Wastewater Treatment

Field of the Invention

The present invention relates to biological processes for at least partial removal of nitrogen, phosphorus and BOD from wastewaters which have very high levels of nitrogen as well as significant phosphorous levels, such as abattoir wastewaters.

Background to the Invention

The meat processing industry requires large quantities of water, much of which is discharged as high biochemical oxygen demand (BOD) wastewater containing high levels of nutrients such as nitrogen (N) and phosphorus (P).

Similar to high BOD and nitrogen levels in waterways, the presence of excess phosphorus in waterways is also of major concern as it has the potential to promote eutrophication. As major sources of phosphorus include agricultural runoff and a variety of domestic, commercial and industrial processes, the removal of phosphorus during wastewater treatment is of significant environmental importance.

Over the past two decades, BOD and N removal from abattoir wastewater has received much attention, and reliable BOD and N removal systems have been successfully developed and applied to abattoir wastewater treatment using continuous activated sludge systems. However, phosphorous removal has received less attention.

A common means for removing phosphorus from wastewaters is chemical precipitation. Typically this involves the addition of metal salts, such as aluminium sulphate, ferric sulphate and ferric chloride, to react with soluble phosphorus and form solid precipitates that can be removed by solid separation processes.

However, chemical precipitation suffers from a number of disadvantages. Typically, chemical precipitation increases the volume of sludge produced and can produce sludge with poor settling qualities. Further there are environmental concerns associated with the use and release of chemicals.

Nonetheless, phosphorous removal continues to be achieved primarily through chemical precipitation, despite biological P removal being potentially a much cheaper and more environmentally sustainable option.

Enhanced biological phosphorus removal (EBPR) provides a means of removing phosphorus from wastewater through a biological process as an alternative to chemical precipitation. EBPR relies on the ability of polyphosphate-accumulating microorganisms (PAOs) to take up phosphorus in excess of their metabolic requirements.

However, complete nitrification of wastewaters containing high levels of ammonium and other nitrogenous sources produces a high level of nitrate, which interferes with the development of a stable and reliable EBPR process.

Another difficulty in developing an effective biological process for simultaneous removal of nitrogen and phosphorous from abattoir wastewater is that such wastewaters

contain substantial amounts of fat, oil and grease (FOG), which deteriorate the sludge settling properties when directly fed to activate sludge systems. Such wastewaters are therefore typically pre-treated before being treated in biological nutrient removal systems. In Australia, the raw abattoir wastewater is typically pre-treated in anaerobic ponds with a hydraulic retention time ranging between 7 - 14 days. While reducing the FOG content, this anaerobic treatment process also removes a large fraction of the BOD from the wastewater, often resulting in limiting concentrations of available carbon sources (particularly Volatile Fatty Acids - VFAs) required for N removal, and P removal by PAOs.

Thus, an objective of the present invention is to provide an effective biological process for the simultaneous removal of nitrogen and phosphorous from wastewaters comprising elevated levels of nitrogen and phosphorous sources which overcomes or ameliorates one or more of the above identified difficulties.

Summary of the Invention

Through the present studies, it was found that nitrogen and phosphorous in wastewaters comprising elevated levels of nitrogen can be efficiently removed using a biological process employing a combination of biological nitrogen removal and enhanced biological phosphorus removal (EBPR) employing activated sludge comprising nitrifying and denitrifying organisms and polyphosphate accumulating organisms (PAOs), by feeding the wastewater into a reaction vessel in steps and manipulating the aeration conditions in such a manner that significant nitrate and nitrite levels do not accumulate, thereby ensuring anaerobic conditions appropriate for efficient polyphosphate accumulation by the PAOs.

Thus, according to an aspect of the invention, there is provided a biological process for reducing the levels of nitrogen and phosphorous in wastewater, wherein said wastewater comprises at least 100mg/L total nitrogen, wherein said process comprises feeding said wastewater into a reaction vessel in at least two steps, wherein said reaction vessel comprises an active biomass comprising nitrifying and denitrifying organisms and polyphosphate accumulating organisms (PAOs), wherein at least the first feed step is followed by a non-aerated period of sufficient duration to result in sufficiently low concentrations of NO _x species in the wastewater to allow for accumulation of polyhydroxyalkanoates inside the PAO cells, and at least the first non-aerated period is followed by an aerated period of sufficient duration to allow for ammonium oxidation by the nitrifying organisms and assimilation by the PAOs of at least a portion of the phosphorous in the wastewater.

According to an embodiment, the first aerated period is followed by at least one cycle of a feed step, a non-aerated period and a subsequent aerated period.

According to another embodiment, the wastewater is fed into said reaction vessel in at least three steps, wherein each feeding step is followed by a non-aerated period and a subsequent aerated period.

At least the first aerated period may be of sufficient duration to ensure substantially complete ammonium oxidation by the nitrifying organisms and, in a specific embodiment, each aerated period is of sufficient duration to ensure substantially complete ammonium oxidation by the nitrifying organisms.

In such an embodiment, about 50% of the volume of wastewater to be treated may be fed into the reaction vessel in the first feeding step, about 30% of the volume of wastewater to be treated may be fed into the reaction vessel in the second feeding step, and about 20% of the volume of wastewater to be treated may be fed into the reaction vessel in the third feeding step.

The reaction vessel contents may be allowed to settle after the third aerated period, and treated wastewater and settled sludge separated.

A source of volatile fatty acids may also be fed into the reaction vessel or added to said wastewater before feeding into said reaction vessel. The source of volatile fatty acids may be co-fed into the reaction vessel with the wastewater.

The source of volatile fatty acids may comprise a pre-fermented high BOD waste comprising elevated levels of acetic and propionic acids, such as at least 100mg/L of each of acetic and propionic acids.

The source of volatile fatty acids may be fed into said reaction vessel or added to said wastewater in an amount such that the overall soluble COD per litre of influent into said reaction vessel is from about 500 mg COD/L to about 600 mg COD/L.

Alternatively, the source of volatile fatty acids may be fed into said reaction vessel or added to said wastewater in an amount such that the overall ratio of total COD to total nitrogen in the influent to said reaction vessel is from about 5 to about 10, and the overall ratio of soluble COD to phosphorous in said influent is about 15.

In an embodiment, each feeding step may comprise distributing wastewater into settled sludge at the bottom of the reaction vessel. In such an embodiment, although the reaction vessel contents may be mixed during the feeding, it may be advantageous if the contents of the reaction vessel are not mixed during at least a portion of the feeding step. Similarly, although the reaction vessel contents may be mixed during the non-aerated period following feeding, it may be advantageous if they are not mixed during at least a portion of the non-aerated period following the feeding step. If the feeding step is carried out slowly, then there may be no need for a non-mixed, non-aerated period after the feeding step.

The concentration of NO _x species in wastewater undergoing treatment may be measured with an on-line NO _x sensor or the NO _x depletion point may be estimated by measuring the oxidation reduction potential of the reaction vessel contents.

The dissolved oxygen concentration of the reaction vessel contents may be monitored during an aeration period, and maintained within predetermined concentrations, for example between about 4mg/L and about 0.3mg/L, by adjustment of the rate of aeration. For example, air may be provided intermittently during an aeration period using an on/off control system in communication with a dissolved oxygen probe in contact with the reaction vessel contents.

According to another embodiment of the present invention, the first aerated period may be followed by at least one cycle of a feed period and an aerated period during which the level of dissolved oxygen is controlled to allow for simultaneous nitrification and denitrifϊcation in the contents of said reaction vessel. In such an embodiment, the dissolved oxygen level may be maintained between about 0.5mg/L to about 0.3mg/L.

According to another embodiment of the invention, the duration of an aerated period in a process of the invention is determined based on the rate of change of the pH (for example, change in pH over a window of 5 minutes) of the reaction vessel contents due to ammonium oxidation. For example, an aerated period may be stopped when the rate of decrease of the pH of the reaction vessel contents decreases to at least a predetermined value, for example to about 10% or less of the maximum rate of decrease of the pH of the reaction vessel contents due to ammonium oxidation, or when the rate of decrease of the pH of the reaction vessel contents decreases to about 0.01 pH units per five minutes or less. Alternatively, or as well, the duration of an aerated period may be determined by the oxygen uptake rate of the reaction vessel contents. For example, an aerated period may be stopped when the oxygen uptake rate of the reaction vessel contents drops below a predetermined value, for example when the oxygen uptake rate of the reaction vessel contents drops below 80% or less of the maximum oxygen uptake rate of the reaction vessel after introduction of said aerated period, or when the oxygen uptake rate of the reaction vessel contents drops below about lmg/min/L of the reaction vessel contents. The duration of the aerated period may be controlled to promote nitrite production rather than nitrate production during an aerated period, such that nitrogen removal from the wastewater during a non-aerated period occurs predominantly through direct denitritation of nitrite, with reduced need for nitrate reduction and requirement of BOD.

In an embodiment in which the treated waste may be used for land irrigation, a process according to the invention may comprise two feed steps only. Such a process may result in a treated wastewater suitable for land application, which may comprise less than about 50mg/L total nitrogen and less than about 15 mg/L total phosphorous.

Brief Description of the Drawings

Figure 1 shows a schematic diagram of a sequencing batch reactor set up to carry out a process according to the invention.

Figures 2a to 2d show characteristics of the influent (2a and 2b), effluent nutrient levels and the Accumulibacter-P ¹ AOs population (2c), and MLSS and the VSS/MLSS ratio in a reactor (2d) in which a process according to the invention is carried out.

Figure 3 shows nitrogen and phosphorus profiles during a study of a cycle of a process according to the invention.

Figure 4 shows concentration of the main VFAs, PO ₄ ^3" and NH ₄ ⁺ in raw abattoir wastewater before and after pre-fermentation, and after one week storage at 4°C. Other VFAs include iso-butyric, butyric and iso-valeric acids.

Figure 5 shows the concept of a process according to the invention for treatment of abattoir wastewater for land irrigation (two feed steps)

Figures 6 A and 6B show influent and effluent concentrations of N and P species over 7 months operation of a two step feed sequencing batch reactor (SBR) process according to the invention: A.- Nitrogen species: • N-NH ₄ influent, ▼ NH ₄ effluent, 0 nitrite effluent, α nitrate effluent. B.- Phosphorus: • P-PO ₄ influent-, T P-PO ₄ effluent. The solids lines represent the upper and lower discharge limits.

Figures 7A and 7B show carbon evolution in pond wastewater used in the process for which the influent and effluent concentrations of N and P specie ^' s are shown in Figures 6A and 6B. A.- T Total COD; • Soluble COD. B.- Volatile Fatty Acids: • acetate; T propionate.

Figure 8 shows experimental profiles obtained for the different compounds analysed along a cycle of the SBR process for which the influent and effluent concentrations of N and P species are shown in Figures 6 A and 6B: o N-NH ₄ , V N-NO ₃ , T N-NO ₂ , • P-PO ₄ ^3"

Figure 9A and 9B shows experimental profiles obtained for the different compounds analysed for a later, more optimised cycle of the SBR process for which the influent and effluent concentrations of N and P species are shown in Figures 6 A and 6B: TN-NH ₄ , O N-NO ₃ , D N-NO ₂ , • P-PO ₄ ^3"

Figure 1OA shows a schematic representation of a normal denitrifϊcation process via nitrate reduction. Figure 1OB shows a schematic of denitrification via nitrite reduction

Figure 11 illustrates a control strategy employed to cease aeration when NH ₄ ⁺ is fully oxidised in an SBR process according to the invention. The 1 ^st condition to be met is based on the pH slope (1), the 2 ^nd is based on the length of time the valve is in an "off state which is directly related to the OUR in the case that aeration is controlled in an on- off manner (2) and the 3 ^rd condition is based on minimum duration of aeration (3).

Figure 12A shows the degree of nitrite accumulation in the three stages and the abundance of NOB Nitrospira (FISH probe Nitspa-662). The NOB quantification shown is an average (error bars=S.E., n=3). Figure 12B shows the ammonium, oxidised nitrogen and phosphate in the effluent and VFAs in the influent.

Figure 13 shows an example of pH, DO, OUR, nitrogen and phosphorous profiles during a SBR cycle when aeration was manually extended to destabilise nitrite pathway (black arrow). White arrows indicate feeding times.

Figure 14 shows an example of pH, DO, OUR, nitrogen and phosphorous profiles during a SBR cycle after the automatic aeration control was implemented. Vertical dot lines indicate when the aeration was automatically stopped and black arrows represent the anoxic time gained by stopping the aeration. White arrows indicate feeding times.

Figure 15 is a photograph of a lab-scale SBR unit as used in the experiments as described in the examples.

Figures 16A to 16C show nitrogen and phosphorous removal from abbattoir wastewater over almost 7 months operation of a three step pilot scale feed sequencing batch reactor (SBR) process using a gentle, uniformly distributed non-stirred bottom feeding system according to the invention: A. - Treated wastewater effluent nitrogen (as ammonium species - ♦ - and NOx species - D) and phosphorous (as PO ₄ - A). B.- Nitrogen removal: ♦ soluble inorganic nitrogen removal, as a percentage; and o total nitrogen removal , as a percentage. C- Phosphorus: ♦ removal of phosphorous (as phosphate), as a percentage; and o total phosphorous removal , as a percentage.

