Principal wastewater treatment processes used in the meat processing industry 

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Investigation of the feasibility of COD, N and P removal from abattoir wastewater using granular sludge technology.

As discussed in Chapter 2, the excellent settleability of aerobic granular sludge allows for more biomass to be maintained in a relatively small reactor volume, enhancing the ability of the reactor to withstand high loading rates. This is of great interest for the treatment of high nutrient containing industrial wastewater such as abattoir wastewater compared to conventional floccular sludge systems. To date, aerobic granular sludge technology has mainly been studied with synthetic wastewater, with the only real wastewater studies being performed using domestic wastewater. The feasibility of achieving high-levels of COD, N and P removal from abattoir wastewater using granular sludge technology is investigated in this thesis. In addition, the microscale structure of these aerobic granules is examined using a multi-disciplinary approach.

Research Methods

In this thesis, three different lab-scale SBRs were operated to demonstrate the feasibility to achieve high-levels of biological COD, N and P removal from abattoir wastewater. The first SBR, referred to as RivD-SBR, was employed to experiment novel strategies, which can easily be implemented to current water treatment facilities used by the meat industry, to produce an effluent suitable for river discharge (>95% TCOD, TN and TP removal). RivD-SBR was used to address research objectives 1, 2 and 3. The second SBR, referred to as SNDPR-SBR, was operated to provide a platform for an in-depth investigation of the previously proposed simultaneous nitrification, denitrification and phosphorus removal (SNDPR) process, which has the potential for application to the treatment of abattoir wastewater. SNDPR-SBR was first employed to identify the causes of N2O emission in lab-scale SNDPR bioreactors fed with synthetic wastewater (research objective 4). The operation of this reactor was then modified to transform its floccular biomass into granules, which are believed to reinforce the SNDPR process. The microbial community structure in this new granular SNDPR-SBR was then examined via a newly developed method (research objective 5). The third SBR, referred to as Granular-SBR, was operated for the treatment of the high nutrient-containing abattoir effluent (research objective 6).
The operations of the three SBRs are briefly described below, which are followed by an overview of the key analytical methods employed in this thesis to address all of the above research objectives.

Operation of lab-scale SBRs used in this thesis

RivD-SBR

RivD-SBR had a working volume of 7 l (Figure 9) and was operated with a cycle time of 6 h in a temperature-controlled room (18-22°C). Each cycle, 1 l of abattoir wastewater collected weekly from a local abattoir (mixture of primarily treated and anaerobically treated wastewater) and stored in a cold room at 4°C was pumped into the reactor over 3 feeding periods. Each feeding period was followed by an anaerobic/anoxic period and an aerobic period (Table 2). During the aerobic periods, air was provided intermittently using an on/off control system to keep the DO level between 1.5 and 2 mgO2 .l-1. The HRT and SRT in the SBR were kept constant at 42 h and 15 days, respectively. Full description of the SBR operation is given in Appendix A.
A 5 l SBR performing SNDPR, which was previously designed and operated at the AWMC lab, was used in this thesis (Figure 10) to identify the causes for N2O accumulation in lab-scale SNDPR processes (research objective 4). Initially, the SBR 6h cycle consisted of a 90 min anaerobic, 220 min aerobic, 40 min settling, and 10 min decanting period. In each cycle,
3 l of synthetic wastewater (230 mgCOD.l-1 as acetate, 23 mgN.l-1 as NH4+ and 18 mgP.l-1 as PO43-) was pumped into the reactor resulting in an HRT of 10 h. The SRT was kept at 20 days. Aeration was provided intermittently using an on/off control system to keep the DO level relatively low between 0.35 and 0.5 mg.l-1. Full description of the initial SBR operation is given in Appendix D.
The SBR operation was then modified to promote the formation of aerobic granules. The settling time was gradually reduced from 40 min to only 5 min and the nutrient concentration in the synthetic wastewater was increased to 350 mgCOD.l-1 as acetate, 35 mgN.l- 1 as NH4+ and 23 mgP.l-1 as PO43-. The cycle time was reduced to 4 h and consisted in 55 min anaerobic period followed by 170 min aeration, 5 min settling, and 10 min decant. The DO was kept between 1.3-1.7 mgO2.l-1. Full description of the new SBR operation is given in Appendix E.

