WO2014142762A1

WO2014142762A1 - Combined ultrasonication and enzymatic pretreatment of waste activated sludge prior to anaerobic digestion

Info

Publication number: WO2014142762A1
Application number: PCT/SG2014/000131
Authority: WO
Inventors: Antoine Prandota TRZCINSKI; Chong Wang; Xinbo TIAN; Maszenan Bin ABDUL MAJID; Wun Jern Ng
Original assignee: Nanyang Technological University
Priority date: 2013-03-15
Filing date: 2014-03-17
Publication date: 2014-09-18
Also published as: CN105189366B; CN105189366A

Abstract

There is herein disclosed a method of pretreating organic waste comprising the steps of (a) providing an organic waste; (b) subjecting the organic waste to ultrasonication; and (c) subjecting the ultrasonicated organic waste to a heat treatment step at a temperature of from 35°C to 85°C. There is also disclosed a waste treatment plant comprising a pre-treatment apparatus that comprises an ultrasonicator apparatus and a heating apparatus adapted to act in a batch or continuous fashion on an organic waste, wherein the ultrasonicator apparatus is placed upstream of the heating apparatus and is in fluid communication therewith.

Description

Combined Ultrasonication And Enzymatic Pretreatment Of Waste Activated Sludge Prior To Anaerobic Digestion

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

A large amount of surplus biological sludge is generated during the activated sludge process. The costs involved in sludge treatment and disposal may be as high as 50% of the total operating costs for a wastewater treatment plant. Anaerobic digestion is commonly accepted as an ideal method to stabilize sludge for safe disposal and utilization. It has the advantages of providing a low biomass yield and a high degree of stabilization, as well as producing methane gas. It is known that the digestible organic fraction in Waste Activated Sludge (WAS) is only about 30-45% (w/w) of the total biomass in a conventional anaerobic treatment, while it is also known that methane production can be improved markedly by disintegrating cells in WAS to release intracellular organics (e.g. chemicals and enzymes) using chemical or mechanical disruption methods.

WAS mainly consists of intact microorganisms and their secretions, which together form particles larger than 0.1 pm that cannot be directly assimilated by the microorganisms. Cell lysis of the microorganisms is the rate limiting step of the process, because it limits the rate of hydrolysis which in turn limits the rate of the whole anaerobic process. Furthermore, during the activated sludge process, bacterial cells form floes, which structure is enhanced by extracellular polymeric substances (EPS). These floes protect microorganisms from being degraded and make cell lysis even harder.

Pretreatment of WAS has been proven to disrupt sludge structures, causing solubilisation of organics and accelerate subsequent anaerobic digestion.

Biological treatment encompasses a broad range of processes that can include both aerobic and anaerobic processes. Biological pretreatment aims at intensification by enhancing the hydrolysis process in an additional stage prior to the main digestion process. One of the pre-treatment methods is an anaerobic or aerobic biological method that requires either thermophilic (around 55°C) or hyper-thermophilic (between 60 and 70°C) conditions which typically result in an increase in hydrolysis activity, and an increase biodegradable COD and pathogen destruction.

There have been a number of configurations tested, including short pretreatment prior to mesophilic digestion, dual digesters: thermophilic and mesophilic, single stage digesters and, recently, temperature co-phase processes. Thermophilic conditions generally result in an increase of the organic solids destruction rate, attributed to increased hydrolytic activity, as summarized in Table 1 below.

Substrate Treatment Anaerobic Results Reference conditions digestion

conditions

Activated 70°C CSTR, HRT: Increase of CH₄ production (Skiadas et sludge 2 days 13 days (15 from 40 to 55 mL.L^"1 d^"1 al., 2005) days without (+28%)

pretreatment)

55°C

Primary 70°C CSTR, HRT: Increase of CH₄ production Skiadas et sludge 2 days 13 days (15 from 146 to 162 mL.day^'1 al., 2005b) days without (+11%)

pretreatment)

55°C

Activated 70°C Batch Increase of biogas (Climent et sludge 9h 55°C production +58% al., 2007)

Mixed 70°C CSTR, HRT: Increase of CH₄ production (Ferrer et sludge 9, 24, 48 h 10 days from 0.15 to 0.18 mL.g^"1 al., 2008,

55°C VSin (+20%) Ferrer et

Increase of energy al., 2009) production (+60-100%)

Primary 70°C CSTR, HRT: Increase of CH₄ production (Lu et al., sludge 2 days 13 days (15 from 13.6 to 20.1 mmol.g^-1 2008) days without VSin (+48%)

pretreatment)

55°C .

Primary 50-65°C CSTR HRT: Increase of CH production (Ge et al., sludge 2 days 13-14 days (+25%) compared to 35°C 2010)

35°C pretreatment

Table 1. Biological pre-treatment methods

In order to improve the degradation of recalcitrant organic matter, aerobic treatments have also been evaluated, as there are materials that can be degraded under aerobic (but not anaerobic) conditions. Hyper-thermophilic aerobic treatment is also an option. Destruction of 75% organic solids from excess waste activated sludge was obtained at full scale, by combining a conventional municipal activated sludge process with a thermophilic aerobic sludge digester (65°C, H T of 2.8 days). However, depending on the type of sludge (primary, secondary or mixture of both) the residence of this type of treatment is generally 2 days or longer.

Hyper-thermophilic aerobic microbes have been identified as belonging to Bacillus with a predominance of Geobacillus stearothermophilus. These are protease- excreting bacteria, which are present in untreated sludge and can survive under anaerobic mesophilic conditions. Therefore, the potential for increased performance is inherent in the sludge itself. An increase of 50% in biogas production was observed using a hyper-thermophilic aerobic reactor as the first stage of a dual process (with an anaerobic digester as the second stage).

Recently, a combined aerobic hyper-thermophilic (AHT) process (65°C, HRT of 1 day) coupled to conventional mesophilic digester (HRT of 21 and 42 days) has been shown to increase the intrinsic biodegradability of sludge to between 20 and 40% . The AHT cotreatment allowed an increase in COD removal by 30% for an overall process retention time of 42 days. Nevertheless, this COD was oxidised in the aerobic stage, and therefore the methane production yield was not improved. Compared to a conventional mesophilic digester, the same quantity of COD was degraded with AHT treatment at 21 days HRT than without AHT treatment at 42 days HRT. Therefore, the AHT treatment enables one to reduce the HRT or digester volume by half. An increase in the release of soluble mineral fraction (from 6% to 10%) was also observed under these conditions.