Figures 17A to 17C show nitrogen and phosphorous removal from abbattoir wastewater over almost 7 months operation of a three step pilot scale feed sequencing batch reactor (SBR) process using a bottom feeding system according to the invention (employing mixing and aeration): A. — Treated wastewater effluent nitrogen (as ammonium species - ♦ - and NOx species - o) and phosphorous (as PO ₄ - A). B.- Nitrogen removal: ♦ soluble inorganic nitrogen removal, as a percentage; and o total nitrogen removal , as a percentage. C- Phosphorus: ♦ removal of phosphorous (as phosphate), as a percentage; and o total phosphorous removal , as a percentage.

Figure 18 shows NOx species concentrations and phosphorous (as phosphate) concentrations) and pH profile for a representative cycle of the SBR process represented in Figures 16A to C, carried out on 3 September 2007.

Figure 19 shows NOx species concentrations and phosphorous (as phosphate) concentrations) and pH profile for a representative cycle of the SBR process represented in Figures 17A to C, carried out on 3 September 2007.

Abbreviations and Definitions

The following abbreviations are used herein:

AOB ammonia oxidising bacteria

BOD biochemical oxygen demand

COD chemical oxygen demand

DO dissolved oxygen

EBPR enhanced biological phosphorous removal

FOG fat, oil and grease

GAO glycogen accumulating organism

HRT hydraulic residence time

MLSS mixed liquor suspended solids

N nitrogen

NH ₄ ammonium

NO ₂ nitrite

NO ₃ nitrate

NO _x sum of nitrate and nitrite

NOB nitrite oxidising bacteria

OUR oxygen uptake rate

P phosphorous

PO ₄ phosphate

PAO polyphosphate accumulating organism

PHA polyhydroxyalkanoate

SBR sequencing batch reactor

SRT sludge retention time

TKN total Kjedahl nitrogen

TP total phosphorous

TSS total suspended solids

VFA volatile fatty acid

VSS volatile suspended solids

As used herein, the term "comprising" means "including principally, but not necessarily solely". Variations of the word "comprising", such as "comprise" and "comprises", have correspondingly similar meanings.

As used herein, the term "phosphate accumulating organism" means any organism capable of taking up phosphorus in excess of its metabolic requirements and accumulating it intracellularly as a phosphate rich species.

Detailed Description of the Invention

Efficient simultaneous removal of phosphorous and nitrogen from wastewaters containing elevated amounts of nitrogen, such as abattoir wastewaters, which contain at least 100mg/L and often more than 150mg/L total nitrogen, and at least 20 mg/L total phosphorous in the form of phosphate and organic phosphorous is complicated by the fact that the high levels of NO _x resulting from nitrification of the biologically available nitrogen thwart phosphate uptake by polyphosphate accumulating organisms (PAOs).

Another problem that faces treatment of such wastewaters is lack of BOD, and particularly volatile fatty acids (VFAs) in the wastewater to be treated. PAOs require VFAs during an anaerobic period to store polyhydroxyalkanoates to provide energy for phosphate uptake during an aerobic period. Although raw abattoir wastewater has a high BOD due to elevated levels of fats oils and grease (FOG), these wastewaters are typically pre-treated to improve the settling properties of these wastes, resulting in significant depletion of biologically available carbon sources.

The present invention provides processes for simultaneous removal of BOD, nitrogen and phosphorous from wastewaters having high N levels using a sequencing batch reactor (SBR) system employing an active biomass comprising nitrifying and denitrifying organisms as well as polyphosphate accumulating organisms.

In the current studies it has been found that a step-feed SBR scheme, characterised by alternating aerobic and anoxic phases in a SBR cycle allows timely removal of nitrate or nitrite so that, when an adequate amount of COD is available, nitrate or nitrite build-up is avoided.

A process of the present invention for at least significantly reducing the levels of nitrogen, phosphorous and BOD in a wastewater containing elevated total nitrogen, such as abattoir wastewater, involves feeding said wastewater into a reaction vessel in at least two separate steps. The wastewater may contain at least 100mg/L total nitrogen, such as at least about 150mg/L total nitrogen, at least about 200mg/L total nitrogen, at least about 250mg/L total nitrogen, at least about 275mg/L total nitrogen, at least about 300mg/L total nitrogen, at least about 325mg/L total nitrogen, or even at least about 350mg/L total nitrogen. The total nitrogen content of the wastewater may be significantly higher than 350mg/L - this may require feeding the wastewater into the SBR system in more than three feeds, allowing for longer process steps (such as nitrification and/or denitrification), reducing the volume of wastewater fed into the SBR system each cycle, or any combination thereof.

The reaction vessel contains an active biomass including nitrifying and denitrifying microorganisms and polyphosphate accumulating organisms (PAOs). At least the first feed step is followed by a non-aerated period of sufficient duration to result in sufficiently low concentrations of NO _x species in the wastewater to allow for

accumulation of polyhydroxyalkanoates in the PAOs, thereby allowing for phosphate accumulation by PAOs in a subsequent aerated/aerobic period.

At least the first non-aerated period is followed by an aerated period of sufficient duration to allow for ammonium oxidation by the nitrifying organisms and assimilation by the PAOs of at least a portion of the phosphorous in the wastewater. Depending on the quality of effluent desired from the process, at least the first aerated period may be of sufficient duration so as to allow for substantially complete oxidation of ammonium introduced into the SBR system by the feed step. Subsequent aerated periods may also be of sufficient duration to allow for substantially complete ammonium oxidation by the nitrifying organisms after each feeding step.

Referring to Figure 1, an embodiment of a process according to the invention may be carried out in a sequencing batch reactor system comprising a reaction vessel 10 containing a biologically active sludge 20.

In a first feeding step, a portion of wastewater to be treated is fed into reaction vessel 10 from wastewater reservoir 30 by pump 40 via conduit 50. The amount of wastewater to be fed may depend on the extent of P and N removal to be achieved - if the treated wastewater is to meet standards for discharge into waterways, the wastewater may be fed into the reaction vessel in three steps, or more. Although the amounts of wastewater fed at each stage may be the same, they may also be of increasingly smaller volume, increasingly larger volume, alternating larger and smaller volumes, or any permutation thereof. However, a large final feed step may result in significant NO _x levels in the reactor discharge, and therefore according to an embodiment feed steps of progressively smaller size are employed. In a specific embodiment, about 50% of the wastewater to be treated may be fed into the reaction vessel in a first feed step, about 30% in a second feed step, and about 20% in a third feed step.

Although the wastewater may be introduced into the reaction vessel in any appropriate manner, feeding using the UniFED™ process, as described in international patent publication WO 95/24361, or an adaptation thereof may be used. Briefly, the sludge in reaction vessel 10 may be allowed to settle before any or each feeding step, and feeding may comprise distributing the wastewater into the bottom of the reaction vessel, into the settled sludge, without aeration or stirring. This allows for intensive contacting of all biomass with the fresh feed stream entering the reactor, avoidance of mixing of the biomass with supernatant water from a previous process cycle, which often contains nitrates which can be detrimental to the performance of the phosphorous removal processes, and quickly established anaerobic conditions favourable to VFA uptake by PAOs.

The feeding step may be followed by a non-mixed, non-aerated period or, if the feeding step (which is non-mixed, non-aerated) is carried out slowly, a subsequent non- mixed non-aerated period might not be necessary: due to efficient contact between the

wastewater and settled sludge when feed is distributed into settled sludge, if the feed rate is sufficiently slow, all NO _x species present in the settled sludge may be denitrified, and volatile fatty acids taken up by PAOs soon after the feeding step is completed. Slower feed rates also result in less disturbance of the settled sludge, and therefore better contact of the feed with the sludge.

'Sufficiently slow' feed rates may comprise inflow rates into reaction vessel 10 of from about 20% to about 1% of the original, uncharged volume per hour, such as from about 15% to about 2% of the uncharged volume per hour, from about 12% to about 4% of the uncharged volume per hour, from about 10% to about 5% of the uncharged volume per hour, about 10% of the uncharged volume per hour, about 9% of the uncharged volume per hour, about 8% of the uncharged volume per hour, about 7% of the uncharged volume per hour, about 6% of the uncharged volume per hour, or about 5% of the uncharged volume per hour.

After a sufficient non-mixed, non-aerated period or, if the feed step is carried at a sufficiently slow inflow rate, once the feeding step is over, the contents of reaction vessel 10 may optionally be mixed by any appropriate means, without aeration. For example, mixing may be by an impeller 60 driven by motor 70.

During or after the feeding step, the concentration of NO _x species in the reaction vessel contents may be monitored by monitoring the oxidation/reduction potential (ORP) and/or pH of the reaction vessel contents, by using an online NO _x sensor, or any combination thereof. ORP may also be monitored to assess uptake of volatile fatty acids from the contents of reaction vessel 10 - as VFAs are taken up by organisms from the extracellular contents of reaction vessel 10, the ORP signal decreases, and as the VFAs are depleted from the extracellular contents of reaction vessel 10, the rate of decrease of the ORP signal slows and may plateau or even rise depending on the complexity of the contents of reaction vessel 10.

Oxidation/reduction potential may be assessed using an ORP meter 80 communicating by any appropriate means with an ORP probe 90 which is in contact with the contents of reaction vessel 10. ORP meter 80 may be connected by conductive lines 100 to ORP probe 90. pH may be determined using a pH meter 110 communicating by any appropriate means with a pH probe 120 which is in contact with the contents of reaction vessel 10. pH meter 110 may be connected by conductive lines 130 to pH probe 120.

The concentration of NO _x (and oxygen) in the reaction vessel contents after at least the first non-aerated period needs to be sufficiently low before uptake of VFAs from the extracellular medium and intracellular accumulation of polyhydroxyalkanoates by the PAOs (to provide energy for phosphate uptake during the subsequent aerobic phase) will occur. Once at least most of the VFAs have been depleted from the extracellular contents

of reaction vessel 10, which may be determined by a break in declining ORP slope observed at ORP meter 80, a period of aeration may be started.

Alternatively, for example for a SBR process operating industrially for the treatment of wastewaters, each cycle of wastewater treatment (that is, from first feed to treated effluent discharge) may be of a substantially fixed timing, for scheduling purposes. In such a case, at least the first non-aerated period, and possibly other non- aerated periods may be of a fixed length of time, which may be of sufficient time to ensure sufficiently low NO _x concentrations and depletion of the VFAs (based on the ongoing performance of the SBR) before commencing an aerated period. For example, for a three feed step process having a cycle time of approximately 6 hours, the first non- aerated period may be fixed at, say, about 20 minutes to about 1.5 hours duration (depending on the ongoing performance of the SBR system), such as about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes or about 90 minutes.

During an aerated period, air is pumped into reaction vessel 10 from blower 140 through aerator device 150 (such as, for example, an air diffuser), via conduit 160. Other possible aeration means/configurations, as are known in the art, such as a surface aerator (which does not require the use of a blower), may be used.

Although aeration may be uncontrolled, control of aeration may be required to avoid excessive dissolved oxygen in the contents of reaction vessel 10, which could require a longer subsequent non-aerated period to provide sufficiently anoxic conditions for subsequent PHA storage by PAOs in the active sludge. In addition, excessive DO during, and particularly towards the end of an aerated period may promote nitrate accumulation, rather than nitrite accumulation. Removal of nitrogen using complete nitrification (to nitrate) consumes 33% more oxygen than oxidation to nitrite alone, and overall carbon consumption for N removal via the nitritation/denitritation process is about 40% lower than for the nitrification/denitrification process (see Figures 1OA and 10B). Thus significant savings on aeration and BOD can be made by promoting N removal by nitritation/denitritation rather than nitrification/denitrification.

Thus, the amount of dissolved oxygen in the contents of reaction vessel 10 may be controlled during an aerated step. In order to do so, the dissolved oxygen content of the wastewater may be monitored through a DO meter 170 communicating by any appropriate means with a DO probe 180 which is in contact with the contents of reaction vessel 10. DO meter 170 may be connected to DO probe 180 by conductive lines 190. A flow meter 200 and/or a valve 210 may be used to monitor and/or regulate aeration, and may be positioned in line with conduit 160 to monitor and/or control the air flow respectively so as to maintain the DO level in the contents of reaction vessel 10 within desired ranges. The valve 210 may be any appropriate type of valve capable of providing

the type of air flow control desired, such as an on/off valve, or a mass flow controller, and may be in communication with a suitable controlling module, such as a programmable logic controller (PLC) unit, which may also be in communication with DO meter 170. The controlling module may also be in communication with flow meter 200 for feedback control of air flow rate via valve 210. Alternatively, air flow rate may be controlled by other means, such as by appropriate control of blower 140 and monitoring of air flow by flow meter 200. In such an arrangement, DO meter 170, blower 140 and flow meter 200 may be in communication with a controlling module.

The contents of reaction vessel 10 may be mixed during the aerated step. This may be achieved by any appropriate means known in the art. For example, mixing may be achieved by the aeration itself, or as well as by an impeller 60 driven by motor 70.

