Effect of sludge cell disruption on compactibility of biological sludges

A. Erdincler* and P.A. Vesilind**

*Bogazici University, Institute of Environmental Sciences, 80815 Bebek, Istanbul, Turkey **Duke University, Department of Civil and Environmental Engineering, Durham, NC 27708-0287, USA Abstract Biological sludge contains various fractions of water associated with sludge solids which are mostly microorganisms. These water fractions affect the liquid-solid separation of sludge. A considerable amount of sludge water is trapped either inside the sludge microorganisms or within the oc structure and this is labeled interstitial water. Release of interstitial water held inside the cell structure involves disruption of sludge cells and this does not occur during conventional dewatering. In this study, sludge cell disruption is introduced as a new method to improve the compactibility of sludge. Biological sludge cells are disrupted by different methods including alkali treatment, NaCl treatment, heat treatment, and sonication. The effect of cell disruption on compactibility of biological sludge is investigated. The results of the study indicate that the disruption of the sludge cells changes the water distribution in sludge and improves the compactibility of sludge. Disruption apparently releases from 60% to 80% of interstitial water, depending on the disruption method used. On the other hand, it causes creation of extra surfaces for water binding and leads to an increase in the unfreezable water content (vicinal water, water of hydration and a fraction of interstitial water) of sludge. The cell disruption increases the solid content of compacted sludge up to 87% depending on the cell disruption method used. Keywords Biological sludge; cell disruption; compactibility; liquid-solid separation; water distribution

Water Science and Technology Vol 42 No 9 pp 119126 2000 IWA Publishing and the authors

Introduction

Today the most challenging aspect of sludge treatment is to reduce enough of its liquid portion in an effective manner so that sludge behaves as a solid. Biological sludge contains various fractions of water, including free water, interstitial water, vicinal water, and water of hydration, associated with its solids. These water fractions affect the liquid-solid separation of sludge. Biological sludge contains 0.25 to 12% solids depending on the wastewater processes. The big portion of sludge solids are organic in nature and a complicated mixture of bacteria, viruses, protozoa, and other microorganisms which exist in either unicellular or in floc forms. A considerable amount of sludge water is trapped either inside these sludge microorganisms or within the floc structure. It is impossible to extract cell associated water from sludge cells by conventional dewatering processes. Conventional dewatering processes can only remove free water that is not attached to sludge solids, and a part of interstitial water that is trapped in the crevices and interstitial spaces of the flocs. Release of interstitial water held inside the cell structure involves disruption of sludge cells and this does not occur during dewatering. In this study cell disruption has been introduced as a new method to improve the compactibility of sludge. Sludge cells were disrupted by different disintegration methods including alkali treatment (chemical), NaCl treatment (osmotic), heat treatment (thermal) and sonication (mechanical). The effect of cell disruption on the compactibility of biological sludge was investigated by observing the various fractions of water associated with sludge cells.
Water distribution in sludge

Commonly, water in sludge is divided into two categories: free (bulk) and bound (non-free) water. However, the bound water term does not have a uniform meaning, and its operational definition depends upon the measurement method of the bound water.