An industrial process combined with the aerated sludge process, Biolysis® E, is being commercialised by Ondeo-Degremont (Suez). Thickened sludge is introduced into a thermophilic reactor where enzymes (proteases, amylases, lipases) are produced by specific microorganisms (Bacillus stearothermophillus). According to the company, this process allows from 40% to 80% reduction of excess sludge production, without deteriorating the wastewater quality.

Enzyme pre-treatment of WAS can also increase the anaerobic gas production. A mixture of two glycosidic enzymes has been reported to increase the daily biogas production by 10-20%. A more than 50% daily biogas increment has also been achieved by either adding Alpha-amylase or Beta-glucanase into anaerobic digesters. Nevertheless, enzyme pre-treatment has its limitations. Due to the complexity of WAS, addition of enzyme may not always achieve the expected performances. The optimum temperature for hydrolytic enzymes is around 50°G, so the optimum performance of enzymes may not be obtained under mesophilic conditions.

A huge fraction of the organic matter in WAS (30-85%) is formed by particles larger than 0.1 μηη that cannot be directly assimilated by the microorganisms. These microorganisms degrade the organic matter by producing hydrolytic enzymes that are released into the media. However, it has been demonstrated that the free enzymatic activity present in the liquid phase is almost negligible, as it is generally immobilised on floes (connected to the polymeric extracellular substances) or attached to the cellular walls by ionic and hydrophobic interactions. Therefore, a physical treatment is useful to disrupt the floes and release the enzymes.

The use of a mill to disrupt excess sludge has been investigated and it was found that among protease, amylase, glucosidase, lipase and dehydrogenase, protease was the one with the highest activity. Almost 69% of the protease activity was recovered by protein precipitation using ammonium sulphate, while the recovered enzyme solution lost its activity by 32% after preservation at -20°C for 1 month. After the recovery of protease the authors studied the applicability of the protease using milk as a model. A maximum protease activity of 2210±308 U/g MLSS was reported for a municipal wastewater sludge application at a temperature of 50°C, while for a laboratory cultivated sludge the maximum activity was 3450±124 U/g MLSS at 75°C. This suggested that each sludge has different microbial populations, therefore different protease activity and optimum temperatures. It has also been found that protease activity of about 4000 U/g MLSS can be obtained with enzymes extracted from WAS using Triton (0.5%). A high WAS solubilisation ratio of about 53%, defined by soluble TOC per total carbon has been reported by the use of a mill with glass beads to disrupt a laboratory-cultivated WAS. The WAS solubilisation by the continuous mill disruption was successfully analysed with a kinetic model consisting of first-order disruption kinetics.

A simple heat-treatment process (700 ml was incubated at 60 °C, 120 rpm for 24 h in a 1 I Erlenmeyer flask) for the reduction of excess sludge has also been investigated. This process showed there was rapid increase in population of thermophilic bacteria at the early stage of heat-treatment and the emergence of protease-secreting bacteria. Culture-independent analysis by denaturing gradient gel electrophoresis (DGGE) revealed that Bacilli, which include most of thermophiles, became the dominant class in the community with the treatment. The protease activity in supernatant of the sludge increased instantly after 1 h heat-treatment, which was considered to be released from microbial cells by lysis. The protease activity succession was correlated with the microbial succession and also with the change in MLSS and TOC concentrations during heat-treatment, suggesting that the protease activity played an important role in the lysis-cryptic growth induced by heat-treatment. The TOC increased rapidly to the maximum value (355 mg/L) after 3 h, and then decreased gradually to 146 mg/L by the end of treatment. Heat-treatment combined with thermophilic protease treatment has already been applied to engineering processes in Japan. However, the biological response of the sludge matrix induced by heat-treatment was poorly understood.

The reduction of excess sludge by heat-treatment is induced by sludge lysis and further cryptic growth (lysis-cryptic growth). In the lysis-cryptic growth, sludge reduction is achieved because some portions of lysates are consumed for the catabolism and finally emitted as CO2. Consequently, the microbial community succession in the sludge should occur during heat-treatment. Proteolytic cleavage of peptide bonds by protease is considered to be the main enzymatic reaction in the digestion or lysis of excess sludge. Therefore, protease activity in the sludge should be an important factor for the sludge reduction efficiency during heat-treatment.

In another study, proteases and lipases were extracted from WAS using ultrasound (24 kHz, 3.9 W/cm², 30 min, 5°C using water-ice bath) combined with a non-ionic detergent (Triton X100). It has been suggested that the recovery of valuable products from sludge could be used in the sludge degradation itself, but this has not been attempted. In the study, it was also found that the activity of extracellular protease in activated sludge tank was much lower than that of intracellular protease.

Ultrasonication (ULS) has been used previously to treat activated sludge by reducing its amount, achieving a better dewaterability, increasing the soluble chemical oxygen demand and destroying the floes. Besides this, ultrasonication alone or combined with detergents or ion exchange resins was one of the methods allowing recovery of these enzymes and maintaining their activity. Some studies have been carried out to evaluate the influence of an enzymatic pretreatment step prior to the anaerobic digestion of domestic or industrial wastewater. The results showed that it was possible to remove solids, decrease the COD level and improve the biogas production during the anaerobic digestion. Huge hydro-mechanical shear force generated by cavitation bubbles during ULS is believed to be the predominant effect for sludge disintegration. ULS is comparatively a very rapid method that causes solubilization of both extracellular and intracellular substances. Microorganisms in WAS degrade the organic matter by producing hydrolytic enzymes that are released into the media. Therefore, a physical treatment such as ULS should be useful to disrupt the floes, release the enzymes and at the same time improve the thermophilic enzymatic pre-treatment, but information about a combination of these two pre- treatment is still scarce in the literature. Since ultrasonication is an energy-intensive process, its major disadvantage is its high energy consumption.

Summary of Invention

In a first aspect of the invention, there is provided a method of pretreating organic waste comprising the steps of:

(a) providing an organic waste;

(b) subjecting the organic waste to ultrasonication; and

(c) subjecting the ultrasonicated organic waste to a heat treatment step at a temperature of from 35°C to 85°C.