The DO level in the contents of reaction vessel 10 may be maintained at any desired level during the aerated period. However, to facilitate rapid achievement of anoxic/anaerobic conditions before or during a subsequent feeding step and/or to promote nitritation/denitritation rather than nitrification/denitrifϊcation, dissolved oxygen levels may be maintained at limiting levels throughout an aerated step. Thus, according to an embodiment the DO levels in the contents of reaction vessel 10 are maintained at a level between about 5mgθ ₂ /L and about 0.1mgO ₂ /L, such as between about 4mgO ₂ /L and about 0.1mgO ₂ /L, between about 4mgO ₂ /L and about 0.3mgθ ₂ /L, between about 3mgO ₂ /L and about 0.5mgO ₂ /L, between about 3mgO ₂ /L and about lmgO ₂ /L, between about 3mgO ₂ /L and about 1.5mgO ₂ /L, or between about 2mgO ₂ /L and about 1.5mgO ₂ /L

In an alternative aeration regime, the first aerated period may be followed by at least one cycle of a feed period and an aerated period during which the level of dissolved oxygen is controlled to allow for simultaneous nitrification and denitrification in the contents of said reaction vessel. This is possible as, if the dissolved oxygen level (DO) is kept low enough, anoxic zones may develop within the reaction vessel 10, such as within floes or other aggregates that may form in the contents of reaction vessel 10, allowing for NO _x reduction within those zones, and ammonium oxidation within oxic zones. Suitable DO levels at which this may be achieved, if suitable aeration monitoring and control is available, may be from about lmgO ₂ /L to about 0.1mgO ₂ /L, about 0.8UIgO ₂ ZL to about 0.2mgO ₂ /L about 0.8mgO ₂ /L to about 0.3mgO ₂ /L about 0.7mgO ₂ /L to about 0.3mgO ₂ /L or about 0.5mgO ₂ /L to about 0.3mgO ₂ /L.

The duration of an aerated period may be determined based on the average rate of change of the pH in a moving window of the mixed liquor. The pH of the contents of reaction vessel 10 typically increases quickly as soon as aeration is introduced but then decreases due to ammonium oxidation until nitritation is complete, after which the pH starts to rise again or decrease more slowly. This turning point is referred to as the ammonia valley (the point at which substantially all ammonium has been oxidised), characterised by a reduction in rate of pH decrease, possibly followed by a pH increase.

Thus an aerated period may be completed when the ammonia valley for the contents of reaction vessel 10 is approached or has passed.

If aeration is allowed to continue beyond the ammonia valley, accumulation of nitrate at the expense of nitrite may occur in reaction vessel 10. Thus, if N removal by nitritation/denitritation is to be promoted rather than nitrification/denitrification, the aerated period may be ended once the ammonia valley is being approached or has just passed, and therefore may be ended when the rate of change of pH of the contents of reaction vessel 10 has reached to a predetermined value. The predetermined value may be, for example, a rate of decrease of pH which is about 20% or less of the maximum rate of decrease observed earlier in the same aerated period (not having regard to any pH changes observed immediately after introduction of aeration, such as within 5-10 minutes after introduction of aeration, such as about 20% or less, about 15% or less, about 10% or less, about 8% or less, about 6% or less, about 4% or less, about 2% or less, or about 0% of the maximum rate of decrease observed. Alternatively, the predetermined value may be an absolute value for the rate of change of pH of the contents of reaction vessel 10, such as a rate of pH decrease of about 0.05pH units or less per five minutes (not having regard to any pH changes observed immediately after introduction of aeration, such as within 5-10 minutes after introduction of aeration), such as a rate of pH decrease of about 0.04pH units or less per five minutes, 0.03pH units or less per five minutes, 0.02pH units or less per five minutes, 0.0 IpH units or less per five minutes, or OpH units per five minutes, but this value may differ widely for a given active sludge composition. The predetermined value may also comprise a positive rate of change of pH, such as the first sign of a positive rate of change of pH of the contents of reaction vessel 10, or soon thereafter (again, not having regard to any pH changes observed immediately after introduction of aeration, such as within 5-10 minutes after introduction of aeration).

Alternatively, or as a complimentary mechanism for detection of the end-point of an aerated period, the duration of the aerated period may be determined based on the oxygen uptake rate (OUR) of the contents of reaction vessel 10 - when nitrification is complete, oxygen demand by the active sludge decreases markedly - a point also known as the 'DO elbow'. The oxygen uptake rate may be estimated by any appropriate method as is known in the art. For example, OUR may be estimated by the amount of aeration required to maintain the DO level at a given value, or within a given range of values. Alternatively, if valve 210 is an on/off valve, the OUR may be indirectly estimated by the amount of time valve 210 is in an "off state (this time is inversely proportional to the OUR). End of nitrification may also be detected by a sudden rise in DO in the contents of reaction vessel 10, especially if constant aeration is employed using a variable throughput valve 210.

In addition, oxygen demand during oxidation of nitrite to nitrate is lower than oxygen demand during oxidation of ammonium to nitrite, and this can be detected as a

drop in OUR as well. Thus, an aerated period may be stopped when the oxygen uptake rate of the contents of reaction vessel 10 drops to or below a predetermined value. The predetermined value may be, for example, an OUR which is about 80% or less of the maximum OUR observed earlier in the same aerated period (not having regard to any OUR values observed immediately after introduction of aeration, such as within 5-10 minutes after introduction of aeration, such as about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 50% or less of the maximum OUR observed. Alternatively, the predetermined value may be an absolute value for the OUR, such as about 1.5 mgO ₂ /min/L, about 1.2 mgθ ₂ /min/L, about lmgθ ₂ /min/L, about 0.9 mgO ₂ /min/L, about 0.8 mgθ ₂ /min/L, about 0.7 mgθ ₂ /min/L, about 0.6 mgO ₂ /min/L, or about 0.5 mgO ₂ /min per litre of the contents of reaction vessel 10, but this value may differ widely for a given active sludge composition.

As the nitritation and/or nitrification endpoint is approached, reached or passed, aeration may be stopped, and the contents of reaction vessel 10 optionally mixed without aeration prior to carrying out the second step of wastewater feed into the reaction vessel 10. If nitrogen removal by the nitritation/denitritation pathway is to be promoted, aeration may be stopped once the nitritation endpoint is approached or reached.

Without wishing to be bound by theory, it is believed that by turning off aeration as soon as nitritation is complete, or nearing completion, nitrite oxidising bacteria (NOBs) are limited for nitrite, and therefore being disadvantaged compared to ammonium oxidising bacteria (AOBs). Over many cycles, this may lead to washing out of the NOB population within an active sludge, which in turn is believed will strengthen/further promote the nitritation/denitritation pathway (that is, reduce the amount of nitrate produced, and subsequent need for denitratation) from within the sludge. This in turn will return reduced aeration and COD requirements/costs, as described previously.

Second and third, and optionally further cycles of feeding, non-aerated periods and aerated periods may be carried out substantially as described above for the first feed step, although the feed may be introduced while the contents of reaction vessel 10 are being mixed.

After the final aerated period is completed, the biomass and at least a portion of the treated wastewater may be separated by any appropriate means known in the art, such as by filtration, centrifugation or settling and decanting/discharging the supernatant. Where the active sludge is to be re-employed for several consecutive processes according to the invention, the contents of reaction vessel 10 may be allowed to settle after the final aerated period, and supernatant, treated wastewater decanted via conduit 220, controlled by valve 230.

In order to control the level of solids/sludge in the SBR over a number of cycles or processes according to the invention, at least a portion of the contents of reaction vessel 10 may also be removed as waste during each cycle, or between cycles by any appropriate

means, such as by pump 310 via conduit 320 to waste receiver 300. Control of the solids in the SBR is necessary as excessive solids accumulation, as a result of, for example, multiplication of organisms within the contents of reaction vessel 10, may result in excessive settling times before decanting treated waste and/or a first feed step of a subsequent treatment cycle. In addition, solids removal from the system is also necessary for removal of phosphorous accumulated in the PAO population. Insufficient solids retention may result in washout/depletion of the organisms required for the treatment process. The amount of wastage during or between cycles may depend on the temperature at which the process is carried out, and may be determined so as to allow a sludge retention time (SRT) of from about 5 days to about 30 days. A lower SRT may be applicable when organisms have higher specific growth rates (shorter doubling times) due to for example a high temperature, while a longer SRT may be required when the specific growth rates of the microorganisms required have lower specific growth rates caused by, for example, a lower temperature. Under normal operating conditions (such as a temperature of about 2O ⁰ C), the SRT may be from about 10 to about 20 days, such as about 15 days.

For a given SRT, which is determined by the specific growth rates of microorganisms, the hydraulic retention time (HRT - the average time that a soluble compound remains in the reaction vessel 10, or reactor volume/influent flow rate) may be designed such that the resulting sludge concentration in the reactor would have a reasonable settling rate, for example, so as to allow decanting of treated wastewater to start after 30min - 1 hour settling. Typically, the higher the sludge concentration is, the longer the settling time required would be. For a given SRT, the sludge concentration in a reactor is determined by two factors, namely HRT and the solids and COD concentrations in the wastewater. The shorter the HRT is, the higher the sludge concentration in the reactor will be. The higher the COD and solids concentrations in the wastewater are, the higher the sludge concentration in the reactor will be. Nitrogen supports the growth of nitrifϊers and therefore has some impact on the sludge concentration as well. However, nitrifϊers typically represent a small percentage of the bacterial population in treatment systems receiving wastewaters containing high levels of COD and solids such as domestic and abattoir wastewaters.

For treatment of wastes with high nitrogen loads (such as from about 200mg/L nitrogen or higher, the HRT may vary from about 24 hours to about 72 hours, such as about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours or about 72 hours. According to a specific embodiment, the HRT is about 42 hours or more, especially if the nitrogen levels are 250mg/L or higher. The HRT also may need to be balanced against a target SBR cycle schedule - when using an SBR process, the HRT is directly related to the length of each

cycle. Increasing the SBR cycle time will increase the HRT which means that less wastewater is treated per day.

The treated wastewater resulting from a process as described above may comprise less than about 2mg/L total phosphorous and less than about 20mg/L total nitrogen and, with proper tuning of the system, may produce effluent comprising less than about lmg/L total phosphorous and less than about 10-15mg/L total nitrogen, which would meet most Australian standards for discharge into waterways. Total phosphorous in effluent obtained from such processes of the present invention may be expected to even be lower than about 0.8mgP/L ₃ such as less than about 0.6mg/L, less than about 0.5mg/L, less than about 0.4mg/L, less than about 0.3mg/L, or less than about 0.2mg/L. Total nitrogen in effluent obtained from processes of the present invention may be expected to even be lower than about lOmgN/L, such as less than about 9mg/L, less than about 8mg/L, less than about 7mg/L, less than about 6mg/L, or less than about 5mg/L.

In contrast to waterway discharge, although disposal of wastewaters by land irrigation requires a high level of biological oxygen demand (BOD) removal (>95%), only medium levels of nitrogen and phosphorus removal are required. The presence of total phosphorus at a level of up to about 10-20mgP/L and total nitrogen, preferably mostly in the form of ammonium, at up to about 50-100mgN/L in the treated effluent is considered appropriate for this purpose. To meet these objectives, a process according to the invention may produce effluent with some presence of nitrogen (primarily ammonia nitrogen) and phosphorus. Such a process may be effectively similar to that described above, although only two feed steps are required, and an aerated period after the second non-aerated period is only optional, as phosphorous removal is not as important. If total nitrogen in the process effluent is to be predominantly ammonium, any aeration after the second feed step may be kept to a minimum, although a brief aeration step may be desirable to strip the effluent of any nitrogen gas formed by denitrification, and thereby improve the settling properties of the sludge in reaction vessel 10. The treated wastewater resulting from such a process will typically comprise up to about 20mgP/L and total nitrogen at up to about 100mgN/L, such as less than about 50mg/L total nitrogen and less than about 15 mg/L total phosphorous. Total phosphorous in effluent obtained from such a process may be between about 10mgP/L and about 15mgP/L, although values below 10mgP/L may occur. For example, total phosphorous in the resulting effluent may be less than about 12mgP/L, such as less than about 10mg/L, less than about 8mg/L, less than about 7mg/L, less than about 6mg/L, or less than about 5mg/L. Total nitrogen in effluent obtained from such a process may be expected to be between about 20mgN/L and about 50mgN/L, although values below 20mgP/L may occur. For example, total nitrogen in the resulting effluent may be less than about 40mg/L, less than about 35mg/L, less than about 30mg/L, less than about 25mg/L, or less than about 20mg/L.

BOD supplementation

Due to pre-treatment of raw wastewaters containing high FOG levels, there are often insufficient carbon resources in the pre-treated wastewater for efficient or complete phosphorous uptake by PAOs or denitritation and/or denitrification by denitrifiers.

To address this, a process according to the invention may comprise supplementation of the wastewater to be treated, or being treated with a source of COD, such as VFAs (which are most readily used by PAOs for intracellular PHA storage) when the wastewater to be treated does not contain a sufficient amount of these for biological phosphorus and nitrogen removal.

For wastewaters comprising from about 200-300mg/L total nitrogen, if necessary, the wastewater being fed into reaction vessel 10 may be supplemented with extra COD, or a source of COD may also be added to reaction vessel 10, to provide a total influent COD (CODt) concentration of from about l,000mg/L to about 3,000mg/L. This value will also depend on whether the process is using nitrification and denitrification predominantly via nitrate, or via the nitritation/denitritation pathway, which uses approximately 40% less carbon sources. In addition, if the PAOs utilised are capable of denitrification as well as phosphate accumulation (as appears to be the case for, for example, Candidatus Accumulibacter phosphatis), further COD economies may be achieved.