Granular-SBR

The design of granular-SBR was similar than SNDPR-SBR (Figure 10) with a working volume of 5 l. Granular-SBR was also operated in a temperature-controlled room (18-22°C). The reactor was seeded with the granular sludge obtained from the lab-scale SNDPR-SBR fed with synthetic wastewater. During the first 4 months of operation, the SBR was fed with a mixture of anaerobically treated abattoir effluent and synthetic wastewater and the cycle time varied between 4-8h. After the initial 4 month adaptation period, only anaerobically treated abattoir effluent was fed into the SBR. Once steady state was established, the cycle time was fixed at 8 h and consisted of an 18 min feeding, 60 min anaerobic/anoxic, 315 min aerobic, 80 min post-anoxic, 2 min settling and 5 min decanting period. During the aerobic period the DO level was kept between 3.0 and 3.5 mgO2.l-1 . The HRT gradually increased from 6.7 h during the adaptation period to 13.3 h once stable operation was reached (3 l fed each cycle). Full description of the granular SBR operation is given in Appendix F.

Analytical Methods

A wide range of techniques was employed to address the research objectives defined in this thesis. It includes reactor process studies, microbial investigations (fluorescent in -situ hybridisation – FISH combined with confocal laser scanning microscopy – CLSM) and several micro-scale techniques (e.g. micro-sensors, electron microscopy and light microscopy). The combination of these multi-disciplinary techniques has helped deliver significant outcomes.

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Chemical analysis

The ammonia (NH4+ + NH3), nitrate (NO3- ), nitrite (NO2-) and phosphate-P (PO43-P) were analysed using a Lachat QuikChem8000 Flow Injection Analyser (Lachat Instrument, Milwaukee). Dissolved nitrous oxide (N2O) was measured on-line with a N2O microsensor constructed according to Andersen et al. (2001). Total and soluble COD (TCOD and SCOD), soluble BOD5, total and soluble Kjeldahl nitrogen (TKN and SKN), total phosphorus and total dissolved phosphorus (TP and TDP), mixed liquor suspended solid (MLSS) and volatile MLSS (MLVSS) were analysed according to the standard methods (APHA, 1995). The major ions present in the SBRs bulk liquid (Ca2+, Fe2+, K+, Mg2+, Na+ and HS-) were measured by Inductively Coupled Plasma – Atomic Emission Spectrometry (ICP-AES Varian Vista-PRO, Varian, Inc.). VFAs were measured by Perkin-Elmer gas chromatography with column DB-FFAP 15 m x 0.53 mm x 1.0 µm (length x ID x film) at 140°C, while the injector and FID detector were operated at 220°C and 250°C, respectively. High purity helium was used as carrier gas at a flow rate of 17 ml.min-1 . Polyhydroxylalkanoates (PHA=PHB+PHV+PH2MV) and glycogen were determined using the method described in Oehmen et al. (2005). The phosphorus fractionation in granules was determined using the cold perchloric acid (PCA) extraction procedure developed by De Haas et al. (2000) as detailed in Appendix F.