In certain embodiments, the organic waste of step (a) is split into a first portion and a second portion, only the first portion is subjected to step (b) and is then combined with the second portion so that the combined first and second portions are subjected to step (c). For example, the first portion may be from 0.5 wt% to 99 wt% of the organic waste (e.g. from 1 wt to 80 wt%, such as from 2.5 wt to 60 wt%, from 5 wt to 50 wt%, 7 wt to 40 wt% or 10 wt to 25 wt%). In particular embodiments, the first portion may be 20 wt% or 50 wt%. In further embodiments, the first portion may be 50 wt% and the heat treatment of step (c) may be performed at 65°C for 24. hours.

In further embodiments, the organic waste may be subjected to ultrasonication of from 10 seconds to 1 hour (e.g. from 30 seconds to 30 minutes).

In yet further embodiments, the ultrasonicaton may use a power of from 0,5' to 3 kWh/m³ of organic waste treated, such as from 1 to 2 kWh/m³ of organic waste treated. In still further embodiments, the ultrasonication step may use a SEI of from 2500 to 7500 kJ/kg TS, such as a SEI of 5000 kJ/kg TS.

In yet still further embodiments, the ultrasonication step may be conducted at a frequency of from 19 kHz to 200 kHz, such as at a frequency of about 20 kHz.

In further embodiments, the temperature of step (c) may be from 40°C to 80°C (e.g. from 45°C to 75°C, such as from 50°C to 70°C). In particular embodiments, the temperature of step (c) may be 65°C.

In yet further embodiments, the heat treatment of step (c) may be from 30 minutes to 24 hours, such as from 45 minutes to 10 hours (e.g. from about 1 hour to about 6 hours). In particular embodiments, the heat treatment of step (c) may be about 24 hours.

In further embodiments, the heat treatment of step (c) may be conducted without mixing and aeration or with agitation of the organic waste.

In further embodiments, the method may further comprise feeding the pre-treated organic waste into an aerobic or an anaerobic digester. The anaerobic digestermay be, for example, a mesophilic anaerobic digester or, particularly, a thermophilic anaerobic digester.

In certain embodiments, the organic waste comprises biodegradable solids. For example, the organic waste may have a total solids concentration of from greater than or equal to 3 g/L to less than or equal to 50 g/L, such as from 5 g/L to 45 g/L.

For example, the organic waste may be a sludge (e.g. a waste activated sludge), food waste, oily waste, solid waste with a high organic content or any combination thereof.

In a further aspect of the invention, there is provided a waste treatment plant comprising a pre-treatment apparatus that comprises an ultrasonicator apparatus and a heating apparatus adapted to act in a batch or continuous fashion on an organic waste, wherein the ultrasonicator apparatus is placed upstream of the heating apparatus and is in fluid communication therewith. In certain embodiments, the ultrasonicator apparatus and the heating apparatus may be adapted to act in a batch fashion.

In further embodiments, the pre-treatment apparatus is adapted so that a portion of the organic waste is fed to ultrasonicator apparatus and the remainder is fed directly to the heating apparatus. In further embodiments, the waste treatment plant may be a wastewater treatment plant.

Figures

The invention will now be described in further detail below, with the aid of the following figures.

Figure 1: Effect of ultrasonication Specific Energy Input (SEI) on (A) soluble proteins, carbohydrates and COD, (B) soluble phosphorus concentration, (C) soluble ammonia concentration, (D) evolution of various groups of particles based on the size.

Figure 2: Effect of ultrasonication of 0, 25, 50, 75 and 100% of sludge (30 sec, -5000 kJ/kg TS) followed by thermal treatment at 30°C (top) and 55°C (bottom). Figure 3: Effect of ultrasonicating 0, 25, 50, 75 and 100% of WAS (30 sec, 3500J) followed by thermal treatment at 55°C.

Figure 4: Effect of the incubation temperature on the SCOD during the enzymatic treatment following ultrasonication of 25% of sludge (30 sec, -5000 kJ/kg TS).

Figure 5: Effect of the incubation temperature on the SCOD produced by indigenous enzymes during thermal treatment of WAS following ultrasonication of 25% of WAS (30 sec, 3500J).

Figure 6: Evolution with time of the SCOD (A), soluble proteins (B) and carbohydrates (C) during the enzymatic treatment at 55°C following the ultrasonication of 0, 5, 10, 20, 50 and 100% of sludge (30 sec, -5000 kJ/kg TS). Figure 7: Evolution with time ^' of the SCOD (A), soluble proteins (B) and carbohydrates (C) during the enzymatic treatment at 65°C following the ultrasonication of 0, 5, 10, 20, 50 and 100% of sludge (30 sec, -5000 kJ/kg TS). Figure 8: Growth of microorganisms from WAS onto an agar-skimmed milk Petri dish after 24 hours and evidence of proteolytic bacteria (10³ dilution).

Figure 9: Growth of proteolytic bacteria from WAS after 48 hours. The Petri dish on the right-hand side was sealed with parafilm.

Figure 10: Growth of proteolytic bacteria from WAS (non-diluted) after 48 hours. Figure 11: Isolation of a proteolytic colony at 55°C. The colony did not grow, but the enzymes were active.

Figure 12: Isolation of several colonies onto new Petri dishes.

Figure 13: (A) Samples taken after 6 hours of hyper-thermophilic enzymatic pre- treatment at 65°C with and without ultrasonication and pipetted into wells on Petri dishes placed at 55°C. The top half of the Petri dish contains replicated wells with sludge samples.- The bottom half contains wells with only the clear supernatant from the sludge. (B) Samples taken after 6 hours of enzymatic pre-treatment at 65°C with and without ultrasonication and pipetted into wells on Petri dishes placed at 37°C. (C) Samples taken after 24 hours of hyper-thermophilic enzymatic pre-treatment at 65°C with and without ultrasonication and pipetted into wells on Petri dishes placed at 37°C.

Figure 14: Evolution of soluble biopolymers (top: COD, Middle: proteins, bottom: carbohydrates) during incubation at 65°C following the ultrasonication of a small percentage of WAS. ^' Figure 15: Cumulative biogas (top) and methane (bottom) production using the combined ultrasonication and thermal pre-treatment of WAS.

Figure 16: Cumulative methane production after the combined ultrasonication and hyper-thermophilic enzymatic pre-treatment of the sludge used in this study.

Description of Invention

There remains a need for improved pre-treatment processes that enhance the solubility of organic waste in waste water (e.g. sludge) before being fed into an anaerobic or aerobic digester for further processing. With this in mind, although heat treatment is beneficial for solubilisation, long thermal treatments are not interesting from a process point of view because some of the lysate is consumed by thermophilic bacteria and lost as CO2, and so cannot be used to produce methane. Therefore, there is a need to shorten thermal treatment and ULS is a possible solution to quickly release intra-cellular enzymes. The combination of ULS before heat treatment may be particularly beneficial.