The ratio of CODt to total influent nitrogen may be from about 5 to about 15, such as from about 5 to about 12, from about 5 to about 10, from about 6 to about 10, from about 7 to about 10, from about 8 to about 10, from about 5 to about 9, from about 5 to about 8, or from about 5 to about 7.

For phosphorous removal from wastewater, VFAs are important, being a preferred substrate for intracellular storage of PHAs by PAOs. For wastewaters comprising from about 30-50mg/L total phosphorous, if necessary, the wastewater being fed into reaction vessel 10 may be supplemented with extra VFAs, or a source of VFAs may also be added to reaction vessel 10, to provide a total influent VFA concentration of from about 300mg/L to about l,000mg/L, such as from about 350mg/L to about 900mg/L VFAs, from about 350mg/L to about 800mg/L VFAs, from about 350mg/L to about 700mg/L VFAs, from about 400mg/L to about 650mg/L VFAs, from about 400mg/L to about 600mg/L VFAs, from about 450mg/L to about 600mg/L VFAs, from about 450mg/L to about 550mg/L VFAs, about 250mg/L VFAs, about 300mg/L VFAs, about 350mg/L VFAs, about 400mg/L VFAs, about 450mg/L VFAs, about 500mg/L VFAs, about 55Omg/L VFAs, about 600mg/L VFAs, about 650mg/L VFAs, or about 700mg/L VFAs. VFAs typically make up the majority, but not all of soluble COD, and therefore, if considering soluble COD levels instead of VFA concentrations, the amount of soluble COD will be to be fed in an SBR process of the invention will be commensurately higher than the values provided above for VFAs.

The ratio of total influent VFAs to total influent phosphorous may be from about 5 to about 30, such as from about 10 to about 25, from about 12 to about 25, from about 13 to about 20, from about 14 to about 18, from about 14 to about 17, from about 14 to about 16, about 14, about 15, about 16, about 17, about 18, about 19 or about 20.

A convenient source of VFAs may comprise pre-fermented raw wastewater.

Although the additional source(s) of COD/VFAs may be added to reaction vessel 10 in any appropriate manner and at any appropriate time, for ease of operation and timing of the various steps/periods during the process, including feeding steps, non- aerated periods and aerated periods, the additional COD/VFAs may be co-fed into reaction vessel 10, or may be added to the wastewater to be treated before feeding into reaction vessel 10.

Having reference to Figure 1, raw wastewater with a high BOD (such as raw abattoir wastewater, with a high FOG level) may be pre-fermented and then held in a reservoir 240. The pre-fermented raw wastewater reservoir 240 may be linked to wastewater conduit 50 via conduit 260 and co-fed into reaction vessel 10 by pump 250 with the wastewater during a feed step. VFAs may be further supplemented during a process of the invention, if necessary, by pumping VFAs into reaction vessel 10 from a VFA reservoir 270 via conduit 290 by pump 280, independently of wastewater feeding.

The source(s) of volatile fatty acids may comprise elevated levels of acetic and propionic acids, such as at least 100mg/L of each of acetic and propionic acids, and may be co-fed into said reaction vessel with said wastewater at the desired ratio to provide the desired CODt: total nitrogen ration and VFA: total phosphorous ratio. For example, where a pre-fermented raw abattoir wastewater is used to supplement the CODt/VFA of anaerobic abattoir pond wastewater (which is typically low in CDOt and VFAs), the ratio of pre-fermented waste to abattoir pond wastewater may be from about 1:20 to about 1:1, such as about 1 :15, about 1:10, about 1 :8, about 1:7, about 1 :6, about 1 :5, about 1:4, about 1:3, about 1:2 or about 1:1.

Excess use of pre-fermented high FOG waste should be avoided due to the possibility of impaired settling ability of the resulting sludge.

Other process parameters a) Organisms i) PAOs

Polyphosphate accumulating organisms for use in active sludges in processes according to the invention may be any appropriate known PAO, or combination of PAOs. The PAO(s) may be obtained from purified/isolated cultures, or may be part of a consortium of organisms enriched from naturally occurring sources, such as wastes.

A non-exhaustive list of PAOs considered to be useful for the purposes of the invention includes Actinobacteria and the Rhodocyclus group of organisms, including

Candidatus Accumulibacter phosphatis. The latter bacterium has also been shown to be capable of denitrification, and may be beneficial in further reducing carbon requirements in processes of the invention. ii) Nitrifying and denitrifying organisms

Many nitrifying, nitriting, denitrifying and denitriting organisms are known in the art, and are typically present in wastewaters naturally. Any suitable combination of such microorganisms which will provide at least nitritation and denitritation in a process according to the invention may be used. Such microorganisms may be obtained from purified/isolated cultures, or may be part of a consortium of organisms enriched from naturally occurring sources, such as wastes.

A non-exhaustive list of nitrifying and denitrifying microorganisms considered to be useful for the purposes of the invention includes:

Nitriting organisms (ammonia oxidisers) Nitrosomonas spp. Nitrosococcus spp. Nitrosospira spp. Nitrosolobus spp.

Nitrifying organisms (nitrite oxidisers) Nitrobacter spp. Nitrospina spp. Nitrococcus spp. Nitrospira spp.

Denitrifying organisms (nitrate and nitrite reducers): a wide range of facultative anaerobes, including:

Achromobacter spp.

Alcaligenes spp.

Comomonas denitrificans

Eschericia spp.

Micrococcus denitrificans

Pseudomonas spp. (such as P. aeruginosa)

Paracoccus spp. (such as P. denitrificans)

Serratia spp.

Thiobacillus spp. (such as T. denitrificans)

b) Temperature (see components 350, 360 and 370 in Figure 1)

The operating temperature for processes of the invention is not crucial, but may be kept below 4O ⁰ C, as many of the bacteria important to the process may perish at such temperatures. The temperature may also be maintained above at least 5° C. For practical process turnover times, the temperature at which the process is carried out may be at least 10°C, such as at least 15 ⁰ C, at least 18 ⁰ C, at least 2O ⁰ C, at least 22°C at least 24 ⁰ C, at least 26°C, at least 28°C, at least 30°C, about 20°C, about 22°C, about 24°C, about 25, about 26 ⁰ C, about 28 ⁰ C, or about 30°C.

The temperature of the contents of reaction vessel 10 may be monitored at temperature meter 350, communicating with temperature probe 350 by any appropriate means, such as conductive line 360. If necessary, reaction vessel 10 and its contents may be heated or cooled by any appropriate means known in the art. c) pH Control

The optimum pH for Nitrosomonas and Nitrobacter is between 7.5 and 8.5 and nitrification by these organisms has been reported to stop at a pH at or below 6.0. However, there has also been a recent report of nitrification at pH 4.0. pH of the contents of reaction vessel 10 may be monitored as described previously. Although in most cases the pH of the reaction vessel contents will self-regulate to within pH values at which the biological processes necessary for processes of the present invention will take place, if necessary the pH of the reaction vessel contents may be adjusted by any appropriate means. For example, an alkaline agent, such as a carbonate or bicarbonate salt, or even a hydroxide, such as sodium hydroxide may be added to the reaction vessel contents to raise the pH if necessary, or an acid such as hydrochloric or sulphuric acids may be added to the reaction vessel contents to reduce pH. Such additions may be controlled automatically by a controlling module, such as a PLC, in communication with pH meter 110 and a pump controlling flow of acid or alkali from suitable reservoirs.

Preferred forms of the present invention will now be described, by way of example only, with reference to the following examples, which are not to be taken to be limiting to the scope or spirit of the invention in any way.

Examples

Example 1 - Treatment of abattoir wastewater for discharge into waterways

Reactor set-up and operation

A lab-scale SBR (set up as per Figure 1 and as described above. See also Figure 15) with a working volume of 7L was used in these studies. The SBR was seeded with non-EBPR (enhanced biological phosphorus removal) sludge from a full-scale SBR treating abattoir wastewater in Queensland, Australia. IL of EBPR sludge (MLSS around 4g/L) enriched in a lab reactor was added on Day 60 to initiate the EBPR process in the reactor as there seemed to be no EBPR organisms present in the initial seed sludge used. The SBR was operated with a cycle time of 6h in a temperature-controlled room (18- 22° C). In each cycle, IL of abattoir wastewater (more details given below) was pumped into the reactor over the three filling periods with a volume distribution of 0.5 L, 0.3 L and 0.2 L respectively. Each filling period was followed by non-aerated (either anoxic or anaerobic depending on when the oxidized nitrogen was completely consumed) and aerated periods (Table 1). During aerated periods, air was provided intermittently using an on/off control system to keep the DO level between 1.5 and 2mg/L. After the settling period, IL supernatant was removed from the reactor resulting in a HRT of 42 h. 115mL of mixed liquor was wasted every cycle resulting in a SRT of 15 days. The pH in the system was recorded, ranging between 7.1-7.9, but not controlled. The ORP signal was also recorded to give indications of the nitrate levels in the reactor during the anoxic periods. The reactor was mixed with an overhead mixer except during the settling, decanting and first filling periods.

Table 1: Operating conditions of lab-scale SBR (6h cycle)

HRT = 1.75 days SBR sequences duration (min) DO levels (mg O ₂ /L)

Fill no-mix 1 5 ~0

No-aerated mix 1 (anoxic or anaerobic*) 30 ~0

Aerated mix 1 (no aeration in the last 5 min) 55 1.5-2

Fill mix 2 3 ~0

No-aerated mix 2 (anoxic or anaerobic*) 70 ~o

Aerated mix 2 (no aeration in the last 5 min) 35 1.5-2

Fill mix 3 2 ~0

No-aerated mix 3 (anoxic or anaerobic*) 60 ~0

Aerated mix 3 (sludge wasted at the end) 20 1.5-2

Settle 70 ~0

Decant 10 ~0

* when nitrate and nitrite depleted

Wastewater

The wastewater used in this study was from a local abattoir in Queensland, Australia. At this site, the raw effluent passes through four parallel anaerobic ponds before being treated in a SBR for biological nitrogen and COD removal. The anaerobic ponds serve to reduce FOG and COD, and also to produce easily biodegradable COD, particularly VFAs, to facilitate the down-stream biological nitrogen removal. The pond from which wastewater was sourced for these studies (pond A) was under-loaded, leading to much lower COD and VFAs concentrations in comparison to other ponds (see Table 2). Therefore, extra VFAs were added to this wastewater to simulate the higher VFA levels present in other ponds, as will be detailed in Table 3.

Raw wastewater and anaerobic pond effluent from the abattoir were collected on a weekly basis and stored at 4°C.

Raw wastewater was subjected to one-day pre-fermentation before being pumped into the SBR with anaerobic pond effluent. The pre-fermentation was performed in a 5OL tank continuously mixed with a submersible pump. No inoculum was introduced in the pre-fermenter, and hence the microbial population present in the raw abattoir wastewater was used to carry out the fermentation. The temperature inside the tank was kept at 37°C by a heating probe, but would not require a special heating system in a full- scale installation due to the temperature of the abattoir raw wastewater (typically around 40 ⁰ C). The aim of this pre-fermentation step was to increase the level of easy biodegradable COD, in particular VFAs, which is critical for bio-P removal. The characteristics of the pre-fermented raw wastewater and the anaerobic pond effluent are compared in Table 2.

Table 2. Characteristics of the different types of wastewater used in this study. The intervals represent the mid-95% range.

^a Acetate and propionate only ^b Pond effluent used in these studies; additional acetate and propionate was added (see Table 3) to simulate Pond B effluent, which was non-accessible for wastewater collection on site.

Table 3. Characteristics of the SBR influent during its nine-month operation.

Influent Parameters Day 0-30 Day 30-80 After day 80

Ratio VFA:TP 3.7 12.2 15.1

Ratio CODtTN 5.5 8.7 12

% pre-fermented raw 15% 15% 25% wastewater in influent

VFAs added to Pond A No Yes Yes to simulate other ponds (250 mgCOD/L acetate, (250 mgCOD/L acetate, and l00 mgCOD/L and l00 mgCOD/L propionate) propionate)

Analyses

The ammonia (NH ₄ ⁺ ), nitrate (NO ₃ ^" ), nitrite (NO ₂ ^" ) and orthophosphate (PO ₄ ^3" -P) were analysed using a Lachat QuikChem8000 Flow Injection Analyser (Lachat Instrument, Milwaukee). Total and soluble chemical oxygen demand (CODt and CODs, respectively), total Kjeldahl nitrogen (TKN), total phosphorus, mixed liquor suspended solids (MLSS) and volatile MLSS (MLVSS) were analysed according to standard methods (APHA (1995). Standard methods for the examination of water and wastewater. Washington, DC, American Public Health Association). VFAs were measured by Perkin- Elmer gas chromatography with column DB-FFAP 15m x 0.53mm x l.Oμm (length x ID x film) at 140°C, while the injector and FID detector were operated at 220°C and 250 ⁰ C, respectively. High purity helium was used as carrier gas at a flow rate of 17mL/min. 0.9mL of the filtered sample was transferred into a GC vial to which O.lmL of formic acid was added.

