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    Understanding the mechanisms involved in Escherichia coli decay during wastewater treatment in High Rate Algal Ponds : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Chambonnière, Paul
    Little is known about the mechanisms and magnitude of pathogen disinfection in High Rate Algal Ponds (HRAPs). However, maturation ponds are used worldwide for wastewater disinfection, and pathogens can experience similar environmental conditions in maturation ponds and HRAPs. The literature suggests that pathogen removal in maturation ponds is primarily supported by sunlight-mediated mechanisms (direct DNA damage, endogenous photo-oxidation, and exogenous photo-oxidation), and a range of poorly characterized ―dark‖ mechanisms. Based on this evidence, and knowing HRAPs are specifically designed to optimize light supply into the broth, there is reason to believe sunlight mediated disinfection mechanisms should be significant in HRAPs. This thesis therefore aimed at identifying and quantifying the mechanisms responsible for Escherichia coli (E. coli) decay in HRAPs under the hypothesis that understanding the mechanisms involved in disinfection during wastewater treatment in HRAPs can provide the scientific foundation needed to optimize the design and operation for this critical wastewater treatment service. E. coli was selected for being an established indicator of the removal of faecal contamination during wastewater treatment. Two pilot scale HRAPs (0.88 m3) were commissioned and monitored over 1-2 years, showing a mean E. coli decay coefficient of 11.90 d-1 (std = 24.05 d-1, N = 128), equivalent to a mean E. coli log removal of 1.77 (std = 0.538, N = 128) when operated at a hydraulic retention time (HRT) of 10.3 d (std = 2.01 d, N = 139). Hourly monitoring showed high daily variations of E. coli log removal (up to 2.6 log10 amplitude) during the warmest summer days, with the lowest E. coli cell counts observed in the late afternoon, when the broth pH, dissolved oxygen concentration, and temperature typically reached peak values in the HRAP. No mechanisms driving E. coli removal in HRAP could be identified during the monitoring of pilot scale HRAPs so a mechanistic study of E. coli decay was performed at laboratory and bench scale to individually quantify potential mechanisms. At laboratory scale under various conditions (e.g. darkness vs sunlight exposure, neutral pH vs alkaline pH, RO water vs filtered HRAP broth), direct DNA damage, endogenous photo-oxidation, and high-pH toxicity were identified as the main mechanisms contributing to E. coli decay. Exposure to potentially toxic algal metabolites and exogenous photo-oxidation were not found to be significant under the conditions tested. Natural decay (i.e. decay in conditions identified not to be detrimental to E. coli survival) was never significant. The impact of predation could not be investigated due to technical challenges although pilot scale observations suggested this mechanism may be significant in certain conditions. Subsequent bench-scale tests conducted in HRAP broth indicated that temperature-dependent uncharacterized dark decay (i.e. decay in conditions not known to be detrimental to E. coli survival) was likely to be the dominant mechanism of E. coli removal under conditions relevant to full-scale operation. Temperature-dependent high-pH toxicity was confirmed to further increase E. coli decay at pH levels commonly reached in HRAPs. The contribution of sunlight mediated mechanisms was however not significant. Exposure to toxic algal metabolites was suspected to cause significant E. coli decay at times of extreme photosynthetic activity, but more research is needed to confirm this mechanism and its true significance. Results from laboratory scale and bench scale experiments enabled the development of a model capable of predicting E. coli decay in HRAP broth according to pH, temperature, and sunlight intensity distribution. A model predicting HRAP broth temperature and pH according to design and weather data was also developed and validated against data from the pilot scale HRAPs monitored during this study for temperature (average absolute error of predictions 1.35°C, N = 25,906) and pH (average absolute error of predictions 0.501 pH unit, N = 23,817). Coupling the E. coli decay model with the environmental model enabled long term predictions of E. coli removal performances in HRAP for various weather conditions, design, and operational regimes. Simulations predicted that a 3-HRAPs series would sustain average yearly E. coli log-removal of 3.1 in Palmerston North, New Zealand when operated in conditions similar to the pilot scale HRAPs used in the present study. Such performance would deliver year round compliance with local microbial quality guidelines. Disinfection performance could be further improved by increasing the hydraulic retention time, lowering the depth, or collecting the effluent once daily in the late afternoon while letting HRAP depth fluctuate. Overall, this research challenges the common belief that sunlight mediated disinfection mechanisms contribute the most to pathogen removal in HRAPs. Instead, uncharacterized dark decay was predicted to cause 87% of the total E. coli decay over one year simulation. High-pH toxicity may significantly contribute to overall E. coli decay in specific conditions (e.g. low depth where high-pH toxicity was predicted to account for 33% of total yearly E. coli decay), while sunlight mediated disinfection was limited under all simulated designs and operations (highest contribution predicted being 16% of total yearly E. coli decay). Because this study also confirmed the potential of HRAP to achieve sustained wastewater disinfection, further research is needed to better characterize dark decay mechanisms (for E. coli and other key indicators) as this knowledge has the potential to further improve HRAP design and operations for wastewater disinfection.