Microbial analysis

Sludge samples were fixed and FISH probed as previously described (Amann, 1995). Oligonucleotide probes used in this thesis were the combination of EUB338 i-iii (EUBmix) for the detection of all bacteria (Daims et al., 1999), the combination of PAO462, PAO651 and PAO846 (PAOmix) for Accumulibacter spp. (Crocetti et al., 2000), the probe combination (GAOmix) of GAOQ989 (Crocetti et al., 2002) and GB_G2 (Kong et al., 2002) for Competibacter spp., NTSPA662 (Daims et al., 2001) for Nitrospira spp., NIT3 (Wagner et al., 1996) for Nitrobacter spp. and NSO1225 (Mobarry et al., 1996) for most of the ammonia oxidising bacteria (AOB) from the Betaproteobacteria. Additionally, probes for the proposed Actinobacterial PAOs (Kong et al., 2005) and Defluviicoccus spp.-related GAOs (Meyer et al., 2006) were also used. Fluorescently labelled oligonucleotides were purchased from Thermo (Ulm, Germany) with fluorescein isothiocyanate (FITC) or one of the sulfoindocyanine dyes indocarbocyanine (Cy3) or indodicarbocyanine (Cy5). FISH images were collected with a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) CLSM using an argon laser (488 nm), a helium neon laser (543 nm) and a red diode laser (633 nm) fitted with 515-565 nm BP, 590 nm LP and 660-710 nm BP emission filters, respectively. Two different Zeiss oil immersion objectives were used, a Plan-Neofluar 40x/1.3 and a Plan-Apochromat 63x/1.4. FISH quantification was performed according to Crocetti et al. (2002) where the relative abundance of each group was determined as mean percentage of all bacteria based on pixel area counting.
Nile-Blue A staining (Ostle and Holt, 1982) was used to determine cells containing intracellular PHA and visualised on the confocal laser scanning microscope (see above).
Fixed granule samples for FISH and Nile Blue A staining were embedded in optimum cutting temperature (OCT) compound (TissueTek, Sakura, USA) for cryosectioning as previously described (Meyer et al., 2003). Embedded granules were then frozen and sectioned into 10 µm thick slices using a cryotome operated at -20°C (Kryo 1720, Leitz, Germany). The granule sections were collected on SuperFrost Plus microscope slides (Menzel-Glaser, Germany). Finally, the slides were dehydrated by sequential immersion for 3 min in 50%, 80% and 98% ethanol, followed by air-drying.

Physical analysis

To monitor the granule structure and characteristics, granule size distribution and density were measured. To determine the size distribution of the granules, 30 ml of well mixed granular sludge was sampled from the SBR at the end of the aeration period and pumped through a Malvern laser light scattering instrument, Mastersizer 2000 series (Malvern Instruments, Worcestershire, UK). The granule density, defined as the quantity of dry mass per biomass volume, was measured by the blue dextran method adapted from Di Iaconi et al. (2004) and further described in Appendix E.
The gradient of oxygen in granules was measured with oxygen microsensors (tip diameter <10 µm), which were constructed as described by Revsbech et al. (1989). Granules were transferred to a flow-cell with an upward flow where replicate oxygen profiles were then measured and averaged as described in Meyer et al. (2003). The pH gradient in granules was also measured with pH microsensors using the same experimental set-up.

Table of contents :

1.0 Introduction
2.0 Literature Review
2.1. Introduction to biological nutrient removal processes
2.1.1. Nitrogen removal process
2.1.2. Biological phosphorus removal process
2.1.3. Advanced biological nutrient removal processes
2.1.4. Achieving BNR using sequencing batch reactor technology
2.1.5. SBR control strategy
2.2. Abattoir wastewater treatment
2.2.1. Abattoir wastewater characteristics
2.2.2. Principal wastewater treatment processes used in the meat processing industry
2.2.3. SBR technology for abattoir wastewater treatment
2.2.4. Challenges facing abattoir wastewater treatment
2.3. New possible BNR technologies
2.3.1. Simultaneous Nitrification, Denitrification and Phosphorus Removal
2.3.2. Aerobic Granular Sludge technology
3.0 Thesis Overview
3.1. Research Objectives
3.2. Research Methods
3.2.1. Operation of lab-scale SBRs used in this thesis
3.2.2. Analytical Methods
3.3. Research Outcomes
4.0 Conclusions and Recommendations for Future Work
4.1. Main Conclusions of the Thesis
4.2. Recommendation for Future Research

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