Fluorescence in situ hybridisation (FISH) was performed as specified in Amann R. I. (1995) ("In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes" Molecular Microbial Ecology Manual. Dordrecht, Holland, Kluwer Academic Publications. 3.3.6: 1-15).

Oligonucleotide probes used in this study were: EUBmix for the detection of all Bacteria (Daims H., Bruhl A., Amann R., Schleifer K. H. and Wagner M. (1999), "The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and evaluation of a more comprehensive probe set", Systematic and Applied Microbiology. 22(3): 434-444); PAOmix for detection of Accumulibacter (Crocetti G. R., Hugenholtz P., Bond P. L., Schuler A., Keller J., Jenkins D. and Blackall L. L. (2000), "Identification of polyphosphate-accumulating organisms and design of 16S rRNA- directed probes for their detection and quantitation", Applied and Environmental Microbiology. 66(3): 1175-1182); GAOQ989 and GB_G2 for detection of Competibacter (Crocetti G. R., Banfield J. F., Keller J., Bond P. L. and Blackall L. L. (2002), "Glycogen accumulating organisms in laboratory-scale and full-scale activated sludge processes",

Microbiology. 148(11): 3353-3364; Kong Y., Ong S. L., Ng W. J. and Liu W.-T. (2002), "Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic-aerobic activated sludge processes", Environmental Microbiology. 4(11): 753- 757); DFlmix (TFO_DF218 plus TFO_DF618) for detection of Cluster 1 Deβuvicoccus vanus-rύateά Alphαproteobαcteriα (Wong M. T., Tan F. M., Ng W. J. and Liu W. T. (2004), "Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic-aerobic activated sludge processes", Microbiology-Sgm. 150: 3741-3748); and DF2mix (DF988, DF1020 plus helper probes H966 and Hl 038) for detection of Cluster 2 Defluvicoccus vαnus-rolated Alphαproteobαcteriα (Meyer R. L., Saunders A. M. and Blackall L. L. (2006), "Putative glyco gen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing", Microbiology-Sgm. 152: 419-429). FISH quantification was performed as described in Crocetti G. R., Banfield J. F., Keller J., Bond P. L. and Blackall L. L. (2002) ("Glycogen accumulating organisms in laboratory-scale and full-scale activated sludge processes", Microbiology. 148(11): 3353-3364.

RESULTS

Figure 2 presents the influent and effluent COD, N and P concentrations, along with the MLSS concentration in the reactor and its volatile fraction, during the nine months operation of the SBR. Also presented in Figure 2 is the fraction of Accumulibαcter-V AO in the system. Potentially competing glycogen accumulating organisms (GAOs) Competibαcter-GAO and the putative Defluvicoccus vαrøws-related GAO (Cluster 1 and 2) were negligible in this reactor (<1% of the total microbial population at all time). According to the effluent and MLSS data (Figs. 2c and 2d), the SBR reached a steady state around day 100. The study can be divided into two periods: the start-up period from day 0 to 100 and the steady state period from day 100 to 275.

Start up period (day 0 to 100)

Complete nitrification was achieved in the SBR after less than one week of operation as shown by the absence of NH ₄ ⁺ in the effluent (Fig. 2c). However, denitrification was incomplete and NO _x ^" accumulated in the reactor reaching 60 mgN/L in the effluent during the first 30 days of operation (Fig. 2c). In order to improve the denitrification, more COD was needed during anoxic periods. Therefore, extra VFAs (i.e. acetate and propionate) were added to pond A effluent on day 30 in order to simulate the concentration in the other ponds (typically 250 mg COD/L acetate and 100 mg COD/L propionate). These additional VFAs improved the denitrification and the level of NO _x ^" in the effluent dropped to 15 mgN/L (Fig. 2c). The similar levels of PO ₄ ^" measured in the influent (Fig. 2a) and effluent (Fig. 2c) indicate that phosphorus removal was negligible during the first 60 days. P removal was likely limited by the slow development of PAOs, which was possibly inhibited by the level of nitrate present during most of the time over a

cycle. The fact that non-EBPR sludge was used to seed the reactor could have also contributed to the slow development of PAOs.

After the introduction of 1 L lab-scale EBPR sludge enriched in Accumulibacter- PAO on Day 60 (details of the culture can be found in Lemaire R., Meyer R., Taske A., Crocetti G. R., Keller J. and Yuan Z. G. (2006) "Identifying causes for N ₂ O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal", Journal of Biotechnology. 122(1): 62-72), P removal improved dramatically, and consistent high-level of P removal was achieved and maintained since then. The process data suggests that the seeded PAOs managed to survive and develop in a very different environmental setting. This is confirmed by the FISH quantitation results (Fig. 2c) and the decrease of the organic fraction in the biomass due to poly-P storage in the PAO cells (Fig. 2d).

However, Figure 2c also shows that while P removal was improving, NO _x ^" started to accumulate again in the system. This was believed to be due to a shortage of easily biodegradable COD in the reactor as the PAOs and denitrifiers were now competing for the same carbon sources, hi order to further increase the amount of VFA available for P and N removal, the amount of pre-fermented raw wastewater in the influent was increased from 15% to 25% on day 80 resulting in a higher VFA:TP ratio and CODt:TN ratio in the influent (Table 3). Denitrification improved immediately and from day 100 onwards, less than 10 N-mg/L was present in the effluent.

Steady state period (from day 100 to 275)

Following the addition of extra VFAs in the pond effluent on day 30, the introduction of EBPR sludge in the reactor on day 60 and the increase of the pre- fermented raw wastewater proportion in the influent on day 80, the reactor reached a steady state around day 100 with excellent removal of COD, nitrogen and phosphorus. There was one interruption to the reactor operation between day 125 and 160, when the abattoir closed down and no wastewater could be supplied to the SBR (Fig. 2). The reactor performance quickly recovered (within four days) after the starvation period, as indicated by the low nutrient level in the effluent shortly after the normal SBR operation was resumed (Fig. 2c). The reactor biomass concentration decreased by 30% during this long starvation period but returned to its previous level after only 2 weeks and then remained relatively constant around 5g/L with an organic fraction fluctuating between 0.7 and 0.75 (Fig 2d).

During the starvation period, the reactor was put into a 'sleeping mode' in a five- week period when the abattoir, where the wastewater was sourced, was closed down for annual maintenance. The 'sleeping mode' operation consisted of 15 minutes aeration in a 6 hour SBR cycle, which has been shown to provide significantly lower decay of nitrifying bacteria populations than anaerobic conditions only. The sludge was allowed to

settle in the remaining time of the cycle. The decay rates for ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) were determined to be 0.017 day ^"1 , and 0.004 day ^"1 , respectively, through monitoring the ammonia- and nitrite-oxidation rates over the starvation period on a weekly basis. These decay rates were supported by the FISH (Fluorescence In-Situ Hybridization) quantification data. The decay rate of the polyphosphate accumulating organisms (PAOs) could not be determined, but phosphate release was observed throughout the five-week period. Three different phosphate release rates were clearly visible on the measured phosphate profile, suggesting different processes were dominating in the three periods. A resuscitation period with a gradual increase of the wastewater load was applied during the re-startup of the reactor. The performance of the reactor in terms of nitrogen and phosphorus removal fully recovered in just four days after the re-startup.

Table 4 details the SBR effluent quality after the starvation period, between day 170 and 275. For comparison, the COD and nutrient levels in the influent are also presented in the table. The SBR process consistently achieved 95, 97 and 98% of COD, TN and TP removal, respectively. The remaining COD in the effluent could be regarded as non-biodegradable and represented about 5% of the total COD initially present in the influent. It was observed that the sludge velocity index (SVI) was relatively high throughout the study period, between 180 and 250 mL/gMLSS. This could have partially been caused by the remaining high fat/oil/grease content of the pre-fermented raw wastewater. However, the suspended solids concentration in the effluent was lower than 25mg/L at all times (data not shown).

Table 4. Influent and effluent characteristics between day 170 and 275 (n represents the number of samples analysed between day 170 and 275)

Parameter Influent (N=13) Effluent (N=32) Removal of

(mg/L) mid-95% range mean mid-95% range mean COD, N & P

CODt 2600-3120 2870 129-151 140

95 % CODs 1150-1320 1240 114-127 121

TKN 236-277 256 5.3-7.7 6.5

N-NH ₄ 196-215 206 0.2-0.8 0.5 97 % N-NO _x not detected 1.9-2.8 2.3

TP 38-41 40 0.7-1.4 1.0

98 % P-PO ₄ 35-38 37 0.04-0.09 0.06

Figure 3 shows the nitrogen and phosphorus transformations in a typical process according to the invention during the steady state period. At the end of each aerated period, NH ₄ ⁺ was fully oxidised, and the low level of NO _x ^" accumulated was then removed in the following non-aerated period. It can be seen that a very low level of NO _x ^"

was carried over to the next cycle, and was denitrified very quickly at the beginning of the first non-aerated period. PO ₄ ^3" levels increased during each non-aerated period due to both anaerobic P release and wastewater feeding (containing approximately 40mgP/L), but most P release occurred in the first non-aerated period. PO ₄ ^3" was then fully taken up during the subsequent aerated periods.

Performance of the pre-fermenter

The impact of the one-day pre-fermentation performed on raw wastewater is depicted in Figure 4. The overall VFA concentration more than doubled as a direct result of this pre-fermentation. Acetate and propionate were the most abundant VFAs in the raw abattoir wastewater before and after pre-fermentation, with propionate having a slightly higher production rate than acetate. Also shown in Figure 4 is the impact of pre- fermentation on the NH ₄ and PO ₄ ^" concentrations. While PO ₄ ^" concentration stayed constant, NH ₄ ⁺ concentration doubled due to partial mineralisation of the organic nitrogen, which represents around 75% of the raw wastewater total nitrogen. One week storage of the pre-fermented raw wastewater in the cold room at a temperature of 4°C affected VFAs levels more than nutrient levels, with a 20% reduction of acetate and propionate concentration.

DISCUSSION

Multi-feed strategy to promote biological P removal

Biological phosphorus removal from wastewaters containing a high level of nitrogen, such as abattoir wastewater, is challenging. Large accumulation of nitrate or nitrite must be avoided in order to secure anaerobic conditions required by the PAOs.

The use of a multi-feed strategy in this study aimed to limit the level of NO _x ^" recycled to the anaerobic period. Figure 2 shows that the strategy was very successful. The NO _x ^" level was limited to below 8mgN/L throughout the cycle, despite the high level OfNH ₄ ⁺ and organic nitrogen in the wastewater (over 250 mgN/L, see Table 4). P release occurred in all three non-aerated periods. According to the amount of wastewater fed over the 3 feeding steps in the SBR cycle (i.e. 0.5 L, 0.3 L and 0.2 L, respectively), the increases in PO ₄ ^3" concentration in the reactor directly attributable to the wastewater feeding were estimated to be 2.7, 1.5 and 1.0mgP/L, respectively. The P release following the three feeding periods was thus estimated to be 25.3, 4.5 and 1.0mgP/L, respectively. The considerably higher P release observed after the first feeding step compared to the second and third feeding steps indicates that the first non-aerated period is crucially important for P removal. This would not have been achieved if a high level of NO _x ^" was allowed to accumulate in the reactor.

Pre-fermentation of raw wastewater

The performance of a biological nutrient removal system depends greatly on the availability of easily biodegradable carbon sources in the wastewater, particularly VFAs. Considering the fact that it is difficult to control the VFAs content in large anaerobic ponds, a more controllable VFA source may be necessary for reliable biological nutrient removal from abattoir wastewater. In this study, a high-rate pre-fermentation step was demonstrated to be a cheap and effective option for providing sufficient VFAs for N and P removal where the wastewater to be treated has insufficient levels of these carbon sources. Table 3 shows that the VFA:TP ratio increased from 12.2 to 15.1 when the pre- fermented wastewater fraction in the SBR influent increased from 15% to 25% on day 80. This caused an immediate reduction in the nitrate level, with a drastic improvement to the reliability of P removal (Figs 2b and 2c). The results show that it is both necessary and practically feasible to include a high-rate pre-fermenter to generate VFAs that may be supplemented to the nutrient removal SBR when an inadequate amount of VFAs is present in the pond effluent.

However, it should be highlighted that the use of raw wastewater should be minimised. There is evidence suggesting that a high fraction of raw feed deteriorates the sludge settling properties (data not shown) likely due to its higher FOG content compared to pond effluent. An oversupply of carbon sources through this stream would also increase aeration costs and sludge production in the SBR system. Controlled addition of this stream using an on-line control system would be highly beneficial. However, the control of VFAs supplement to biological phosphorus removal systems in accordance to the actual demand for VFAs (varying with time) is still unresolved.

An alternative solution that is being investigated is to reduce the demand for carbon sources by achieving nitrogen removal via nitrite instead of nitrate. This strategy, if successful, would reduce the carbon demand for denitrification by 40%. This would therefore reduce the amount of additional carbon supply, which in turn will also reduce the overall oxygen requirement. Such an improvement would have significant benefits for the operation of large-scale wastewater treatment facilities. On-line control systems based on simple pH and DO signals are being developed to achieve this nitrite pathway in our lab-scale SBR and this is described in Example 3.