Such a method of pre-treating organic waste can comprise the steps of:

(a) providing an organic waste;

(b) subjecting the organic waste to ultrasonication; and

(c) subjecting the ultrasonicated organic waste to a heat treatment step at a temperature of from 35°C to 85°C. As noted in Example 1 below, the use of ultrasonication alone can be used to increase various parameters, such as soluble phosphorus, ammonia, carbohydrates and proteins and SCOD (see Figure 1 ). However, the method alone is not efficient because it requires a significant input of power to achieve the desired efficacy. The ultrasonication may be conducted using pulses of ultrasound or, more particularly, using ultrasound continuously.

The ultrasonication may be conducted for 30 seconds. The ultrasonication is generally conducted using a frequency of from 19 kHz to 200 kHz (e.g. 20 kHz). The ultrasonication is also generally conducted using 5,000 kJ/kg TS.

As noted above, the ultrasonication is conducted before the application of a separate heat treatment. However, the process may also reverse the order of steps (b) and (c). However, it is noted that the application of a heat treatment followed by ultrasonication of the entire batch of material may not be as efficient because the presence of colloids in the organic waste following the heat treatment step may hinder the subsequent ultrasonication treatment.

When the process is conducted such that step (b) is before step (c), the organic waste of step (a) may be split into a first portion and a second portion, where only the first portion is subjected to the ultrasonication of step (b) and this first portion is then combined with the second portion so that the combined first and second portions are subjected to step (c). For example, the first portion may contain from 0.5 wt% to 99 wt% of the organic waste (e.g. from 1 wt to 80 wt%, such as from 2.5 wt to 60 wt%, from 5 wt to 50 wt%, 7 wt to 40 wt% or 10 wt to 25 wt% (e.g. 20 wt%)).

The heat treatment of step (c) may be conducted at a temperature of from 40°C to 80°C (e.g. from 45°C to 75°C, such as from 50°C to 70°C, for example 65°C). The heat treatment step may be conducted for from 30 minutes to 24 hours (e.g. from 45 minutes to 10 hours, such as from 1 hour to 6 hours).

Further to the completion of the pre-treatment, the pre-treated organic waste may be fed into an aerobic or anaerobic digester. For example, the method may further comprise feeding the pre-treated organic waste into a mesophilic anaerobic digester or, more particularly a thermophilic anaerobic digester. It will be appreciated that the organic waste mentioned herein generally comprises biodegradeable solids. For example, the organic waste may be. a sludge, food waste, oily waste, solid waste with a high organic content or any combination thereof. In certain embodiments, the sludge is a waste activated sludge. In general, the organic waste contains a total solids concentration of from greater than or equal to 3 g/L to less than or equal to 50 g/L. At above a total solids concentration of 50 g/L, ultrasonication is not likely to be feasible because the sludge would be too thick. While it is technically possible to use an organic waste with a total solids concentration of 3 g/L, it would not be economically sensible to do so.

It will be apparent that the method may be employed in a waste treatment plant. As such, there is also provided a waste treatment plant comprising a pre-treatment apparatus that comprises an ultrasonicator apparatus and a heating apparatus adapted to act in a batch or a continuous fashion on an organic waste, wherein the Ultrasonicator apparatus is placed upstream of the heating apparatus and is in fluid communication therewith. For example, when the pretreatment apparatus is run in a continuous fashion, the heat treatment can be carried out in an insulated pipe that acts as a tubular plug flow reactor.

The pre-treatment apparatus may be adapted so that a portion of the organic waste is fed to ultrasonicator apparatus and the remainder is fed directly to the heating apparatus. In certain embodiments, the waste treatment plant may be a wastewater treatment plant.

Experimental Section

1.1. Sludge Samples

A mixture of primary sludge and thickened waste activated sludge (ratio around 1:1 based on dry solids) were collected from a municipal wastewater reclamation plant. Properties of the sludge used in this study are listed in Table 2. Parameters (acronym, unit) WAS Anaerobic Inoculum

5.9-6 7.3

Soluble Chemical Oxygen Demand 670 - 1440 454 ± 8

(SCOD, mg/L)

Total Chemical Oxygen Demand 18 - 25 13.75 ± 0.53

(TCOD, g/L)

Total Solids (TS, g/L) 13.6 -17.2 9.5 ± 0.3

Volatile Solids (VS, g/L) 10.7 - 13.4 7.1 ± 0.3

Total Suspended Solids (TSS, g/L) 12.4 - 15.9 9.3 ± 0.2

Volatile Suspended Solids (VSS, 10.3 - 13.0 7 ± 0.3

g/L) . ^'

Ammonia (mg N/L) 122.97 ± 2.72 NM

Phosphate (mg P0₄ ³7L) 24.11 ± 4.71 NM

Table 2. Properties of the sewage sludge used in this study. NM= not

measured.

Analytical methods

The measurement of pH (Jenway pH meter) was accurate to within ±0.02 units. The Total Solids (TS), Volatile Solids (VS), Total Suspended Solids (TSS), Volatile Suspended Solids (VSS), Soluble Chemical Oxygen Demand (SCOD) and Total Chemical Oxygen Demand (TCOD) were measured in triplicate as described in Standard Methods (APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington D.C, 1999). Their coefficient of variation (COV) for ten identical samples was 2.7%, 3.8%, 2.8%, 4.8%, 1.9 and 1.6%, respectively.

