soluble that are dispersed and able to move freely in sludge whereas eEPS are attached and bounded to the sludge flocs. In order to perform a detailed analysis upon EPS, the first step is to perform an extraction protocol so that it is separated from the cell plus the EPS are quantified and prepared for further analysis [9]. This review paper focused on the mechanisms, conditions and efficiencies of the commonly used chemical extraction methods by researchers.

Extraction Protocol
A standard protocol is yet to be determined, this has caused results unable to be compared as there is no absolute right or wrong in any extraction protocol [11,13]. Despite without having a standard extraction protocol, there are some requirements that needs to be followed during the extraction procedures. Selection of extraction protocol should impose as much isolation and separation as possible for both the functional EPS and the sample to prevent the damaging of the structure and causes the occurrence of cell lyses [12,14]. Restriction of cell lyses must be done to prevent intracellular materials such as humic acids to cause interference in the subsequent analysis [9,14]. DNA [13], ATP [15], nucleic acids [16] and Glucose-6phosphate dehydrogenase (G6PHD) [17] are all indicators that can be measured to detect cell lyses towards the biopolymers. However, even if considerations are taken, it has been known that there is a high chance of underestimation or overestimation [8,18].
The extraction protocol is classified into three main steps: (a) pretreatment of sample, (b) extraction method, and (c) purification and analysis step. EPS has a great diversity, therefore the selection of the extraction method used changes everything. Pretreatment is carried out to remove soluble EPS and achieved homogeneity. This helps to prevent any cases of contamination and ensure better analysis [12]. Pretreated samples are then ready for the extraction process, physical and chemical methods are the two main approaches that are being applied in the extraction protocol. Physical methods focused on using external forces to extract the EPS [10,12]. Chemical method, uses either acidic or alkaline chemicals, detergents and/or ionic to destroy the connection between the cells and biopolymers. Purification step is taken to remove any remains of unwanted substances that might affected the EPS analysis [12]. Protein and carbohydrate are the dominant macromolecules that are normally being analyzed using colorimetric method through electromagnetic spectrum. The commonly used calorimetric methods for protein measurement are Lowry, modified Lowry and Bicinchoninic Acid (BCA); whereas for carbohydrate measurement are Anthrone, Dubois, phenol-sulphuric and sulphuric-UV.

Chemical Extraction Methods
Chemical extraction method works by the involvement of various types of chemical into the EPS matrix aims to destroy or destabilize their linkage among molecules and thus freeing the bound-EPS [19,20]. Many chemical extraction methods rely on the breakage of the electrostatic interactions, therefore promoting an extraction of water soluble compounds. Research and studies have been done and found that while comparing with physical extraction protocol, chemical extractions yield higher EPS concentration [21,22].
Comte et al. [10] showed a range of 17-47% yield achieved through chemical extraction while physical extraction achieved within the range of 2-4%. Another explanation on chemical extraction having high efficiency is because chemical agents added into EPS changes the solvents' pH which improves the efficiency [3]. Common chemical agents extractants are acidic or alkaline solution [8], mineral salts [15], ethylenediamine tetraacetic acid (EDTA) [6], formaldehyde [16], cation exchange resin (CER) [23], hydrophobic [1], enzymatic and ethanol extraction. Although chemical extractions show promising results, considerations need to be taken as the involvement of chemicals bring damage to the structure and contaminates the biopolymer substances through cell lyses [12]. Moreover, some chemicals used for extraction would be remained within the EPS solution, making an extra purification step is required to prevent interferences [15,22].

Formaldehyde
Formaldehyde works by destabilizing the structure of granules by creating a reaction with the amino-polysaccharide, hydroxyl or amino groups that are present inside protein to form alkylate cell wall molecules [16].The destabilization process helps to improve EPS yield, at the same time to prevent the destruction of cell membrane and stops intracellular substance to leach out [21]. The downside is the reaction with amino groups would be remain and affect the properties of biopolymers [16].