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Many researchers have noted the existence of bound water in sludges (Heukelekian and Weisberg 1956, Javaheri and Dick 1969, Vesilind 1974, Muller 1983, Katsiris and Kouzeli-Katsiri 1987, Smollen 1988, Lee and Lee 1995). Most of them agree that the bound water content is directly related to the dewaterability of sludge and it therefore affects the moisture content of the sludge cake. Vesilind (1994) defined four categories of water in sludge: Free water: water that is not associated with and not influenced by the suspended solid particles. Interstitial water: water that is trapped in the crevices and interstitial spaces of the flocs and microorganisms. Vicinal water: water that is associated with the multiple layers of water molecules held tightly to the particle surface. This water can be within cells as well, as long as it is associated with a solid surface. Water of hydration: water that is chemically bound to the particles and can only be removed by thermal destruction of the particles. Based on Vesilinds definition it is appropriate to assume that the term bound water used in the environmental engineering literature is actually a gross estimate of several states of water including interstitial water, vicinal water, and water of hydration. Smollen (1988) used the drying technique to quantify the distribution of water in sludge, and found that bound water content of sludge does not affect the solids content of sludge cake. Her result supports the idea that sludge cake contains both vicinal water and interstitial water that can not be removed by conventional dewatering processes. Robinson and Knocke (1992) used constant temperature (35C) dilatometry at various solids concentrations and concluded that the bound water content of sludge decreases with increasing sludge solids content. They suggested that increasing solids concentration progressively increases the amount of interstitial water expelled from the flocs. Eventually, all interstitial water is expelled so that sludge cake contains only vicinal water and water of hydration at the end of dewatering. They appeared not to take the cell associated water into consideration, however, and it is likely that a portion of interstitial water was still trapped inside the sludge solids. Several researchers have studied cell-associated water. Cooke and Kuntz (1974) stated that most of the water in a cell is interstitial water which has the same physical properties as bulk water. On the other hand Ling (1979) reported that the water molecules in close vicinity to biological macromolecules like proteins, DNA, etc., exist in a physical state different from that of bulk water. Therefore, it would not be wrong to say that some of the vicinal water exists within the cell structure of microorganisms as well on their surfaces. Ling (1979) stated that in a living cell almost all water molecules are in the form of polarized multilayers oriented on the surfaces of macromolecules such as cell proteins due to the fixed charges on the macromolecules and other associated counter ions. He suggested that in the presence of a hydrated ion, the water molecules in the hydrated shell and in the polarized multilayer merge and are reinforced by making lots of stronger H-bonds for the hydrated ion than in bulk water. Clegg (1979) believes that a substantial amount of water in cells differs in its properties and behavior from ordinary bulk water. He studied intracellular water in eukaryotic cells and found that the amount of vicinal water changes roughly from 15 to 50% of the total cell water in most eukaryotic cells. In the case of sludge cells, prokaryotic cells, due to their simple structure they have a smaller intracellular surface area leading to less intracellular vicinal water content and higher amount of interstitial water within the cells. It is then appropriate to suggest that some part of intracellular water is interstitial water and it can behave like free water only if the sludge cells are disrupted.

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Based on all of these studies it is reasonable to expect that sludge has a substantial amount of vicinal water and interstitial water associated with its solids.
Disruption of sludge cells

Cell disruption is a method for the release of intracellular material and so the water trapped within the sludge cells. Chisti and Moo-Young (1986) pointed out that the microorganisms are very robust and their disruption is not a very easy process. Cell disruption affects the liquid-solid separation properties of sludge in different ways including release of interstitial water trapped inside the sludge cells and promotion of the flocculation. Vallom and McLoughlin (1984) studied the relationship between levels of extractable exopolymers and flocculation in activated sludge and found a relationship between lysis of sludge and flocculation as a decrease in cell dry weight by the release of exopolymers, leading to an increase in flocculation. They demonstrated that the lysis of cells normally causes the release of polymers into the medium. These polymers include proteins, RNA, DNA, carbohydrate, etc., which are all high molecular weight polymeric materials that possibly act as polyelectrolytes and thereby promote flocculation.
Materials and methods

A. Erdincler and P.A. Vesilind

The observation of the cell disruption and control of the biological parameters becomes extremely difficult in waste activated sludge (biological sludge), due to its complex nature. To overcome this difficulty, all of the experiments were carried out with a pure culture of Flavobacterium aquatile. Flavobacterium aquatile ATCC 11947 (American Type Culture Collection) is selected as the representative bacteria of waste activated sludge. Flavobacterium aquatile is one of the most predominant microorganisms in the waste activated sludge of domestic origin (van Gils 1964; Breed et al., 1957). Flavobacterium aquatile is gram-negative, non-motile, rod shaped, and 0.5 to 0.7 by 1.0 to 3.0 microns in size. This microorganism is found naturally in water. The culture was centrifuged at 2800 g for 15 minutes and resuspended in 0.06% NaCl solution, which has the same osmotic pressure of 17 mOsm/kg as that of fresh waste activated sludge, to obtain 0.3% biomass concentration. After the cell disruption process cells were washed two times in sterile 0.06% NaCl solution by centrifugation and resuspension. Control samples had biomass concentration of 0.3% in 0.06% NaCl solution, and pH value of 6.9. All of the experiments were done at room temperature. Total sludge solids were determined by using standard evaporation technique (Standard Methods, 1995).
Sludge cell disruption