Proteins concentration was determined in triplicate by Lowry's method (O. Lowry, N. Rosebrough, A. Farr, R. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951 ) 265-275) using bovine serum albumin (Sigma-Aldrich) as standard and a UVA IS scanning spectrophotometer (Shimadzu, UV-1800) against the blank at a wavelength of 750 nm. The coefficient of variance was within 2.8% for ten identical samples. As the precise chemical formula of the proteins detected was not determined, the percentage of soluble COD represented by protein had to be estimated by assuming a, stoichiometric conversion factor of 1.5 which is derived from the typical formula of proteins (C16H24O5 4) presented in Rittmann and McCarty (B.E. Rittman, P L. McCarty, Environmental biotechnology: Principles and applications, McGraw-Hill Int. Editions, London, 2001 ). Carbohydrates concentration was determined in triplicate by sulphuric-phenol method (M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric Method for Determination of Sugars and Related Substances, Analytical Chemistry, 28 (1956) 350-356) using D-Glucose (Merck) as standard and the same UV spectrophotometer against the blank at a wavelength of 485 nm. To convert into COD, 1g carbohydrates assumed as C6H12O6 is equivalent 1.07 g COD (W.T.M. Sanders, Anaerobic hydrolysis during digestion of complex substrates, in: Department of Environmental Technology, Wageningen University, The Netherlands., Wageningen, 2001 ). The coefficient of variance was within 6.8% for ten identical samples. Soluble fractions of above-mentioned parameters were obtained from by filtrating supernatant fraction of centrifuged sludge (10,000 rpm for 10 mins) through a 0.45 μιτι membrane filter. Ammonia-Nitrogen was measured in triplicate using Nessler method (APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington D.C, 1999) by reading the absorbance at 425 nm. The COV was equal to 6.6% for ten identical samples. Soluble Phosphorus (as PO4^3") was analyzed using the vanadomolybdophosphoric acid colorimetric method described in Standard Methods (APHA, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, Washington D.C, 1999). The absorbance was read on the same spectrophotometer at 470 nm and the coefficient of variation for three replicates was 0.6%. Particle size distribution was measured with particle size analyzer (Shimadzu, model SALD-3101 ) according to laser diffraction. The median diameter was used to quantify the particle size distribution. By definition it is the particle size such that 50% of the particles are larger and 50% are smaller than that value.

Sludge disintegration degree (DDCOD) was used in this study to express the ratio of solubilized COD to the maximum possible COD solubilization and can be used to quantify the sensitivities of different sludge to ultrasound attack (J. Muller, G. Lehne, J. Schwedes, S. Battenberg, R. Naveke, J. Kopp, N. Dichtl, A. Scheminski, R. Krull, D.C. Hemper, Disintegration of sewage sludges and influence on anaerobic digestion, Water Science & Technology, 38 (1998) 425): Where SCODjis the Soluble COD of treated sample, SCOD_NaoH is the Soluble COD of sample immersed in 1M NaOH (ratio 1 :1 ) at 90°C for 10 minutes and SCODo is the soluble COD of the raw sample.

Example 1

Preliminary tests on the effects of ultrasonication (ULS) alone

We present preliminary data on the effects of ultrasonication alone. Various parameters, such as soluble phosphorus, ammonia, carbohydrates and proteins and SCOD are shown in Figures 1 and 2.

Figure 1A shows that ULS has a significant impact on soluble biopolymers with an increase in SCOD, proteins and carbohydrates concentrations to 5.5 g/L, 1.6 g/L and 500 mg/L, respectively. Figure 1B shows that ultrasonication had a significant effect on the soluble phosphorus concentration, which means that ULS was able to release phospholipids from cell membrane and phosphorus from the DNA into the bulk liquid. The concentration of ammonium in the supernatant was also analyzed, and it was found that it slightly increased from 120 to 170 mg/L during the first 5 minutes of treatment, but afterwards it remained constant. It is possible that some proteins in the sludge were broken down or that ammonium from the cytoplasm was released in the supernatant due to the action of ULS.

Figure 1 D shows the evolution of various groups of particles based on their size. Cavitation bubbles caused by ultrasound are known to disrupt floe structures and reduce floe size. Particles larger than 100 pm or cells floes and aggregates are readily disrupted by ULS within the first minutes with the number of large floes reduced from 26% to 12% and then below 5% as the SEI reached 10,000 kJ/kg TS. At the same time, the number of colloidal particles or small floes (13-100 pm) also dropped significantly due to physical disruption, while the amount of single cells, small colonies and possibly cell debris (2-13 pm) started to increase markedly from 10% to 50%. Overall, it can be concluded that ULS was more efficient towards large floes. Example 2

Preliminary tests on the Combination of ULS and thermophilic enzymatic treatments

In our preliminary tests it was observed that the temperature could increase up to 70°C during ULS if a small volume of sample (<50 mL) was used and if the sample was not cooled down. Using a pulse mode could reduce the heat generated, but was not efficient to solubilise more COD (data not shown). It was then decided to investigate the effect of ULS and heat separately and by combining ULS and thermal treatment in sequence. The sequence thermal-ULS was tested and it was found that the thermal energy during enzymatic treatment could lyse cells which release soluble materials such as colloids and proteins (data not shown). However, because of these colloids the propagation of ultrasound waves was hindered during the subsequent ultrasonication treatment which rendered the ULS inefficient. In other words energy was wasted on soluble materials and dissipated before it could reach intact cells. Therefore, we focused on the ULS-thermal sequence.

In this section we investigated the effect of combining ultrasonication and thermal treatment in sequence. First we ultrasonicated a specific percentage of WAS (0-25- 50-75-100%) and then mixed with the non-ultrasonicated remaining fraction and placed it in a water bath to study thermal treatment. That is, a specific percentage of the sludge was ultrasonicated and the SCOD concentration was measured after mixing with the non-treated fraction. The sludge was then incubated at a specific temperature and it can be seen in Figure 2 (top) that after 24 hours incubation at 30°C the SCOD increased to ~3 g/L for the 25% ULS-treated sludge and decreased for the higher ratios meaning that SCOD was consumed by mesophilic microorganisms at 30°C, and converted to CO2. This experiment indicated that enzymes were not very active at 30°C.

The situation was very different at 55°C as shown in Figure 2 (bottom) and Figure 3. When the sludge was not ultrasonicated and placed at 55°C (referred to as "100% raw") the SCOD increased from 750 mg/L to 5.35 g/L, whereas with 25% ULS- treated sludge the SCOD increased to 7.1 g/L (+560%). When all the sludge was ultrasonicated (referred to as "100% ULS-treated"), the SCOD increased from 4 g/L to 7.8 g/L (+96%) showing that ultrasonication of all the sludge did not result in a marked increase. This demonstrated the presence of powerful proteolytic enzymes active at thermophilic temperatures. Moreover, the use of ultrasonication on 25% of the sludge improved even further the performance of indigenous enzymes. This was due to the disruption of floes containing active extra-cellular enzymes and the breakdown of cells containing intra-cellular hydrolytic enzymes. Moreover, at higher ratios of ULS- treated WAS the advantage of using ultrasonication became less interesting as the percentage of SCOD increase dropped to +100%. However, at 100% ULS-treated sludge the final SCOD concentration reached almost 8 g/L showing that the activity of enzymes was not affected by ultrasonication, which is an interesting feature if ultrasonication is used in conjunction with indigenous enzymes.