A further opportunity to reduce the demand for carbon sources is to enhance denitrification by PAOs. It has been found that Accumulibacter-ε AOs are capable of taking up phosphorous under anoxic conditions. This is particularly attractive as the same carbon could be used for both denitrification and P removal. However, the exact conditions necessary to promote this type of denitrification are still unclear and further investigations are needed.

The low abundance of GAOs in the sludge

Competibacter-GAOs have been widely reported to be abundant in both lab-scale EBPR reactors and full-scale EBPR plants. Surprisingly, in this study, Competibacter- GAOs were scarcely present in the reactor, representing always less than 1% of the total microbial population. The Defluvicoccus vanus-xelated Alphαproteobαcteriα organisms, a new putative GAO recently reported in literature, was also found to be in very low abundance in the reactor.

Several factors may influence competition between PAOs and GAOs. For example, pH has a significant impact on PAO and GAO competition with Accumulϊbαcter-Y AOs possessing advantages over Competibαcter-GAOs for anaerobic carbon uptake at relatively high pH (>8). The pH in the study fluctuated between 7.1 and 7.9 during a cycle (uncontrolled), which is unlikely to have provided any selective advantages to PAOs over GAOs. The presence of nitrite in the anaerobic or aerobic periods may inhibit PAO activity, and could therefore enhance the presence of GAOs in the system. However, from the results, the presence of nitrite in the reactor during all three aerated periods and during the second and third non-aerated periods apparently did not promote the growth of Competibαcter-GAOs. Some studies have also shown that better EBPR performance may be achieved at relatively low temperature (5-15° C) due to a shift in the microbial community from GAOs to PAOs. The temperature used in this study, controlled between 18-22°C, is very similar to many reactor studies where GAOs appeared to be a problem, and is therefore not believed to be a significant contributor to the low abundance of GAOs. A more likely reason for the limited growth of GAOs in this reactor could be the large fraction of propionate present in the influent (propionate to acetate COD ratio was 0.8) - propionate as a carbon source may provide selective advantage to PAOs. The pre-fermenter used in this study largely contributed to the increase of the propionate fraction. If this hypothesis is true, the operation of the pre- fermenter should be optimised to not only maximise the total amount of VFAs produced but also to control the VFAs composition and particularly the acetate to propionate ratio.

CONCLUSION

A sequencing batch reactor system was demonstrated to effectively remove nitrogen, phosphorus and COD from abattoir wastewater. This provides a more cost- effective and environmentally friendly alternative to chemical phosphorus removal, which is a common practice at present. Each 6h cycle contained three anoxic/anaerobic and aerobic sub-cycles with wastewater fed at the beginning of each anoxic/anaerobic period. The following conclusions are drawn:

• It is possible to achieve a high degree (>98%) of biological phosphorus removal from abattoir wastewater in the presence of high levels of nitrogen (200 - 300mgN/L) while simultaneously substantially removing total nitrogen (>97%) and total COD

(>95%). The concentrations of phosphate and inorganic nitrogen in the effluent were consistently lower than 0.2 P-mg/L and 8 N-mg/L respectively.

• The multi-step feeding strategy prevents high-level accumulation of nitrate or nitrite, and hence facilitates the creation of anaerobic conditions. The strategy is strongly recommended for practical use in the biological treatment of abattoir wastewater.

• It is important to incorporate a high-rate pre-fermenter as an integrated component of the nutrient removal system if the wastewater to be treated is deficient in VFAs. This stream, which contains a high-level of VFAs, is effective in providing supplementary carbon sources for both phosphorus and nitrogen removal.

Example 2 - Treatment of abattoir wastewater for land irrigation

This project was designed to develop a strategy for SBR operation to produce effluent with a quality suitable for land irrigation. The same multi-feed concept as used for producing effluent suitable for river discharge is used. The sequence of operation is however different.

Land irrigation requires a high level of biological oxygen demand (BOD) removal (>95%), and medium levels of nitrogen and phosphorus removal. The presence of total phosphorus at a level of 10-20 mgP/L and total nitrogen at a level of 50-100mgN/L in the treated effluent is considered appropriate for this purpose. Ammonia/ammonium as opposed to nitrate is the preferred form of nitrogen in the final process effluent for land application.

The SBR, inoculated in June 2005 with sludge from a local abattoir in Queensland and set up similarly to that described in Example 1, was operated over a 12 month period from the beginning of June 2005 to August 2006, with a 2-step feed in accordance to the concept shown in Figure 5, and in 6h cycles (from June to mid September), 8h cycles (from mid September to mid March) and 4h cycles (from mid March to August), with operational conditions as detailed in Table 5.

The hydraulic residence time (HRT) was kept at 28 hours and the sludge retention time (SRT) at 15 days. Each cycle consisted of a non-aerated feeding period allowing phosphate release, an aerated period allowing nitrification and phosphorus uptake, a non- aerated feeding period for denitrification, a short aerated period, and a settling and decanting period. An aim of the design is complete removal of nitrate at the end of the second feeding period so that phosphorous release will not be inhibited in the first feeding period in the next cycle. The design was expected to produce effluent with some presence of nitrogen (primarily ammonia nitrogen) and phosphorus, with the levels determined by the volume exchange ratio for the second feeding period.

Figure 6A shows the influent and effluent concentrations of nitrogen, and Figure 6B shows the same data for phosphorus removal, during the operation of the SBR.

Figure 7A illustrates the total and soluble COD (TCOD and SCOD) and Figure 7B illustrates VFA concentrations (acetate and propionate) over the same period.

Table 5 Irri ation-SBR c cle confi urations.

As can be seen in Figures 6B and 7B, biological phosphorus removal in the reactor was not very stable at the beginning because P removal was strongly related with the amount of COD in particular volatile fatty acids (VFAs) fed to the reactor. Moreover, the presence of nitrite in the aerobic phase of the reactor was also affecting the P-uptake.

As can be seen from the results, good nitrogen removal was achieved soon after the start up of the reactor but no phosphorus removal was observed. In the early stages nitrate and nitrite were present in the effluent of the system and also in the anaerobic period during the first few weeks of operation. As such, denitrifiers were able to take up the substrate, competing with the PAOs. In order to improve the nitrogen removal and also with the aim of removing the nitrate and nitrite from the effluent, the anoxic part of the cycle was extended and the feed pattern was changed, increasing the volume of wastewater added in the second feed (1.25L in the 6 and 8h cycle) and reducing the volume of wastewater in the first feed (0.75 L in the 6 and 8h cycle). This was done to have higher COD in the anoxic period to be used for denitrification. The reactor performance improved in terms of nitrogen removal achieving 87% of efficiency. On the other hand, phosphorous removal was achieved since the nitrate and nitrite were not present in the effluent. Nevertheless, biological phosphorus removal is a very sensitive process and when minor changes were implemented to improve the nitrogen performance, P-removal from the system was affected. After about two months, the system was achieving 38% of P-removal.

In the three months from July, efforts were focused on improving biological • phosphorus removal from the system, first of all trying to avoid the presence of NO _x at the beginning of the non-aerated periods. When the wastewater presents a low COD (achieving values of 400ppm of COD and 120ppm of acetate) the reduction of NO _x to nitrogen gas could not be achieved completely, remaining at the end of the cycle. Then, in the subsequent cycle, most of the COD is consumed by the denitrifying bacteria and PAOs can not compete for the substrate, decreasing its ability to uptake phosphate. In October, the reactor achieved 90% of nitrogen removal and 85% of phosphorus removal with effluent concentrations of 18ppm NH ₄ -N and 5ppm of PO ₄ -P, and was typically achieving the nutrient levels required for land irrigation (between 20 and 50ppm for nitrogen, and between 10 and 15ppm for phosphorus) thereafter. This achievement was possible by extending the cycle time from 6h to 8h in order to have a longer anoxic period at the end of the cycle, so the slowly biodegradable COD could also be consumed by the denitrifying organisms.

Results of a cycle performed in the SBR reactor on 11 October 2005 are illustrated in Figure 8. In the three months to late December 2005, efforts were focused on improving the enhanced biological phosphorus removal (EBPR) performance in the SBR reactor, without affecting the nitrogen removal ability achieved since the first month of operation. Carbon was the limiting compound to achieve good P removal in this reactor. The amount of COD, particularly the amount of VFAs, present in the pond wastewater was crucial in achieving nutrient standard levels required for land irrigation, particularly as the reactor was being operated with 100% pond wastewater, and no raw wastewater was being added to the influent.

As can be seen from Figures 7A and 7B, the continuous oscillations in the amount of COD and VFAs available in the pond wastewater were probably the reason for the varying P removal efficiency. Nitrogen removal seemed not to be significantly affected by these changes, possibly because the microorganisms involved in the denitrification process are more competitive for the substrate than the PAOs and the anoxic period in the reactor is long enough to allow these bacteria to degrade slowly biodegradable COD. Denitrifier diversity is also greater than PAO diversity. On the other hand, PAO activity is very dependent on the amount of VFAs available during anaerobic conditions. So, if the level of VFAs in the pond wastewater is low, the EBPR performance of the system may be directly affected.

Figure 9A illustrates cycle study data from the 7th of March, when the SBR was working with an 8h cycle. An interesting observation is that all the ammonia from the system was converted to nitrite in the first aerobic period and no nitrate was produced. This process is known as nitritation. In the subsequent anoxic period, most of this nitrite was reduced to nitrogen gas during denitritation. Compared with full nitrification, air

requirements for nitritation are 25% less while carbon requirements for denitritation are approximately 40% lower than for denitrification from nitrate.

Moreover, almost no P-uptake was observed. This was due to the concentration of nitrite present during the aerobic period inhibiting P-uptake by PAOs. To overcome the inhibitory concentrations of nitrite in the SBR, the cycle was reduced to 4 hours, and the P-removal performance immediately recovered (Figure 9B).

Example 3 - Establishment and maintenance of nitritation/denitritation by automatic aeration control

This study focussed on control of aeration to achieve nitrogen removal via nitrite, rather than nitrate (see Figures 1OA and 10B). The control strategy was based on the slope of the pH signal and on the oxygen uptake rate (OUR). During an aerated period, the pH slope is calculated once the maximum pH has been reached and starts decreasing.

The first condition to automatically stop aeration is met when the slope of the pH signal is lower than a minimum value entered by the operator.

The second condition is on the OUR. Due to the on-off aeration control system employed for control DO in the reactor, the OUR is directly proportional to the time the O ₂ valve is in an "off state. The second condition is met when the valve is off for longer than the maximum time entered by the operator.

A third "safety" condition assuring a minimum aeration time of 15 min is applied.

Figure 11 illustrates these 3 conditions during a typical aeration period. It should be noted that the condition on the pH slope is important, due to its flexibility and its practicability, and the 2 ^nd and 3 ^rd conditions were not conservative, playing more of a back-up security role to ensure that enough aeration was provided if the 1 ^st condition was met too early.

Reactor set-up and operation

A lab-scale SBR as described in Example 1 was operated with a cycle time of 6 h in a temperature-controlled room (18-22°C). hi each cycle, 1 L of abattoir wastewater (more details given below) was pumped into the reactor over the three filling periods with a volume distribution of 0.5 L, 0.3 L and 0.2 L respectively. Each filling period was followed by non-aerated (either anoxic or anaerobic depending on when the oxidized nitrogen was completely consumed) and aerated periods. During aerated periods, air was provided intermittently using an on/off control system to keep the DO level between 1.5 and 2 mgO ₂ /L. After the settling period, IL supernatant was removed from the reactor resulting in a HRT of 42h. 115mL of mixed liquor was wasted every cycle to keep a constant SRT of 15 days. The pH in the system was recorded, ranging between 7.0 and 8.0, but not controlled. The ORP signal was also recorded to give indications of the nitrate levels in the reactor during the anoxic periods. The reactor was mixed with an

overhead mixer except during the settling, decanting and first filling periods. The SBR cycle operation was controlled by a programmable logic controller (PLC - Opto Control).

Wastewater

The wastewater used in this study was from a local abattoir in Queensland, Australia. At this site, the raw effluent passes through four parallel anaerobic ponds before being treated in a SBR for biological N and COD removal. The anaerobic ponds serve to reduce FOG and COD, and also to produce some easily biodegradable COD ₃ particularly VFAs, to facilitate the down-stream biological nitrogen removal.

Raw wastewater and anaerobic pond effluent from the abattoir were collected on a weekly basis and stored at 4°C. The raw wastewater was subjected to one-day pre- fermentation before being pumped into the SBR (as per Example 1) to further increase the level of easy biodegradable COD, in particular VFAs, which is critical for bio-P removal. The characteristics of the pre-fermented raw wastewater and the anaerobic pond effluent are compared in Table 6.

The wastewater fed to the lab-scale SBR consisted of a mixture of anaerobic pond effluent and pre-fermented raw wastewater as shown in Table 7 further below. The modification of the fraction of raw pre-fermented wastewater used in the influent did not modify the overall N and P content of the influent due to the identical level of N and P present in both type of wastewater.

Table 6 - Characteristics of the different types of wastewater used in this study. The intervals represent the mid-95% range.