In this study, four different methods of cell disruption including alkali treatment (chemical), NaCl treatment (osmotic), heat treatment (thermal), and sonication (mechanical) were used for the release of water trapped within the cells. Alkali treatment. Alkali treatment is a harsh method. At extremely high pH values of medium, the cell loses its viability and can not maintain an appropriate turgor pressure, and then disrupts (Belter, 1988). Disruption of sludge cells leads to leakage of intracellular material out of the cell and a consequent increase in the protein concentration of the supernatant water. On the other hand, as Katsiris and Kouzeli-Katsiri (1987) stated when the pH of sludge samples increased, the bacterial surfaces become increasingly negatively charged. This creates high electrostatic repulsion which causes desorption of some part of extracellular polymers. Alkali added to the cell suspension reacts with the cell walls in several ways, including the saponification of lipids in the cell walls, which leads to solubilization of membrane. The high alkali concentrations required spark many degradations including

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the protein denaturalization. As a result, this method tends both to disrupt cell walls and destroy the products. In this study pure culture cells were disrupted by direct addition of 1 N NaOH solution into the sample to adjust the pH of the sample to 12.30. A Cole Palmer pH meter was used for pH measurements. NaCl treatment. This is dumping a given volume of cells into a medium where the solute concentration is higher (hypertonic) outside than inside the cell. Water tends to flow out of the cell and the cell tends to shrink (plasmolysis). In this study 2.0% NaCl by weight was used to adjust the solute concentration of the medium. The control medium had 0.06% NaCl concentration. Pure culture samples were centrifuged at 2800 g for 15 minutes and the sediments were washed twice with 0.06 % NaCl solution by centrifuging and resuspending. Washed sediments were then resuspended in 2.0 % NaCl solution. Sonication. Sonication process, a widely used method of cell disruption, causes approximately 100% disruption of the cells. Sludge cells were sonicated by a Branson 450 Ultrasonic Cell Disrupter for four-30 second bursts. Percent disruption was determined by measuring the protein concentrations of sample supernatants after centrifugation at 2800 g for 15 minutes. Heat treatment. Heat treatment employed by heating sludge samples in an autoclave for five minutes at 120C. High heat disrupted the sludge cells and then caused hydrolyzation of proteins, carbohydrates, lipids and other macromolecules secreted from the cells.
Protein determination

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The protein concentration in the sample was measured and used as an indicator of the degree of cell disruption. Protein concentrations of sludge samples before and after cell disruption were determined by the Bio-Rad DC Protein Assay Protocol, based on the Lowry assay (Bio-Rad, 1994). Bio-Rad DC Protein Assay can be used in the presence of sodium hydroxide or detergents, which would otherwise interfere with standard protein, assays. Assay involves the addition of two Bio-Rad DC protein assay dye reagent into the samples (protein solution) and then reading absorbance at 750 nm with a spectrophotometer (Hach DR/2000). Bovine serum albumin was used as protein standard. The percentage cell disruption was calculated as: [Protein]sample [Protein]control Percentage cell disruption = [Protein]total [Protein]control [Protein]sample : Protein concentration in the sample, mg/ml [Protein]control : Protein concentration in the control, mg/ml [Protein]total : Protein concentration in the sample when all of the cells are disrupted.

Sludge compaction

Compaction tests were carried out by use of a centrifuge (International Equipment Company Model PR-6). Samples of 15 ml were centrifuged at 2800 g for 30 minutes and then the degree of compaction was measured as the height of the compacted sludge layer (pellet).
Determination of water distribution in sludge

Water distribution in sludge was determined by two different methods including differential scanning calorimetry and centrifugation.