From Figure 2 (bottom) and Figure 3 it also appeared that treating a greater fraction (50, 75 and 100%) did not result in proportionally greater SCOD levels.

Example 3

Effect of temperature during the ULS-enzymatic pre-treatment of sludge

From the previous experiment, we found that mixing 25% of treated sludge with 75 % untreated sludge was better than treating higher ratios with ultrasonication. Therefore, in the subsequent experiment we used this 25% ratio to determine the optimum temperature for the indigenous enzymes.

The temperatures chosen for the experiment were 25°C (ambient), 35°C, 45°C, 55°C, 65°C, 75°C and 85°C and the results are shown in Figures 4 and 5.

Figures 4 and 5 show that the higher the temperature, the more COD can be solubilized (up to -11 g/L). However, beyond 65°C the increase was marginal. Considering the energy aspect, it was suggested that 65°C was the most suitable temperature for enzymatic experiments. It was hypothesized that the COD increase was the result of two possible mechanisms: 1 , the degradation of organic matter by proteolytic enzymes and ii) thermal energy can cause the cells to lyze.

A higher SCOD could be obtained depending on the initial solid concentration of the sludge (data not shown). It was also found that mixing during the thermophilic enzymatic treatment provided a better enzyme-substrate interaction which resulted in a 20% increase in SCOD concentration (data not shown). W

To investigate further the effect of temperature on enzymes, a sample was autoclaved (121°C for 20 min) and the final SCOD was only 6,700 mg/L. As the temperature rises slowly in an autoclave, the enzymes were still active but were then deactivated at high temperatures (>85^tiC) which limited the extent of solubilization compared to our milder thermal process. This showed further that heat was not the only phenomenon taking place. The solubilization of WAS by heat-treatment can be induced by sludge lysis and further cryptic growth (lysis-cryptic growth). In the lysis- cryptic growth, sludge reduction is achieved because some portions of lysates are consumed for the catabolism and finally emitted as CO2. This was confirmed using our sludge as a CO2 production of 4.4 mL and 6 mL was recorded after 1 hour incubation at 55°C and 65°C, respectively. After 24 hours, the cumulative C0₂ production reached 9.9 mL and 10.2 mL, respectively, indicating the consumption of SCOD for the growth of both thermophilic and hyper-thermophilic bacteria.

Therefore, the potential for increased performance is inherent in the sludge itself and although heat treatment is beneficial for solubilisation, long thermal treatments are not interesting from a process point of view and also because some of the lysate is consumed by thermophilic bacteria and lost as CO2 and cannot be used to produce methane. There is therefore a need to shorten thermal treatment and ULS is a possible solution to quickly release intra-cellular enzymes.

Example 4

Combination of different ratios of sludge treated by ULS and enzymatic pre- treatment at 55°C and 65°C

In this experiment we ultrasonieated a specific percentage of sludge (0, 5, 10, 20, 50 and 100%) and then mixed it with the remaining non-ultrasonicated fraction and incubated it in a water bath to study the kinetics of the enzymatic treatment at 55°C and 65°C. As 75°C and 85°C were shown to result in a marginal SCOD increase in the previous section, these temperatures were not tested further in details in this study. Carbohydrates and proteins are two predominant biopolymers in EPS structure which also contributes a great part of COD to sludge. Therefore, the solubilization of carbohydrates and proteins provide essential information about disintegration of sludge structure. Results at 55°C

The soluble COD, proteins and carbohydrates concentrations obtained at 55°C are shown in Figure 6.

It can be seen that the thermophilic enzymatic treatment alone resulted in a final SCOD of 7.8 g/L, whereas a significant increase to 8 g/L, 8.7 and 9.3 g/L was observed when 20%, 50% and 100% of sludge was ultrasonicated prior to the thermophilic enzymatic treatment, respectively. BeloW, 20% of ULS-treated sludge there was a small effect as indicated by close SCOD values. The results indicated that as the percentage of ULS increases, more cells are broken down and more enzymes are released into the bulk which results in an improved enzymatic hydrolysis. However, the effect of ULS is not linear, meaning that 100% ULS treated did not result in twice the solubilization of 50% ULS-treated sludge. This shows that treating 100% of sludge by ULS is not an interesting option, however, 20% and above had an impact on the subsequent enzymatic treatment.

It was also found that ultrasonication increased the SCOD solubilization kinetic of the subsequent thermophilic enzymatic treatment. For instance, the thermophilic enzymatic treatment took 24 hours to reach 7.8 g/L SCOD, whereas only 3 hours thermal treatment was required when 100% sludge was ultrasonicated. The increase in the kinetics was due to an improved contact between substrate and intra-cellular enzymes as more cells were broken down. The disruption of floes and the release of extra-cellular enzymes by ULS also improved the mass transfer of enzymes to the substrate.

It can be seen from Figure 6B and 6C, that ULS improved the kinetics of proteins and carbohydrates solubilization compared to the thermophilic enzymatic treatment alone. The concentration increased up to 6 hours of thermophilic enzymatic treatment and decreased afterwards due to the consumption of nitrogen and carbohydrates by thermophilic bacteria. This indicates that longer thermophilic enzymatic treatment is not adequate before the anaerobic digestion step as some proteins and carbohydrates are degraded to CO2. Results at 65°C.

The soluble COD, proteins and carbohydrates concentrations obtained at 65°C are shown in Figure 7. As expected, the extent and rates of COD, proteins and carbohydrates solubilization was enhanced at 65°C compared to 55°C. This is due to an improved cell lysis and a higher enzymes activity. In terms of final SCOD concentration, 100%ULS was equivalent to 1 hour hyper-thermophilic treatment. Both conditions resulted in ~5 g SCOD/L. When 100% of sludge was ultrasonicated, less than 1 hr of hyper-thermophilic condition was required to reach 8 g SCOD/L. However, 24 hrs were required to reach that level in individual hyper-thermophilic pre-treatment. Therefore, ULS shortened significantly the hyper-thermophilic enzymatic treatment.