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    The antibiotic sensitivity patterns and plasmid DNA content of gram-negative anaerobic bacteria isolated in Palmerston North, New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Science at Massey University
    (Massey University, 1987) Mooney, Christopher Allan
    One hundred and seven Gram-negative bacteria, including 65 Bacteroides species, 28 fusobacteria and 14 veillonellae were isolated from 17 oral infections treated in two dental surgeries in Palmerston North. These bacteria, plus 37 isolates belonging to the B. fragilis group received from Palmerston North hospital, were surveyed for their antibiotic sensitivity levels, and their plasmid DNA content. The hospital isolates of the B. fragilis group were found to have sensitivity levels comparable with those of B. fragilis group isolates reported in the literature recently. The oral isolates were more sensitive to penicillin, cefoxitin, and tetracycline than isolates of the same species reported in the literature. Half the hospital isolates had plasmids, which were all between 8.5 and 2.7 kilobases (kb) in size except for one 60, and one 43 kb plasmid. Comparatively few of the oral anaerobes had plasmids. One Fusobacterium russii isolate had four plasmids, and five Bacteroides isolates had one plasmid each. These five Bacteroides isolates came from two specimens, R5 and R6. Restriction enzyme analysis of all plasmids revealed that the three 5.6 kb plasmids from sample R5 may be related to a group of 5.8 kb plasmids harboured by four of the hospital isolates. Two different species of Bacteroides isolated from sample R5 harboured the 5.6 kb plasmid, and two species of the B. fragilis group bacteria harboured the 5.8 kb plasmid. Plasmid DNA isolated from two tetracycline resistant hospital isolates was used to transform restriction negative E. coli to a low level of tetracycline resistance.
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    Understanding bacterial adaptation to aerobic and anaerobic environments through experimental evolution and whole genome analysis : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Finn, Thomas
    Facultative anaerobic organisms have the metabolic versatility to grow in both aerobic and anaerobic environments. However, molecular mechanisms that underpin adaptation to anaerobic environments are not well understood. This study aims to understand how the facultative anaerobe, Escherichia coli, adapts to environments that vary in oxygen content. An experimental evolution experiment was conducted in which replicate lineages were established from a preevolved clonal culture of E. coli REL4536. Lineages were serially sub-cultured for 4,000 generations within strict aerobic and strict anaerobic environments, and a treatment that fluctuated between the two environments. Significant increases in the relative fitness of lineages exposed to anaerobic conditions were observed, whereas the relative fitness of lineages in aerobic conditions did not increase, likely as the ancestor had been pre-adapted to aerobic growth. Mutations that arose during evolution were identified by genome sequencing randomly-selected clones from each lineage at 2,000 and 4,000 generations. Traits that contributed to adaptation were predicted via the occurrence of independent mutations affecting common traits among lineages. Adaptation to the anaerobic environment was facilitated by modifications to anaerobic fermentation and the inactivation of virulence genes, whereas in the aerobic environment, mutations predicted to confer a growth advantage in stationary phase were observed. The evolution of generalists involved traits that were similar to those found in both aerobic and anaerobically evolved lineages, as well as the deletion of cryptic prophages from the genome and modifications to amino acid transport. Phenotypically distinct small colony morphotypes (SCM) arose within anaerobic lineages and two separate adaptive pathways are hypothesised for this divergence. SCM1 were capable of stable coexistence with co-evolved cells of typical colony morphotype, most likely through an acetate crossfeeding mechanism. In contrast, SCM2 was able to out-compete the ancestor within 14 days, despite exhibiting a lower growth rate than the ancestor. SCM2 likely evolved the ability to inhibit the ancestral strain through a contact dependent inhibition mechanism, as evidenced by a mutation in glgC. This thesis demonstrates the complex nature of adaptation to anaerobic environments, as revealed by experimental evolution and whole genome sequencing.