Differential Scanning Calorimetry. Differential Scanning Calorimetry is a technique based on the measurement of the difference between the power or heat fluxes into the sample material and the reference material through a heat-insulating layer. Seiko, high sensitivity, heat flux DSC 220C automatic cooling differential scanning calorimeter (DSC) calibrated to work in the temperature ranges from 150C to 725C was used for measurements. An empty open pan was used as a reference. Sample size was selected as 30 mg for all of the measurements. A low cooling rate of 2 min is used to observe the differentiation between free and unfreezable water in the DSC outputs. The calculation of enthalpy change (H) of the sample which is equal to the difference between the heat flow to the sample and the heat flow to the reference material. was done by computer. The DSC was calibrated by obtaining the heat flow difference curve of known weight of deionized water and calculating its peak area. The amount of water was directly proportional to the peak area. The proportionality constant was found to be 0.0028 using the calibration data. The mass of freezable water producing the peak was calculated as: mg freezable water = 0.0028 H The differential scanning calorimeter (DSC) easily differentiated free water (mobile) that froze at normal freezing temperature of bulk water, from the portion of sludge water that did not freeze at normal freezing temperature of bulk water. This portion of sludge water was referred to as unfreezable water that probably included vicinal water and water of hydration. The unfreezable water content was calculated by taking the difference between the total water content in the sample, determined by standard evaporation technique, and the free water content determined by DSC. Centrifugation. In this method water content of sludge was operationally classified in to two groups as easily removable water and trapped water. The easily removable water was defined as the portion of water which was removable after 30 minutes centrifugation at 2800 g. The trapped water was defined as the portion of water retained in the pellet (sediment) after 30 minutes centrifugation at 2800 g. This portion is believed to include vicinal water, water of hydration and some part of interstitial water. The weight of free water was determined by weighing the supernatant. The weight of total water was determined by a standard evaporation technique. The trapped water was calculated by taking the difference between weight of total water in the sample and the weight of free water.
Results and discussion

A. Erdincler and P.A. Vesilind

Four different disruption methods: alkali treatment, NaCl treatment, sonication and heat treatment, were chosen for sludge cell disruption so that they all have different disruption mechanisms (chemical, osmotic/chemical, mechanical, and thermal respectively). Otherwise it would be difficult to differentiate whether the changing sludge properties were due to addition of chemicals or cell disruption itself.
Alkali treatment

When alkali treatment was applied to sludge samples, the cell disruption was observed to be 45% leading to a considerable increase in the protein levels of the samples. This increase can be explained by desorption of extracellular polymers by high electrostatic repulsion, release of proteins due to detergent solubilization (Naglak and Wang, 1992) and leakage of proteinaceous intracellular material. These dissolved extracellular polymers and

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intracellular material increased the dewaterability (CST) of the suspension by promoting flocculation (Vallom and McLoughlin, 1984). Compactibility of alkali treated samples increased 68% in terms of increase in the solid content of compacted samples.
NaCl treatment

NaCl itself is an electrolyte and works as a coagulant. The interaction of NaCl with colloidal particles in sample suspension is purely electrostatic (Weber, 1972). NaCl destabilizes the colloids in the suspension by the attraction of the ions of opposite charge to that of the colloid. This destabilization by counter ions is done by compression of the diffuse layer surrounding the particles. At 2.0% NaCl concentration the degree of cell disruption was only 9%. Compactibility improved by 53% increase in the compacted sludge solid content. Unfreezable water content of the samples also increased. This can be attributed to increase in vicinal water content of the samples due to the interaction of water molecules with Na+ ion. Water is a polar molecule with a dipole and its interaction with Na+ is considered as an ion-dipole electrostatic pair interaction. (Israelachvili, 1985), (Dick, 1979). On the other hand, based on centrifugation results, plasmolysis released a considerable amount of interstitial water trapped inside the cells.
Sonication and heat treatment