Ethylenediaminetetraacetic Acid (EDTA)
EDTA is a chelating agent, when EDTA is added into the biopolymer matrix, calcium ions will undergo calcium chelation which turns EPS solution more soluble as the chelation affects the floc strength [10]. EDTA mainly focus on chelating protein bonding due to the high protein to polysaccharide ratio achieved [24]. However, the application of EDTA is not encouraged because EDTA is unable to be removed by the later purification process which leads to overestimation [15]. In addition, cations removed by EDTA will give rise to the release of intracellular into the EPS solution [13].

Cation Exchange Resin (CER)
CER works with two mechanisms, first, cation mainly Ca 2+ and Mg 2+ that are responsible in joining the EPS matrix will be captured by the resin [10]. the second mechanism of CER is applying shear effect by stirring the destabilized matrix which will then improve the EPS extraction [16]. This extraction method has its advantage in maintaining the original chemical structure after extraction process and is able to achieve higher protein extraction due to the affinity of protein towards multivalent cation, Ca 2+ [9,15]. CER is one of the most preferred extraction method used as it is able to achieve high EPS yield with minimum cell lyses and contamination to liquid EPS [12,14,20]. Furthermore, it does not affect the colorimetric analysis of the soluble EPS [5]. Triton X-100 is known as a non-ionic detergent that applies hydrophobic properties to tackle low-energy interactions and specific linkage to extract EPS [9,25]. Its hydrophobic amino acids are able to extract specific proteins like hydrophobic proteins through hydrophobic interactions [5]. Same as EDTA extractants, Triton X-100 tends to affect the colorimetric test but can be overcome by using Dubois method [9]. Triton X-100 is able to extract both types of EPS: LB-EPS and TB-EPS with minimum cell lyses [19]. Membrane protein is solubilized by interfering the connections at the peptidoglycan layer with the application of Triton X-100 [1]. Studies have showed that Triton X-100 is a trustworthy method to extract biopolymers.

Acid/Alkaline Treatment
NaOH with its high pH, brings about the ionization of charged groups such as carboxylic groups due to the low pH of their isoelectric points [20]. Plus, NaOH causes the detachment of acidic group from the EPS due to repulsive forces [13,25]. Furthermore, NaOH is able destroy strong electrostatic bonds through the increase of solution alkalinity [15]. Sodium pyrophosphate same as EDTA, where it chelates multivalent metallic, but it performs better whereby it makes metallic elements-precipitated organic compounds soluble [26]. It can be a substitute of EDTA because as an organic extractants, no transferring of organic carbon into the biopolymer solution occurs [15]. Sodium tetraborate focus on breaking the Van der Waals and weak electrostatic force between the organic compounds and EPS matrix, thus making the EPS more extractable [26]. Anionic exchange and ionizing functional groups are its extraction mechanism because of its high pH [15]. Sulphuric acid, H 2 SO 4 used for acid extraction works by increasing the repulsing forces between EPS with its cell surface [12]. Among the chemical extraction method, acidic method is often ignored due to its inability to extract EPS effectively [14]. According to tests carried out by Wang et al. [25], acidic extraction method has the least performance, due to its weakness in dissociating acidic group compared to alkaline treatment. Therefore, the literature on acid treatment is scarce and less reported. Table 1 has summarized the different chemical extraction methods (discussed in Section 4) and their respective operating conditions. Efficiency of chemical methods in terms of EPS yield, protein, carbohydrate, DNA yield and colorimetric method used for protein and carbohydrate measurements were tabulated in Table 2. Each of the chemical extractant used were able to achieve high EPS yield surpassing those of physical method, but they do show higher DNA yield due to the variation of its composition and structural damage. CER is the most popular method being applied because of its low cell lyses condition and high solubilization efficiency [12], for instance, Xiao et al. [23] showed that EPS yield for using CER method was able to extract approximately 10 times more than the controlled method with only an increase of 1.44 times of DNA yield. Even so, some researchers disagreed due to its bad performance as an extractant [19]. When CER is compared with Triton X-100, Triton were able to exceed CER due to the floc composition being withdrawn. CER mainly focused in loosely-bound EPS only but Triton takes up both loosely-bound and tightly-bound EPS [9].  Zuriaga et al. [9] investigated the effect of Triton X-100. After extraction, the protein and carbohydrate yielded 87.4% and 12.6%, respectively whereby CER achieved 76.1% and 23.8% of protein and carbohydrate, respectively. Triton X-100 has known to be a non-ionic detergent that extracts better proteins than other ionic detergents [1]. Research done by Noda et al. [1] said that the application of polymerase chain reaction (PCR) has been carried out to detect DNA yield proved that Triton X-100 showed no signs of cell lyses after extraction. However, this detergent extraction method is suggested to be used at low concentration to prevent any possible cell disruption at higher concentration [27]. EDTA extractant showed a vast range of EPS extraction yield, this might be due to the type of samples being extracted. It can be seen that EDTA works better with activated sludge from anaerobic digester because EPS from anaerobic sludge are more degradable when in comparison with aerobic sludge [6]. Furthermore, different percentage of EDTA was tested by Nguyen et al. (2014), it showed 2% EDTA achieved the highest EPS yield among the other 0.1% and 4%. It was expected that the higher the percentage, the better the extraction yield, but 2% and 4% EDTA did not show much difference [7]. According to Caudan et al. [24], 2% EDTA was enough to destroy the divalent bridge of the matrix.

