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temperatures (e.g., from 14°C to 38°C), even above optimal-growth temperatures. A decrease in cytoplasmic membrane fluidity, transforming the liquid crystalline membrane into a gel-phase state, results from cold shocks and induces the increase in membrane unsaturated fatty acids that lower the membrane phospholipid melting point and add membrane flexibility.116

The so-called "compatible solutes" are known to be synthesized or taken up from the environment due to hyperosmotic environmental stress, but also due to cold shocks, desiccation, and heating.118 Although compatible solutes are generally polar and neutral at physiological pH, negatively charged organic osmolytes (e.g., sulfotrehalose, diglycerolphosphate) are used by thermophilic Archaea.118 It is possible that such compounds accumulate in the sludge flocs when released from some cells under heat stress and promote more negatively charged flocs. Increased transport of osmolytes and polymerization reactions are energy demanding cellular processes that would increase respiration rates due to increased energy maintenance.114

17.4.2 Proposed Mechanisms of Sludge Deflocculation

The recent research in our group has identified that temperature shifts from 30°C or 35°C to 45°C cause activated sludge deflocculation, which occurs via polymer solubilization and floc fragmentation, leading to poor treatment performance.109 In this section, several mechanisms that could link the occurrence of sludge defloc-culation with decreased metabolic activity, more negatively charged sludge and increased maintenance respiration rates are discussed. A summary is presented in Figure 17.4.

Sludge deflocculation resulting from a temperature shift from 30°C or 35°C to 45°C could be explained by the abiotic change of the physico-chemical characteristics of the sludge flocs and by microbially mediated mechanisms that reduce microbial substrate removal capacity, increase respiration rates, and decrease sludge surface charge. Metabolic inhibition leading to decreased sludge substrate removal capacity could directly induce deflocculation due to a "flocculating-ability shut-off mechanism." This would be similar to negative cellular control mechanisms. Other physiologically mediated mechanisms could render the sludge surface more negatively charged, lead to EPS solubilization, and to the release of soluble products from the cells.

Microbial metabolism could be an indispensable requirement for sludge floc stability; therefore, a significant reduction in sludge substrate uptake would dissipate the sludge flocculating ability. Whatever the actual cellular mechanisms of metabolic inhibition in deflocculation be, the results from the present study agree with the findings of Schwartz-Mittelmann and Galil83 (2000) and Wilen et al.81,84 (20 00), in that suppression of metabolism leads to deflocculation. Sludge defloccula-tion, however, may be governed by different mechanisms under different stress conditions.

The temperature shifts could also increase the cytoplasmic membrane permeability. Elevated temperatures may increase the cell membrane proton permeability and uncouple oxidative phosphorylation, as reviewed by Low and Chase119 (1999).

Biotic Causes

Metabolic inhibition (Decreased substrate degradation)

Metabolic inhibition (Decreased substrate degradation)

Abiotic Causes

Weakening and disturbance of H bonds with nonpolar or hydrophobic molecules/domains

Rupture of cationic and polyvalent EPS bridging

EPS hydrolysis and solubilization

Melting of secondary EPS protein structures

Abiotic Causes

Weakening and disturbance of H bonds with nonpolar or hydrophobic molecules/domains

Rupture of cationic and polyvalent EPS bridging

EPS hydrolysis and solubilization

Melting of secondary EPS protein structures

Flocculation-ability shut-off mechanism

Flocculation-ability shut-off mechanism

Activation of Na+/K+ extrusion mechanisms via antiporters causing cationic imbalance

Accumulation of "compatible solutes" with (-) charges,e.g., sulfotrehalose

Release of SMP including HSP (HSP preventing aggregation)

Increase in saturated fatty acids with (-) charges in membrane phospholipids

Inhibition of expression of adhesive appendages

FIGURE 17.4 Proposed mechanisms of activated sludge deflocculation under a temperature shift from 30°C to 45°C. SMP — soluble microbial products, HSP — heat shock proteins, EPS — extracellular polymeric substances.