It can be seen that the extent of protein solubilization increased as the percentage of ULS-treated sludge increased. This is in line with our previous observations at 55°C. However, at 65°C, the effect of ULS was more dominant as shown by a significantly higher solubilization rate at percentages as low as 10%. This confirmed the higher protease activity at 65°C. Interestingly, there was no net decrease in soluble proteins concentrations at 65°C in contrast to what was observed at 55°C. This indicates that the rate of proteins solubilization was higher than the rate of proteins degradation and consumption by hyper-thermophilic bacteria. Soluble carbohydrates, however, were consumed by hyper-thermophilic bacteria as indicated by a net decrease in concentration after 6 hours. The net decrease was insignificant for the 100% ULS- treated sample showing that ULS could also inhibit to some extent the growth of the hyper-thermophilic bacteria and avoid the consumption of soluble carbohydrates.

Example 5

Qualitative analysis of indigenous enzymes in WAS

In this section we report some more qualitative evidences of proteolytic enzymes in WAS. For this purpose, we used Petri dishes with agar (10 g/L), potassium

phosphate (50 mM) and 20 mUL of skimmed milk.

In. order to emphasize the existence of these proteolytic enzymes and protease- producing microorganisms we pipetted sludge samples (0.5 pL) after various pre- treatment (ULS, thermophilic enzymatic: 6 and 24 hours in a 65°C water bath) into wells on casein-agar Petri dishes. These Petri dishes contained 50mM potassium phosphate, 20 mUL skimmed milk and 10 g/L agar. These dishes were initially whitish in appearance due to the casein in the milk and clear plaques indicated the degradation of casein by proteolytic enzymes. The casein in skimmed milk gives a whitish colour to the Petri dish. The addition of this protein to the agar medium allows us to observe colonies that are able to degrade the casein. If the colony produces proteases that degrade casein, then there will be a clear patch around that colony. Several dilution of raw WAS were prepared in order to be able to observe single colonies on the Petri dishes which were incubated at 37°C. Figure 8 below shows the growth of several colonies and a few colonies were able to degrade casein leaving a clear patch around it. This shows evidence that WAS contains proteolytic bacteria.

It can be seen from that proteolytic bacteria were able to grow even when the Petri dish was sealed with parafilm in order to reduce the amount of oxygen during the incubation period (Figure 9). This shows that these bacteria are probably facultative and the enzymatic degradation of casein was also not affected by these lower oxygen conditions.

Figure 10 shows that when WAS was not diluted, then almost all the casein in the Petri dish, is degraded showing evidence of the powerful action of proteases in WAS.

Furthermore, a colony from Figure 9 was isolated on a new Petri dish using a sterile loop and then incubated at 55°C in order to reveal if these proteolytic bacteria were able to grow in the thermophilic range. Interestingly, Figure 11 shows that these bacteria could not grow at 55°C, but their enzymes (isolated together with the loop) were still active at 55°C and could degrade casein, leaving a clear patch on the Petri dish.

In the next step, we tried to isolate single colonies onto new Petri dishes (Figure 12). This isolation step was successful except for the bottom left picture of Figure 12 where a fungus was growing together with a bacterium. Interestingly, these 2 microorganisms were still able to degrade casein. Example 6

Qualitative analysis of proteases and protease-producing microorganisms.

In this section qualitative results on the proteases involved during the hyper- thermophilic enzymatic treatment are presented in Figure 13. It can be seen that no protease was detected in the non-ultrasonicated sample when the Petri dish was incubated at 55°C (Figure 13A) showing that the free enzymatic activity present in the liquid phase was negligible. It can be seen that although some proteolytic activity was detected at thermophilic temperature, there was no bacterial growth indicating that the microorganism producing these enzymes were not thermophilic.

In Figure 13B, the samples with and without ultrasonication were pipetted into wells and the Petri dishes were placed at 37°C. It was found that at mesophilic protease- producing bacteria were able to grow well and release extra-cellular proteases during the growth as shown by the clear plaque around the colonies. Moreover, the use of ULS enhanced the growth of these microorganisms as demonstrated by larger colonies and larger plaques of depleted casein. The disruption of bacterial floes by ULS improved the production of proteases by these mesophilic microorganisms. When trapped within floes and EPS structure, these bacteria were not able to produce proteases in optimum conditions.

In Figure 13C, it can be seen that the hyper-thermophilic treatment (24 hours at 65°C) had also an effect on these bacteria as more time spent at 65°C during the pre-treatment means more cell lysis which is why fewer and smaller colonies were observed compared to the 6h sample in Figure 13B. However, the bacteria that survived the hyper-thermophilic treatment were still able to produce proteases and again, the growth and enzymes production was enhanced when ULS was applied beforehand. This qualitative analysis was not possible when the samples were incubated at 55°C (data not shown) due to the growth of other microorganisms (fungi and bacteria) that did not produce proteases. This competition between protease- producers and non-producers was in accordance with the consumption of proteins and carbohydrates observed at 55°C. Example 7

Ultrasonication of a small proportion of WAS during the combined pre- treatment WAS

We found that the temperature increase during ultrasonication also played a role in COD solubilization. It was found that WAS contained proteolytic microorganisms able to produce proteases and these enzymes were active at thermophilic temperatures. Up to 11,000 mg/L SCOD were obtained using indigenous enzymes. In order to gain more understanding a small percentage of WAS (1, 3, 5, 7 and 10%) were ultrasonicated (30 sec, 3500J) and then incubated at 65°C. The evolution with time of soluble COD, carbohydrates and proteins is shown in Figure 14.

Figure 14 shows that more SCOD was solubilized when 5% WAS was ultrasonicated compared to 10%, but the difference is within 500 mg/L. The ultrasonication of a small fraction (1 to 10%) gave similar results overall. Moreover, the kinetic was improved at 5%. These results show that a small amount of WAS needs to be ultrasonicated (at least 10%) to see an effect. For proteins, the greatest solubilization was obtained with 5% after 5 hours, but after 24 hours, the final concentration increased with increasing percentage of ultrasonicated WAS. For carbohydrates, the highest kinetic and final concentration were obtained with 1% ultrasonicated WAS after 9 hours followed by 0, 10, 7 and 5%.

Example 8

TSS and VSS removals during ULS and enzymatic pre-treatment.