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In both sonication and heating methods, proteins, polysaccharides, lipids and other intracellular macromolecules secreted from disrupted sludge cells were hydrated. Consequently vicinal water content of sample sludge increased while the overall free water content increased by release of some interstitial water originally trapped inside sludge cells. Improvements in compactibility of sludge samples were about 56% for heat treatment and 87% for sonication. This very high percent of improvement in compactibility of samples can be explained by the hydrolization of exocellular and intracellular materials leading to destruction of colloidal properties of these macromolecules. Table 1 shows the summary of physical properties of biological sludge samples treated with different methods. The average protein concentration of samples increased in all of the treatment methods confirming the cell disruption. The relationship between protein concentration and CST of waste activated sludge is found to be very low. The unfreezable water content of samples increased in all of the treatment methods. This increase was due to the creation of additional surfaces for water binding. In alkali treated and sonicated samples most of the intracellular and cell wall materials including mainly
Table 1 Summary of physical properties of Flovabacterium aquatile suspension treated with different methods
2% NaCl Sludge properties Control treated Alkali treated Heated Sonicated Range

CST, sec Protein Concentraion % Cell Disruption Unfreezable Water Content % Tot. Water Content Unfreezable Water % DS Solid Content in Sample. % Solid Content in the Compacted Sample, % Increase in the Solid Content, %

19.7 0.050 0 3.00 900 0.3 1.5 0

20.4 0.092 9 10.10 4100 0.3 3.2 53

57.7 0.438 45 10.35 4450 0.3 4.7 68

15.2 0.347 34 10.75 4900 0.3 3.4 56

17.1 0.915 100 10.80 5410 0.3 11.3 87

+20.0, 5 +0.01, 0.02 +3.0, 2.0 +0.3, 2.0. +300.0, 200.0 +0.03, 0.02 +0.2, 0.1 +5.0, 8.0

polysaccharides, proteins, lipids, and other macromolecules were released from the cell. The water molecules bound to these macromolecules were believed to be in a more organized state (Ling, 1979; Dick, 1979). In alkali treated samples in addition to release of intracellular macromolecules OH ions were also hydrated by three water molecules as OH (H2O)3 (Israelachvili, 1985; Dick, 1979). NaCl treatment was an osmotic lysis method. Addition of NaCl in to suspension changed the external osmotic pressure in the suspension leading to change in water content of cells. Another effect of NaCl addition was the hydration of Na+ ion leading to increase in overall vicinal water content of the samples. The relationship between unfreezable water content and compactibility of sludge is shown in Figure 1. Figure 2 shows the effect of disruption on compaction. Compactibility of the sludge increased with increasing sludge cell disruption, no matter which method was used. Table 2 shows summary of water content of biological sludge samples obtained by centrifugation method. In all of the treatment methods overall easily removable water content of samples increased while the trapped water decreased. This can be explained by the release of a considerable amount of interstitial water trapped in the crevices and interstitial spaces of flocs and microorganisms by the disruption process. It seems that although some extra water binding surfaces are created, leading to increase in vicinal water content of sludge samples, release of interstitial water increased the overall easily removable water content of the samples.

A. Erdincler and P.A. Vesilind

Figure 1 Relationship between unfreezable water content and compactibility of Flovabacterium aquatile suspension Table 2 Summary of results by centrifugation method

Figure 2 Effect of cell disruption on compactibility of Flovabacterium aquatile suspension

2% NaCl Sludge Control treated

Alkali treated Heated Sonicated Range

Free water, % DS Trapped water, % DS % Decrease in trapped water % Solid content in the compacted sludge % Disruption % Increase in solid content of compacted sludge

36715 535 0 1.5 0 0

39055 157 66 3.2 9 536

45863 104 80 4.7 45 68

49019 356 63 3.4 34 56

49231 144 85 11.3 100 87

+1000500 +100200 +1010 +10-10 +32 +58

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Conclusions

The purpose of this study was to investigate the effect of sludge cell disruption on compactibility of biological sludges. Based on results of this research the following conclusions were drawn. Sludge cell disruption improves the compactibility of biological sludge. Sludge cell disruption changes the water distribution in the biological sludge by releasing a considerable amount of interstitial water trapped inside sludge microorganisms or within the floc structure. Sludge cell disruption increases the unfreezable water content of biological sludge by creating additional surfaces for water binding. Unfreezable water content does not affect the compactibility of the biological sludge. There is no strong relationship found between CST (dewaterability) and unfreezable water content of the biological sludge.
References
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