Increased membrane permeability could cause Na+ stress due to high internal cellular Na+ concentrations, leading to the activation of Na+ (and even K+) extrusion mechanisms via antiporters, known to be involved in bacterial Na+ and pH stress responses.120,121 This would cause an increase in the local levels of monovalent cations in the EPS, as also proposed by Love and Bott100 (2002) under oxidative stress, and cause sludge floc instability and deflocculation. The activation of respirationcoupled Na+ extrusion mechanisms121 or futile ion cycles would cause an increase in respiration rates providing for increased maintenance energy requirements.114,119 This would explain the higher maintenance respiration rates observed after the temperature shifts during endogenous metabolism.

The accumulation of compatible solutes with negative charges on the flocs and the increase in saturated fatty acids in membrane phospholipids with negative charges have been discussed in Section 17.4.1 above as physiological stress responses of bacteria explaining the decrease in surface charge under the temperature shifts (Figure 17.4). Also, the temperature upshift may inhibit the expression of adhesive appendages in cells (pili and fimbriae) that are responsible for cell attachment and are regulated by several environmental factors, including temperature.122,123 At this point in time, the connecting mechanisms between physiological stress responses to mesophilic-thermophilic temperature shifts and sludge deflocculation remain unknown.

The response of the sludge flocs to the mesophilic-thermophilic temperature shifts could also be abiotic. Sludge physico-chemical changes triggered by the temperature shifts could include the melting of secondary structures of proteins present in EPS and in the cellular membrane, and temperature-induced EPS hydrolysis, destroying the aggregating properties of a fraction of these biopolymers and solubilizing them. Also, EPS bridging could be compromised by the weakening of hydrogen bonds that keep EPS flocculated via bridging, and the weakening and rupture of cationic and polyvalent EPS bridging (e.g., by Mn(II) and Fe(II)). The release of cations from the rupture of cationic EPS bridging could also promote an increase in the relative abundance of adsorbed anionic functional groups onto floc EPS, rendering the sludge more negatively charged. Therefore, the decrease in surface charge could also be a result of a pure abiotic process. More negatively charged cells could cause further desorption of EPS due to high electrostatic repulsion, as suggested by Erdincler and Vesilind124 (2000). Another abiotic explanation is that increased temperature disturbs the orientation of water molecules interacting via hydrogen bonds with nonpolar or hydrophobic molecules or domains, as suggested by Vogelaar et al.77 (2002), which would agree with the decreased hydrophobicity of the sludge.

The relative extent of deflocculation due to biotic and abiotic mechanisms is unknown. Nevertheless, the temperature shifts can be postulated to have a greater impact on deflocculation as EPS solubilization via abiotic mechanisms than biotic mechanisms governed by active metabolism. This idea is supported by the relatively large amount of organic biomass detached from sludge flocs due to the temperature shift from 30°C to 45°C vs that detached due to suppression of aerobic metabolism reported by Wilen et al. (2000).81 Whereas the 30°C to 45°C temperature shift caused an approximate 11% reduction in the total organic biomass (in batch experiments), only 1% to 2% of the total amount of organic sludge matter detached under suppressed aerobic metabolism.81 The abiotic mechanisms of the temperature shifts seem predominant vs those regulated by active metabolism, considering that cell viability was not significantly affected109 and that a low percentage of viable cells is present in activated sludge at high SRTs (10% to 15% of MLSS).125

In general, under microbial stress conditions, sludge bacterial lysis may be involved in the mechanisms of sludge deflocculation as a consequence of inactiv-ation of cell metabolism and cell death, and as a source of biopolymers that decrease sludge stability. Although sludge lysis occurred due to the temperature shift from 30°C to 45°C, it was marginal109 and would minimally affect deflocculation significantly. Under other stress conditions (e.g., toxic shocks or drastic temperature shifts over 55°C to 60°C), however, cell death and lysis may weaken the sludge floc structure and cause deflocculation by directly affecting cell metabolism.126,127

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