Parameter Pre-fermented raw Anaerobic pond

(mid-95% range) wastewater effluent ^b

CODtotai (mg/L) 7,460-9,300 930-1,220

CODsoiubie (mg/L) 2,360-2,840 705-745

VF A ^a (mg COD/L) 699-797 548-601

TN (mg/L) 260-306 235-254

NH ₄ -N (mg/L) 141-157 223-229

TP (mg/L) 44-50 36-39

PO ₄ -P (mg/L) 37-42 34-35 ^a Acetate and propionate only ^b Additional acetate and propionate were added to the anaerobic pond effluent to mimic the effluent of better operated, but physically inaccessible pond

Aerobic phase length control to promote nitrite pathway

The SBR was operated for approximately 18 months. Aerobic phase length control for achieving N removal via nitrite was trialled in the last 13 months. The control strategy employed was based on the slope of the pH signal and on the oxygen uptake rate (OUR). Figure 1 shows that the exact time of complete NH ₄ oxidation in each aeration

periods could be detected through the pH bending point and the sharp OUR drop. During each aeration period, the pH slope was calculated once the maximum pH has been reached and started to decrease. The slope of the pH was determined based on pH values in a 2 minute moving window. Due to the on-off control system of the DO in the reactor, the OUR was calculated during the time the oxygen valve was in an "off state. The aeration length control strategy was based on three different criteria that had to be met to automatically stop the aeration:

• when the slope of the pH signal became less than a minimum value entered by the operator. This was the main criterion and therefore the set-point was relatively aggressive;

• when the OUR became less than a minimum value entered by the operator (typically 1.2 mgθ ₂ /L.min). This was only a safety condition and the set-point were not conservative giving more weight to the pH criteria; and

• when the aeration period length became greater than a minimum aeration time entered by the operator (typically 15 min). This criteria was the less conservative and its role was to secure some aeration periods in the SBR if the pH and OUR criteria failed and were met too early.

The control of the length of each aeration period in the SBR to promote the nitrite pathway was implemented in 3 stages. In the first stage, from Day 160 to 340, the aeration control was performed manually by the operator. Based on the observation of the on-line pH and real-time OUR calculation, the length of each three aeration period was adjusted on a daily basis to ensure that the aeration was stopped immediately after complete oxidation OfNH ₄ ⁺ in the SBR. Then, from Day 340 to 410, the manual aeration control was stopped and fixed aeration lengths that were longer than the time required for complete NH ₄ ⁺ oxidation were applied. The purpose of Stage II was to deteriorate the nitrite pathway previously established under Stage I by promoting the growth of NOBs. In Stage III (from Day 410 onwards), the automatic aeration length control was implemented to re-establish the nitrite pathway. The abattoir closed down from Day 480 to 525 due to annual maintenance. During this period, the SBR cycle operation was modified in order to preserve the reactor biomass as no wastewater was available. As also described in Example 1, to place the reactor into 'sleep mode', the sludge was aerated and mixed for 15 min in each 6h cycle and was allowed to settle for the rest of the cycle.

RESULTS and DISCUSSION

Analyses were carried out as described in Example 1.

Effect of the aeration control strategy on the nitrite pathway

Figure 12a presents the level of nitrite pathway achieved in the SBR, measured as the average amount OfNO ₂ ^" produced (mgN/L) per NO _x ^" produced (mgN/L) during the 3

aeration periods, and the relative abundance of NOBs in the SBR throughout Stage I, Stage II and Stage III. Tests to identify the main NOB species present in the SBR using common FISH probes (i.e. NIT3 for Nitrobacter and NITSP A662 for Nitrospira) showed that only Nitrospira was present in the system (data not shown). Therefore, the quantification of the Nitrospira population was considered to be representative of the total NOB population present in the SBR. The reactor used in this study had been already running for 5 months performing high level of COD, N and P removal (Lemaire et al. submitted) before the aeration length control was implemented. In that time, no nitrite accumulation was observed during the aerobic periods as depicted in Figure 12a.

During Stage I, the manual control of the length of each aeration periods resulted in a rapid accumulation of nitrite reaching 95% of the total amount of NO _x ^" produced on Day 280 (Figure 12a). This high level of nitrite pathway was maintained until the start of Stage II. During that second stage, the implementation of fixed aeration periods deteriorated the nitrite pathway previously established but not completely as 20% of NO ₂ ^' accumulation was still observed 50 days after the start of Stage II (Figure 12a). The implementation of the automatic aeration lengths control strategy during Stage III resulted in the recovery of the nitrite pathway in the SBR. However the level of nitrite accumulation increased at a slower rate than when the aeration length control was performed manually and only reached 85% after 150 days (including 50 days of starvation period). The strategy consisting of stopping the aeration in the SBR immediately after NH ₄ ⁺ was oxidised was therefore successful in controlling the level of the nitrite pathway.

When comparing the level of nitrite pathway in the SBR and the Nitrospira population dynamic it clearly appears that the nitrite pathway was achieve through the elimination or the reduction of the NOB population in the system. However, some delay could be observed between the level of nitrite pathway measured and the abundance of NOB. While the NO ₂ ^" accumulation decreased from 98% to 20% during Stage II, the Nitrospira population only increased from 0.3% to 0.5%, but later increased to 1.2% of the total bacterial population 40 days into Stage III (Figure 12a). The presence of this lag phase could be due to the complex dynamics involved in the NOB growth processes when the availability of their main energy source (i.e. NO ₂ ^" ) is modified.

Effect of nitrite pathway on the overall SBR performance

In order to demonstrate the benefit of the nitrite pathway in COD savings, the COD concentration in the SBR influent was adjusted several times during the experimental period through changing the fraction of fermented raw feed and/or the VFA content in the pond effluent. The resulting VFA concentration profile in the SBR influent along with nutrient levels in the effluent are shown in Figure 12b.

From Day 160 to 250, the level of nitrite pathway increased from 0 to 95% after the length of the aeration periods were manually controlled. At the same time, high levels of COD, N and P removal were consistently achieved, respectively 95%, 97% and 98%. Figure 12b shows that, as the level of nitrite pathway increased the amount OfNO _x ^" in the effluent decreased. This was likely the consequence of the amount of COD saved via the nitrite pathway that enhances the subsequent denitrification process where influent was step-fed into the SBR instead of adding an external carbon side-stream. The period between Day 250 and 280 with stable high level of nitrite pathway is referred as "Period A" and is later described in Table 7.

From Day 280 to 310, the VFA concentration in the SBR feed was gradually decreased by first, reducing the fraction of pre-fermented raw wastewater in the influent from 25% to 15% and then, gradually reducing the VFA concentration in pond effluent by 40%. The reduction of the raw wastewater fraction lowered the amount of FOG and colloidal matter, which are detrimental to good sludge settling properties. The bio-P removal was immediately affected due to this sudden VFA shortage but soon recovered (Figure 12b). As the amount of VFA was further reduced, NO _x ^" started to accumulate in the effluent on Day 300 due to incomplete denitrification. The accumulation of NO _x ^" is very detrimental to bio-P removal as it prevents anaerobic periods to occur in the SBR. As a result, PO ₄ ^3" started to accumulate soon after and the amount of VFA had to be adjusted to provide sufficient amount for both N and P removal (Figure 12b). The stable period from Day 310 and 340 is referred as "Period B" and is later described in Table 7.

With the implementation of fixed length aeration control on Day 340, the effluent NO _x ^" and P levels deteriorated considerably, likely due to the gradual conversion from the nitrite to nitrate pathway. Therefore, more VFAs had to be supplied to ensure that NO _x ^" and P in the effluent were kept at sufficient low concentration to avoid any long term damage of the SBR nutrient removal performance. It took about two weeks for the level of nitrite pathway to start decreasing following the end of the manual aeration control and the introduction of fixed aeration periods. On Day 370, the amount of VFA added to the pond effluent was further increased by 15% to compensate for the rapid deterioration of the nitrite pathway from 95% to 45% which trigger the accumulation of NO _x ^" in the effluent (Figure 12b). The stable period from Day 380 to 420 is referred as "Period C" and is later described in Table 7.

On Day 420, the fraction of pre-fermented raw wastewater and the amount of extra VFA were both reduced (by 5% and 30% respectively) following the implementation of the automatic aeration control strategy and the increase of the degree of nitrite pathway. Once again, NO _x ^" and PO ₄ ^" immediately accumulated in the effluent due to the sudden VFA and COD shortage but promptly recovered (Figure 12b). "Period D" ranges from Day 540 to 600, after the normal wastewater load was resumed in the SBR following the long starvation period.

Table 7 - Comparison of the degree of nitrite pathway, the influent composition and the effluent quality in four distinctive periods during the 15 -month study.

^a Includes additional acetate and propionate

The four distinctive stable periods defined earlier are compared in Table 7 in terms of degree of nitrite pathway, influent composition and effluent quality. The overall amounts of COD and VFAs in the influent were considerably reduced during "Period B" while the average NO _x ^" and PO ₄ ^3" levels in the effluent were kept at reasonable low levels even if they were higher than in "Period A" (Table 7). In addition, Table 7 shows that during "Period C", while the level of nitrite pathway was low, the N removal deteriorated compare to that in "Period B" even if the amount of VFA in the influent was at its highest. This demonstrates the importance of the nitrite pathway in saving COD and also in enhancing the nutrient removal performance. At the same time, it is important to consider both N and P removal when assessing the possible COD and/or VFA saved via the nitrite pathway in a BNR system as P removal depends strongly on the level of N removal achieved. The reduction of the COD and VFAs amounts in the influent in "Period D" did not forfeit the overall N and P removal which indeed improved considerably as shown by the low levels of NO _x ^" and PO ₄ ^" in the effluent reported in Table 7.

The aeration control strategy was successful in achieving stable N removal via the nitrite pathway which clearly benefited the nutrient removal performance of the SBR by efficiency in use of COD and VFAs. As a direct result, the fraction of pre-fermented raw wastewater in the influent was reduced from an initial 25% to 10% without affecting the performance of the SBR. The steep-feed strategy employed in this SBR ensured that no external carbon addition was needed to carry out the post denitrification or denitritation making the overall BNR process more attractive.

Aeration length control strategy based on pH and DO signals

SBRs usually operate with fixed time lengths for the different phases of filling, mixing (anaerobic, aerobic or anoxic), settling and decanting. Due to influent fluctuation and system state variations, it is beneficial to operate a SBR process with varying phase

lengths. Therefore, higher levels of process control and automation may be necessary to optimise the SBR operation. ORP, DO and pH provide means to detect the end of the nitrification process, via the "ammonia valley" (pH), the "DO elbow" (DO) or the "nitrogen break point" (ORP) and the denitrification process, via the "nitrate knee" (ORP) or the "nitrate apex" (pH). In particular, it is possible to achieve nitrite pathway in a SBR by stopping the aeration as soon as NH ₄ was oxidised and start the addition of external carbon (glucose) for the denitritation. In this study, we integrated the control strategy to a more complex system (i.e. COD, N and P removal process) where denitrification was performed through a step-feed strategy suppressing the need of external carbon dosage.

Figure 11 clearly demonstrates the simultaneity between the depletion of NH ₄ ⁺ , the "ammonia valley" and the OUR drop symbolising the "DO elbow" during a SBR cycle where the aeration lengths were not controlled. The aeration length control strategy previously described could therefore be implemented in this complex BNR system. Figure 14 presents the pH, DO, OUR, nitrogen and phosphorus profiles in a SBR cycle after this automatic aeration length control strategy was implemented. This strategy was reliable in detecting the end of the nitritation process and stopping the aeration as indicated by the dot lines on Figure 14. The success of this control strategy was further confirmed by the good long-term performances of the SBR presented previously.

However, some technical issues and possible improvement of the control strategy were identified. The pH and OUR profiles in each of the three aeration periods were quite different (Figure 14) making it difficult to design a universal algorithm for all three aerobic periods based on pre-determined absolute values. The pH profile also changed over time as shown by the difference observed between that depicted in Figure 11 and in Figure 14. This was mainly due to the large difference of the initial pH value observed at the start of each aeration period but also to the unbalanced PAOs activity existing between each aeration period, with most of the activity occurring in the first aeration period as indicated by the high P release and subsequent P uptake in Figure 5. This evolution over time of the pH and OUR profiles in the SBR suggests that it may be preferable to design an algorithm where the different control set-points (i.e. pH slope minimum value and OUR minimum value) are determined based on relative threshold values instead of absolute values pre-determined by the operator. These set-points could have been determined as a percentage (e.g. 20%, 10% or 5%) of the maximum pH slope and OUR values calculated in real-time once the aeration started. Such an algorithm would be straightforward to implement.

Example 4 - Pilot plant studies

Pilot plant studies were undertaken to validate the laboratory-scale studies described above. Two sequencing batch reactors were set up at a local (Queensland, Australia) abattoir, in the wastewater treatment section. The main wastewater source, an anaerobic

pond (AP) effluent, is typically variable in COD and an often insufficient amount of volatile fatty acids (VFAs). Accordingly, a pre-fermenter was employed to provide a feed stream supplement so as to increase the influent VFAs ₅ especially for the purpose of phosphorous removal by PAOs.