Conditions and Efficiencies of Chemical Extraction Methods
Alkaline treatment showed an average performance, deprotonation of compound occurs through the increase of pH hence destabilizing the cell matrix and releasing EPS [15,19]. However, NaOH disrupts the cell based on the IR spectra result from Abzac et al. [16] same goes to EDTA. Formaldehyde was able to prevent cell lyses because of the reaction with protein and nucleic acids from cell membrane [13,21]. Chemical extractants are useful but it is important to minimize chances of overestimation and underestimation [22].

Summary
In general, various chemical extraction methods were applied and proved to extract EPS efficiently. However, it is almost impossible to extract all the EPS components by one single method followed by the analysis of the extracted components. For instance, formaldehyde proved to improve the EPS yield, however it affected the carbohydrate analysis by using phenol-sulphuric acid method due to its alteration of properties. EDTA causes a 17% reduction of protein using Lowry method and lesser interference for BCA method [16]. For CER extraction, there are studies mentioned that this method is unable to solubilize the EPS and it is only suitable for extracting loosely-bound EPS [9,19]. Whereas for alkaline or acid treatment, contamination on the samples is one of the common drawbacks on using salt as an extractant [16]. Triton X-100 were able to excel with its advantageous traits. Although some chemical extraction methods surpasses Triton X-100, they possess a higher threat whereby some has high DNA yield and/or chemicals remains in EPS solution [12,21]. Furthermore, Triton X-100 has shown a good example in being able to extract EPS components without cell lyses as some of the extraction methods were unable to do so [1]. The only drawback for Triton X-100 as it is incompatible with Anthrone method only for carbohydrates measurement [5,9]. Among CER and Triton X-100, both have shown great performance in the extraction process, however by taking every aspects into consideration, Triton X-100 portrays to be better in terms of the types of EPS being able to be extracted.

Conclusions
The chemical extraction methods discussed were mild to prevent cell lyses and the disruption of EPS for an efficient extraction protocol. This has made it a challenge to compare the results from different extraction methods as it portrays different results in terms of EPS yield. Furthermore, the selection of a calorimetric method for EPS analysis is important as well, as some colorimetric methods are not suitable for certain chemical extractants. Therefore, the choice of the chemical extraction method influences not only the quantity, but also the composition of the EPS. In addition, the efficiency of the extraction method greatly depends on the type of activated sludge. It is not possible to have a standard method used for EPS extraction due to their extreme complexity. Triton X-100 appeared to be one of the effective chemical extraction method without overestimation or underestimation in extracting EPS.