Experimental approaches to evaluate the influence of hydrolysis

Morgenroth et al. (2002) provided an overview of experiments that have been designed to evaluate mechanisms or location of hydrolysis and to quantify the influence of hydrolysis on substrate utilization. Four experimental approaches can be differentiated, according to measured parameters:

– measurement of specific hydrolytic enzymes;

– measurement of specific hydrolytic intermediates or specific end products;

– overall mass balances for bulk organic parameters;

– measurement of respiration rates to quantify bacterial activity.

The first two approaches allow to study specific mechanisms involved in hydrolysis but are often restricted to specific substrates (e.g. starch), while two last approaches allow to evaluate the overall processes, but may not allow to study specific mechanisms involved in the hydrolysis process (Morgenroth et al., 2002). The most common, from presented above experimental approaches, used for example to mathematical modeling of activated sludge systems is a measurement of bacterial respiration. Dynamic experiments measuring respiration rates, as it was mentioned before, were the basis for introducing two biodegradable XS and SS organic fractions (Ekama and Marais, 1979; Dold et al., 1980). However, the model parameters for hydrolysis (stoichiometry and kinetics) and the component concentration of XS

cannot be easily estimated, based on the respiration rate experiments, because this fraction in wastewater is composed of a heterogeneous mixture of colloids and particles with variable chemical composition (Spanjers and Vanrolleghem, 1995;

Sophonsiri and Morgenroth, 2004). Hence, other research has aimed at evaluating specific mechanisms of hydrolysis using substrates such as starch (Larsen and Harremoes, 1994), dextran (Haldane and Logan, 1994; Confer and Logan, 1997b), dextrin (Confer and Logan, 1997b), bovine serum albumin (BSA) (Confer and Logan, 1997a), and fats (Sprouse and Rittmann, 1990). These commercially available substrates are well defined, but are only representative of macromolecular or colloidal XS. Therefore, during last decades only a limited number of experiments measuring respiration rates in “real” wastewater has been carried out to determine the amount of XS and evaluate of the hydrolysis rate coefficients (Henze and Mladenovski, 1991; Orhon et al., 1998).

Henze et al., 1987 proposed to measure or estimate all other COD fractions and then determine the XS, based on a mass balance. Kappeler and Gujer (1992) proposed curve fitting of the concentration of XS and the first order hydrolysis rate constant, which was assumed to be comparable to hydrolysis kinetics in ASM1 only for low concentrations of XS. Spanjers and Vanrolleghem (1995) also estimated the

concentration of XS from respiration rate measurements. However, they divided the XS into two fractions. The curve fitting allowed to estimate only the SH, for which the process kinetics was described by the first order expression (Table 2.15, Model No I).

Vollertsen and Hvitved-Jacobsen (1999) divided the XS into three fractions: slowly, intermediate and rapidly biodegradable organic matter that are hydrolyzed in parallel (Table 2.14, Model No 4). Subsequently, they used batch experiments with respiration rate measurements to quantify all biodegradable organic matter fractions and hydrolysis rates for the three parallel hydrolysis processes.

For the estimation of parameters describing the surface saturation type hydrolysis, Ekama et al. (1986) proposed to use OUR measurements in a completely mixed reactor fed continuously under a daily cyclic square wave feeding pattern. In the second phase, after an immediate depletion of the SS, the accumulated XS continues to be used at the same rate for a time period. For the batch experiments, the authors recommended that the value of khyd could be estimated at a high XS/XH ratio or performing simulations for curve fitting of OUR profiles. Sollfrank and Gujer (1991) determined the first order hydrolysis rate constant from the slope of the OURH plot vs. the concentration of degradable matter in a batch experiment. For the time t>t1 (t1

denotes the time when a nearly linear relation is reached), it was assumed that hydrolysis was the limiting process in the degradation of filtered wastewater. The value of khyd was determined from the slope after the net respiration rate becomes proportional to the concentration of biodegradable matter by the following equation:

OURHnet (t>t1) = (1 – YH) * khyd *XS(t) (2.16)

where:

khyd – specific hydrolysis rate constant, T^-1

OURHnet – net heterotrophic oxygen uptake rate, M(O2)L^-3T^-1 XS – slowly biodegradable substrates, M(COD)L^-3

YH – “true” growth yield coefficient for heterotrophic organisms, M(COD)M(COD)^-1

In general, the accepted procedure for the experimental assessment of khyd and KX

under aerobic conditions involves model-based evaluation and curve fitting of OUR profiles in laboratory scale batch or semi-continuous reactors using wastewater containing total COD fractions and heterotrophic biomass (Insel et al., 2003).

Experimental results are then evaluated using a mathematical model to determine both the COD fractions in the wastewater and hydrolysis kinetics (Sollfrank and Gujer, 1991; Insel et al., 2002). Such an experimental approachs have been extensively discussed in literature. Insel et al. (2002 and 2003) demonstrated that the model-based evaluation and curve fitting allows to generate not a unique set, but a relatively large combinations of different pairs of khyd and KX coefficients equally

applicable to the experimental data. Mogenroth et al. (2002) concluded that there is a large uncertainty dealing with model parameters estimated from respirograms.