Significant difficulties were encountered with the reliability and control of an old prefermenter existing on the site. Accordingly, a new prefermenter was installed in February 2007. This is a 7500 L polypropylene tank, with a mixer installed. The mixer runs for 3 min following feed batch preparation (off at all other times) and serves to stir the tank sufficiently to prevent accumulation of a significant crust of fat/ grease. DAF effluent (pre-treated raw abattoir wastewater) was pumped through the prefermenter at a rate of 11 L/min in a cycle of 19 min on/ 41 min off in every hour, which gave a nominal HRT of 1.5 days on the days when fed. The prefermenter was not fed on weekends, when the abattoir does not slaughter cattle and cleaning takes place. This avoided diluting/ washing out the prefermenter and gave a true HRT of 2.2 days on a 7-day basis.

Wastewater from the AP effluent overflow tank and from the prefermenter were pumped into a mixing tank periodically. This feed mixture was then pumped into asynchronously operated SBRs during feed periods in the cycles. The operating volume of each SBR is 6000L.

The SBRs were connected to blowers connected to two variable speed drives (Toshiba VSF9 3.7kW), each having a maximum capacity of 164m /hr at 50Hz. Sampling valves at varying heights on the tank sides are also located within the hut. pH Probes (Burkett #8205) and differential pressure transducers (WIKA SL-I; 0-60mbar) were inserted into the tank sides. DO transmitters (Danfoss #OXY3000) connected to DO sensors (Danfoss OXYIlOO) were also inserted into the tanks, through the top. A PLC (Opto22 with Mistic Controller board), for controlling the whole pilot-plant, was also connected to the SBRs.

The two SBRs were operated identically except for the mode of feed delivery during the first feed period of each cycle. Both SBRs have a UniFed feed delivery system (see Table 8 below). However, for SBR 2, the contents of the reactor were mixed (by 30 second air burst) just prior to feed entering the reactor. This mixing did not occur for SBR 1 on the first feed addition (subsequent feed additions followed the same mixing as for SBR 2).

Table 8 - SBR feedin /mixin re imes

Note: Air burst is 30sec aerator on in every 15 min. UniFed Feeding System (SBR I)

The Unifed system involves evenly distributing the feed through the sludge blanket on the horizontal plane. This was usually performed near the base of the tank. No additional mixing was employed during the feeding period and the total feeding period was extended for some time (i.e. a gradual flow) to achieve an anaerobic zone within the sludge blanket.

Only the first feed period of the cycle for SBR 1 was operated as a traditional UniFed system without mixing. During subsequent feed periods the tank was mixed by a short air burst prior to feeding). This was employed because laboratory scale experiments have shown that only one of the feed periods for each cycle needs to reach anaerobic conditions to support the phosphorus release and uptake from polyphosphate accumulating organisms. While mixing of the SBR contents will occur during the 2 ^nd and 3 ^rd feeding periods, an evenly distributed bottom feed delivery was still be used for these periods as the pipework was already present.

For both SBRs, the feed line divided into 4 vertical pipes with horizontal sections attached. Each horizontal length of pipe had two 0.9mm diameter holes drilled into them at 45° to the bottom.

Mixed Feeding System (SBR 2)

For SBR 2 the feed is delivered through a series of pipes as for the UniFed feeding system. However, the tank has an initial air burst mix during all feeds.

A mixing tank was used to mix the two feed sources, the prefermenter effluent and the anaerobic pond effluent. Initially the two feeds were mixed in the ratio of 90% AP effluent: 10% PF effluent. This was subsequently been changed to 84% AP effluent: 16% PF effluent.

Each SBR cycle had 3 feed periods to give a total feed volume of 857L per reactor per cycle (to give a HRT of 1.75 days). 50% of a cycle's feed occurs in the 1 ^st feed period, 30% in the 2 ^nd feed period and 20% in the final feed period. The respective volumes required for each feed stream and feed period are given in Table 9. The feed for each feed period for both SBRs will be prepared in single batches and the SBRs operated out of sync with each other by 20 minutes, minimizing the length of time the feed mixture is retained in the mixing tank. The asynchronous nature of the SBRs is due to the decant lines combining into 1 decant line and the flow needs to be sufficiently low to handle the flow.

Table 9: Volumes of Prefermenter feed and AP effluent feed required for feed batches.

Prefermenter Feed AP Effluent

Combined Feed (16%) (84%) Single Both Single Both Single Both SBR SBRs SBRs SBRs SBR SBRs

Feed l 69 138 361 722 430 860 Feed 2 41 84 216 432 257 514 Feed 3 27 54 143 286 170 340

Total 137 274 720 1440 857 1714

The mixing tank was a polyethylene tank with dimensions of 1.07m diameter and 1.51m wall height, located on a Im high platform due to possible flooding of the DAF bunded area. The mixing tank operating volumes for the 3 feed batches were under PLC control from specified volumes input from Table 8 (or similar).

A pump was assigned to each feed source (PF or APO/F tank). The volumes pumped were critical and control of these were be achieved using an on-line pressure transducer (WIKA S-10; 0-0.1 bar) attached to the mixing tank.

A Davey DClOA (single phase) submersible pump was used to transfer the AP effluent to the mixing tank. The on-period of this pump needs to be low enough to ensure complete drainage of the anaerobic pond overflow does not occur (this is particularly important for the first feed period when the maximum volume of anaerobic pond effluent is required) and is dependent upon the flowrate from the pond due to the small size of the anaerobic pond overflow.

A Davey DClOA (single phase) submersible pump was located in the prefermenter approximately 0.5m below the surface (supported on a steel frame).

A small on-line controlled, submersible pump (Davey DClOA, single phase) is located in the mixing tank and operated intermittently (during batch feeds preparation) to mix the two feed streams and ensure no settling of solids occurs. This is particularly important for the feeding of the second SBR which is delayed by 20 minutes, relative to the first SBR.

Cycle Periods

Table 10 shows representative cycle periods and associated times used for running the SBRs, at least during early phases of the pilot plant trials. These periods were loosely based on the results from the laboratory-scale reactor with alterations of the times set for the settle, decant and feed periods. Following start-up of the pilot plant, the cycle times were adjusted to suit the observed nutrient removal patterns.

Table 10: Cycle periods for pilot-plant.

Period Length Purpose of Period

Settle 70 min Settle sludge Decant 20 min Remove supernatant

Add feed to reactor. For SBR 1, provide

Feed l 20 min anaerobic conditions for P release by PAOs

Mixed, 'Anoxic' period 1 15 mm Denitrification/ Anaerobic P release

Aerated period 1 (max.) 64 min Nitritation

Idle 1 (mixed) O min Depletion of maximum aeration time

Feed 2 15 min Add feed to reactor.

Denitrification/Anaerobic P release

Mixed, 'Anoxic' period 2 30 min (partial)

Aerated period 2 (max.) 45 min Nitritation

Idle 2 (mixed) 0 min Depletion of maximum aeration time

Feed 3 15 min Add feed to reactor.

Denitrification/Anaerobic P release

Mixed, 'Anoxic' period 3 30 min (partial)

Aerated period 3 (max.) 36 min Nitritation/Sludge wastage (1 cycle/day)

Idle 3 (mixed) O min Depletion of maximum aeration time

Total 360 min 6h cycle period; 4 no. cycles per day

Settle

During the settling period, feed, WAS, decant and mixing pumps and the blowers were switched off to allow the suspended solids to settle, in preparation for decanting. The

settle period was assigned a length of 70 min, which was found to be sufficient, given the relatively good sludge settling rates observed to date.

Decant

During decanting, the decant pump (located alongside the pilot-plant control room) pumped out the supernatant to the Effluent Holding tank. The pump suction line in the SBRs is attached to a float (25L sealed drum) via a flexible pipe, such that the pipe entrance is approx. 150mm below the surface level.

The maximum decant time allowed was 20 min, during which time, the SBR water level is registered by the PLC via a pressure sensor located side of the tank. If the pressure/ water level signal reaches the operator-adjusted setpoint, the decant pumps switch off and the solenoid valves on the decant lines close. The SBR then idles for any remaining minutes available in the decant phase.

Feed Periods

Each SBR had three feed periods per cycle. A fresh batch of feed (to be used for both SBRs) was made for each feed period. The feed was pumped into the tanks by the Mono pumps at a rate of approximately 25L/min. The pumps could be operated as on/off to extend the length of the feed periods if necessary. The total volumes fed into the SBRs were controlled by the on-line pressure transducer located in the mixing tank.

Mixed, Non- Aerated Periods

During the non-aerated periods, the SBRs were mixed. These periods were used for denitrification and also phosphorous release from PAO bacteria, after anaerobic conditions are achieved.

Aerated Periods and Sludge Wastage

During the aerated periods, the SBRs were aerated by the blowers through fined bubble diffusers. The blowers were controlled by a PID loop coupled with the DO sensors for continuous operation. Additional mixing was also be employed by the submersible pumps located in the SBRs.

The aim of the aerated periods was to convert the NH ₄ -N to NO ₂ -N (nitritation), not NO ₃ - N. Since reduction of NO ₂ to N ₂ gas requires less carbon substrates than reduction of NO ₃ -N to N ₂ gas. As the system is carbon-limited, this is the preferred option. In the laboratory-scale reactor, oxidation of the NH ₄ -N to NO ₂ -N (and not NO ₃ -N) was controlled by both the OUR and pH profiles such that the rate of change of the pH drop (and OUR) indicates when complete nitritation has been achieved. However, the

laboratory-scale reactor uses an on/off aeration system as compared to the variable air flowrate to be used on the pilot-plant. Thus, although pH levels were monitored, aeration control was by DO probe, maintaining the dissolved oxygen, ideally, between 1.5 and 1.75mg O ₂ /L. pH profiles of recent runs have shown that this was sufficient.

Aeration phases were stopped once the maximum allocated aeration time had lapsed (see Table 10) and the cycle moved into the next Feed phase.

Waste collected in the waste tank (from AP effluent overflow tank and sludge storage overflow), was pumped periodically (by submersible pump controlled by a float switch) to the existing on-site DAF effluent holding tank. The full-scale wastewater stream in this tank was pumped to the AP. The flow from the pilot-plant was very low compared to flow from the full-scale DAF during normal operating days.

As shown in Figures 16A-C and 17A-C, in both SBRs, early trials provided highly variable results. This was predominantly due to equipment issues (including blockage of lines, unreliable flows) and, especially, inconsistent and insufficient VF A/soluble COD levels which are assumed to have disadvantaged the PAOs in the SBRs. A major source of unreliable VFA levels in the feed was the old prefermenter. Once this was replaced, and the ration of prefermenter to anaerobic pond feed adjusted, the results achieved for nutrient removal stabilised (as apparent from mid to late August 2007), and better than 90% nitrogen and phosphorous were reliably achieved. In addition, as apparent from the low nitrate levels observed (see, for example, Figures 18 and 19), nitrogen removal by the nitrite pathway (rather than via nitrate) was also achieved.

Figures 18 and 19 provide data for representative recent SBR cycles for SBRl and SBR2 (run on 3 September 2007), including ammonium, nitrate, nitrite, phosphorous and pH profiles for the wastewater undergoing treatment. The two pilot-scale SBRs were operated identically except for the mode of feed delivery during the first feed period of each cycle. SBRl has a UniFed feed delivery system but not SBR2.

Both SBRs were operated with a cycle time of 6h (see Table 12). In each cycle, 857L of influent was pumped into each SBR over the three filling periods with a volume distribution of 50%, 30% and 20% respectively. Each filling period was followed by non-aerated and aerated periods. During aerated periods, DO level was kept at 1.5 mgO ₂ /L. The SBRs hydraulic retention time was 42h and the sludge retention time was 10 days.

Table 11 - Characteristics of the two types of wastewater and the combined influent as on 03/09/2007. The combined SBR feed was constituted of 30% of pre-fermented raw wastewater and 70% anaerobic pond effluent (volumetric):

Pre-fermented Anaerobic pond Combined feed

Parameter raw wastewater effluent for SBRs

SCOD/TP 51 4.7 15.2

Table 12 - Cycle periods for SBRs 1 and 2 on 3 September 2007

Period Length Purpose of Period

Settle 30 min Settle sludge

Decant 20 min Remove supernatant

Feed 1 20 min Add feed to reactor. For SBR 1,

Mixed, 'Anoxic' period 1 30 min Denitrification/ Anaerobic P release

Aerated period 1 (DO= 1.5mgO ₂ /L) 70 min Nitritation

Idle 1 (mixed) 15 min Depletion of dissolved oxygen

Feed 2 15 min Add feed to reactor

Mixed, 'Anoxic' period 2 30 min Denitrification/ Anaerobic P release

Aerated period 2 (DO=I .5mgO ₂ /L) _. 43 min Nitrification/Sludge wastage

Idle 2 (mixed) 15 min Depletion of dissolved oxygen

Feed 3 15 min Add feed to reactor

Mixed, 'Anoxic' period 3 26 min Denitrification/ Anaerobic P release

Aerated period 3 (DO= 1.5mgO ₂ /L) 31 min Nitrification

Total 360 min 6h cycle period; 4 cycles per day

As can be seen from Figures 18 and 19, almost complete nitrogen removal was achieved for both of the SBRs, and that phosphorous levels in the effluent for the reactors were less than 2-2.5mg/L.

It will be appreciated that, although a specific embodiment of the invention has been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention as defined in the following claims.