Table 3 shows the TSS and VSS removal during the combined pre-treatment. It can be seen that ULS alone resulted in TSS removals lower than 10%, while the thermal treatment results in TSS removals in the range 20-23%. When 50% of the sludge was ultrasonicated and treated at 65°C, then a maximum of 27% TSS and VSS removal was obtained. Treating 100% of the sludge by ultrasonication did not increase this removal, confirming that ultrasonication of a high proportion is not required. TSS removal % VSS removal %

0 0

ULS 20% (5,000 kJ/kg TS) 4.6 2.36

ULS 50% (5,000 kJ/kg TS) 7.28 5.91

ULS 100% (5,000 kJ/kg TS) 8.62 6.86

raw+55°C for 24hrs 20.5 19.15

raw+65°C for 24hrs 22.22 22.93

ULS 20% + 55°C for 24hrs 21.65 23.4

ULS 50% + 55°C for 24hrs 21.46 20.57

ULS 100% + 55°C for 24hrs 22.8 22.46

ULS 20% + 65°C for 24hrs 23.75 23.4

ULS 50% + 65°C for 24hrs 27.2 26.95

ULS 100% + 65°C for 24hrs 24.33 24.35

Table 3. TSS and VSS removal during the combined ULS/thermophilic and hyper-thermophilic enzymatic pre-treatment.

Example 9

Effect of the sludge pre-treatment on anaerobic biodegradability

Finally, the biodegradability of the SCOD obtained by the novel combined pre- treatment was assessed using the Biochemical Methane Potential test. It was found that biogas and methane production were increased by 15 and 19%, respectively, using the combined ultrasonication and thermal treatment (Figure 15). The final biodegradability of WAS was increased from 259 to 308 ml CH₄/g VS.

Example 10

Effect of the combined sludge pre-treatment on anaerobic biodegradability

Since it has been found that higher SCOD were obtained by combining ULS and hyper-thermophilic enzymatic treatment, a higher methane potential was expected to be found using the combined pre-treatment. BMP Results of the novel combined pre- treatment are shown in Figure 16. It was found that the methane production increased by 25%, while the methane percentage in the biogas was up to 6% higher.

Claims

1. A method of pretreating organic waste comprising the steps of:

(a) providing an organic waste;

(b) subjecting the organic waste to ultrasonication; and

2. The method of Claim 1 , wherein the organic waste of step (a) is split into a first portion and a second portion, only the first portion is subjected to step (b) and is then combined with the second portion so that the combined first and second portions are subjected to step (c).

3. The method of Claim 2, wherein the first portion is from 0.5 wt% to 99 wt% of the organic waste.

4. The method of Claim 3, wherein the first portion is from 1 wt to 80 wt%.

5. The method of Claim 4, wherein the first portion is from 2.5 wt to 60 wt%.

6. - The method of Claim 5, wherein the first portion is from 5 wt to 50 wt%.

7. The method of Claim 6, wherein the first portion is from 7 wt to 40 wt%.

8. The method of Claim 7, wherein the first portion is from 10 wt to 25 wt%.

9. The method of Claim 7, wherein the first portion is 20 wt% or 50 wt%.

10. The method of any one Of the preceding claims, wherein the organic waste is subjected to ultrasonication of from 10 seconds to 1 hour.

11. The method of Claim 10, wherein the organic waste is subjected to ultrasonication of from 30 seconds to 30 minutes.

12. The method of any one of the preceding claims, wherein the ultrasonicaton uses a power of from 0.5 to 3 kWh/m³ of organic waste treated.

13. The method of Claim 12, wherein the ultrasonicaton uses a power of from 1 to 2 kWh/m³ of organic waste treated.

14. The method of any one of the preceding claims, wherein the ultrasonication step uses a SEI of from 2500 to 7500 kJ/kg TS.

15. The method of Claim 14, wherein the ultrasonication step uses a SEI of 5000 kJ/kg TS.

16. The method of any one of the preceding claims, wherein the ultrasonication step is conducted at a frequency of from 19 kHz to 200 kHz.

17. The method of Claim 16, wherein the ultrasonication step is conducted at a frequency of about 20 kHz.

18. The method of any one of the preceding claims, wherein the temperature of step (c) is from 40°C to 80°C.

19. The method of Claim 18, wherein the temperature of step (c) is from 45°C to 75°C.

20. The method of Claim 19, wherein the temperature of step (c) is from 50°C to 70°C.

21. The method of Claim 20, wherein the temperature of step (c) is 65°C.

22. The method of any one of the preceding claims, wherein the heat treatment of step (c) is from 30 minutes to 24 hours.

23. The method of Claim 22, wherein the heat treatment of step (c) is from 45 minutes to 10 hours.

24. The method of Claim 23, wherein the heat treatment of step (c) is from about 1 hour to 6 hours.

25. The method of any one of the preceding claims, wherein the heat treatment of step (c) is conducted without mixing and aeration.

26. The method of any one of Claims 1 to 24, wherein the heat treatment of step (c) is conducted with agitation of the organic waste.

27. The method of Claim 2, wherein the first portion is 50 wt% and the heat treatment of step (c) is performed at 65°C for 24 hours.

28. The method of any one of the preceding claims, wherein the method further comprises feeding the pre-treated organic waste into an aerobic or an anaerobic digester.

29. The method of Claim 28, wherein the method further comprises feeding the pre-treated organic waste into a mesophilic anaerobic digester or a thermophilic anaerobic digester.

30. The method of Claim 29, wherein the anaerobic digester is a thermophilic anaerobic digester.

31. The process of any one of the preceding claims, wherein the organic waste comprises biodegradable solids.

32. The process of any one of Claim 31, wherein the biodegradable solids have a total solids concentration of from greater than or equal to 3 g/L to less than or equal to 50 g/L.

33. The process of Claim 31 or 32, wherein the organic waste is a sludge, food waste, oily waste, solid waste with a high organic content or any combination thereof.

34. The process of Claim 33, wherein the sludge is a waste activated sludge.

35. A waste treatment plant comprising a pre-treatment apparatus that comprises an ultrasonicator apparatus and a heating apparatus adapted to act in a batch or continuous fashion on an organic waste, wherein the ultrasonicator apparatus is placed upstream of the heating apparatus and is in fluid communication therewith.

36. The waste treatment plant of Claim 35, wherein the ultrasonicator apparatus and the heating apparatus are adapted to act in a batch fashion.

37. The waste treatment plant of Claim 35 or 36, wherein the pre-treatment apparatus is adapted so that a portion of the organic waste is fed to uitrasonicator apparatus and the remainder is fed directly to the heating apparatus.

38. The waste treatment plant of any one of Claims 35 to 37, wherein the waste treatment plant is a wastewater treatment plant.