The basic assumption of those experiments using respiration rate measurements to quantify hydrolysis is that hydrolysis determines respiration rates, when the readily biodegradable substrate is depleted. However, in the presence of storage of internal polymers the interpretation of OUR profiles (see Section 2.1.3) becomes confusing and extracellular hydrolysis of slowly biodegradable COD and intracellular degradation of storage polymers cannot be distinguished from the OUR profile (Goel et al., 1998c). In the fallowing study, Goel et al. (1999) suggested an experimental approach to separate hydrolysis from storage by performing and analyzing two parallel OUR measurements: one with filtered wastewater (including soluble COD) and the other with non-filtered wastewater (including total COD). On the other hand, batch respirometric tests were proposed by adjusting the appropriate wastewater/biomass mixtures with different (either high or low) initial ratios between wastewater and biomass (ST/XV) (Orhon et al., 1999; Sperandio and Paul, 2000). They estimated wastewater composition and the hydrolysis rate based on simultaneous parameter estimation from two batch experiments. The substrate observed in short-term respirometric experiments was classified as a “readily hydrolysable” COD fraction and could be modeled by a first order reaction. The hydrolysis of “slowly hydrolysable” COD fraction was correctly modeled with a sequential two-step process (adsorption followed by surface-limited hydrolysis).

Figure 2.12. Diagram of the mechanisms of COD degradation according to the model of Lagarde et al. (2005)

XR,NA XS,NA

XS

XR

SS

XH

O2

GROWTH ADSORPTION

HYDROLYSIS

Legend:

XR,NA – non-adsorbed on XH readily hydrolysable COD

XS,NA – non-adsorbed on XH slowly hydrolysable COD

XR – adsorbed on XH readily hydrolysable COD

XS – adsorbed on XH slowly hydrolysable COD

SS – readily biodegradable COD XH – heterotrophic biomass

Aerobic conditions

Anoxic conditions

Lagarde et al. (2005) used analysis of the experimental data of not settled samples, relied on a three substrate model derived from Sollfrank and Gujer (1991) and Spanjers and Vanrolleghem (1995), with an additional distinction between adsorbed and free substrates according to Sperandio and Paul (2000). In this model, as illustrates Figure 2.12, the fractions were: readily (SS) biodegradable COD; readily (XR) hydrolysable COD (adsorbed on biomass); slowly (XS) hydrolysable COD (adsorbed on biomass); non-adsorbed (XR,NA and XS,NA) COD that were readily and slowly hydrolysable, respectively, and two other variables: dissolved oxygen (O2) and heterotrophic (XH) biomass concentrations.

Henze et al. (1987) used that model as a reference for the respirograms simulations in ASM1. The initial COD was considered as not adsorbed (XR,NA and XS,NA) or under SS, i.e. fractions XR and XS are initially equal to zero. Both substrates XR,NA and XS,NA

progressively adsorb on the biomass and only the resulting fractions, XR and XS, respectively, can be hydrolysed, assuming the mediation of bound exoenzymes. For this fraction, the hydrolysis rate constants, khyd,X, and entrapment saturation coefficients, KX,X, ranged 0.25-1.05 d^-1 and 0.33-0.95 g COD/g COD, respectively. In the study of Orhon et al. (1999), the results for municipal wastewater yielded average khyd,S and KX,S values of 3.1 d^-1 and 0.2 g COD/g COD, respectively, associated with the hydrolysis of SH and significantly lower values of khyd,X = 1.2 d^-1 and KX,X = 0.5 g COD/g COD associated with hydrolysis of XS. It was observed, however, that the discrepancy between measured and predicted OURs was reduced considerably by shifting from a single hydrolysis model to a dual hydrolysis model. It should be noted, that the hydrolysis rate (especially of nitrogenous compounds) depends on electron-acceptor conditions. For example, Henze and Mladenovski (1991) found a significant reduction of hydrolysis rates under anoxic and anaerobic conditions compared to aerobic conditions. The rate at 20°C is high under aerobic conditions (0.12 d^-l) medium under anaerobic conditions (0.06 d^-l) and low under anoxic conditions (0.03 d^-l). The ratio between the hydrolysis rates under aerobic and anoxic conditions are very similar to the respiration rates measured as electron equivalents.

This could indicate that the biomass activity has significant impact upon the hydrolysis rate. However, there is no direct explanation for the high rate under anaerobic conditions, but it could be caused by fermentation processes performed by the heterotrophic microorganisms (Henze and Mladenovski, 1991). On the other hand, indirect evidence suggests that the rate under anaerobic conditions should not be high. If the anaerobic hydrolysis rate was significant, it would make a substantial contribution to EBPR, which is contrary to experimental observations strongly relating EBPR to the SS in influent (Ekama and Wentzel, 1999a). A subject of ongoing

debate, according to Morgenroth et al. (2002), has been reduced to whether and how hydrolysis rates are influenced by electron acceptor conditions.

The basic assumption of experiments using respiration rate measurements to quantify hydrolysis is that hydrolysis is the rate limiting step that determines respiration rates (Dold et al., 1980; Arvin and Harremoes, 1990). Experimental evidence would appear to indicate that hydrolysis may be the rate controlling step even at 20°C. In order to check this hypothesis Tian et al. (1993) investigated the effect of temperature on sludge accumulation. The parallel reactors were run at a sludge age of 10 days under identical conditions with the exception of temperature.

The reactor, which run at 12°C produced 12-20% more sludge than the corresponding reactor run at 20°C. The authors reasoned that the excess sludge accumulation resulted from hydrolysis being appreciably slower than synthesis or decay. Overall, respirometry is a valuable tool to estimate wastewater composition and reaction rates, however, there is a large uncertainty associated with hydrolysis parameters extracted from parameter estimation (Vanrolleghem et al., 1999).

Differences between hydrolysis in traditional activated sludge systems and other treatment technologies (membrane activated sludge system, granular sludge system, biofilm reactors) should be also evaluated (Henze et al., 1995; Barker and Dold, 1997a; Ekama and Wentzel, 1999a, Morgenroth et al., 2002).