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    The disposition of metronidazole in goats and its relevance to the treatment of anaerobic infections : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University
    (Massey University, 1988) Dillon, Elizabeth Amanda
    The recent commercial developments in goat farming in New Zealand, have led to an increase in the value of individual goats and to a growing interest in caprine diseases. The importance of anaerobic bacteria other than the Clostridia, as potential pathogens in humans and animals, has also only recently been recognised, even though anaerobic bacteria have been identified since 1861. Various members of this bacterial group are known to be involved in different conditions of goats, particularly in wound and foot infections. Metronidazole (Flagyl'Flagyl', May & Baker (NZ) Ltd.) is a bactericidal agent which has a specific action against anaerobic micro-organisms. This drug is already widely used in the treatment of selected diseases in dogs, cats and humans, but there was little information available on its use in goats. The study which forms the basis of this thesis, was to investigate the disposition of metronidazole in eight goats. Both IV and IM routes of administration were studied in the form of a cross-over experiment. Silicone tubing "cages" were implanted subcutaneously, so that the metronidazole concentration versus time profile could be determined, both in serum and in interstitial fluid. The analysis of serum and tissue cage fluid samples was undertaken using a high pressure liquid chromatography unit, which proved to be reliable over the range of concentrations tested. The system consisted of a Waters Model 6000 A solvent delivery system, a U6K injector, a Z-module radial compression separation system and a Waters programmable automator, Model 710. The mobile phase used was a 75:25 mixture of aqueous potassium hydrogen phosphate and methyl alcohol; this was adjusted to a running speed of 1.5 mls per min. A 450 variable wavelength detector was set at either 0.01 or 0.04 absorbance units, and a constant wavelength of 312 nm. Given these concentration profiles, a full pharmacokinetic analysis was carried out using standard statistical procedures. The following values were determined: maximum serum and tissue cage fluid concentrations (CmaxIV, CmaxIM, CmaxIVtc), time to maximum serum and tissue cage concentration (TmaxIV, cmaxIMtc TmaxIMtc), serum concentration (extrapolated) at zero time (B, B'), half-life (t1/2), elimination rate constant (B,B'), volume of distribution (Vd(area)), area under the concentration curve (AUC), total body clearance (ClB), absorption rate constant (kab), percentage penetration of metronidazole into tissue cage fluid, percentage of drug absorbed into the systemic circulation following IM administration (F), and the total amount of drug which was absorbed into the systemic circulation (in mg/kg). Following IV administration of a 0.5% w/v solution of metronidazole at a dose rate of 20 mg/kg BWgt, the t1/2 at 0.94±0.08 per hour (n=8) was rapid and consistent with a high figure for the elimination rate constant at 0.79 ± 0.09 per hour (n=8). The total body clearance, a more sensitive indicator of the biotransformation and excretion processes than t1/2, was also rapid (0.32 = 0.06 L/kg/hr) which is in keeping with the efficient drug metabolism of the goat. This may account for the low Vd(area) which was unexpected for a basic drug of this nature in the ruminant. Critical parameters for drug concentrations and durations of effect are summarized in Table I. Metronidazole was rapidly detected in both sera and interstitial fluid (within 0.25 hrs) following the intramuscular administration of a 40% suspension of metronidazole at a dose rate of 20 mg/kg BWgt. The uptake of metronidazole from the injection sites differed markedly between individual goats, resulting in a mean absorption percentage of 42.4%±8.8% (n±8), equivalent to 8.4 mg/kg BWgt. Maximum serum levels were achieved within approximately one hour of IM administration, but the peak was more than ten-fold lower than the corresponding concentration found in serum following IV administration. Peak tissue cage drug concentrations were not achieved until four hours after IM administration. The maximum drug concentration in tissue cage fluid was greater than the MIC upper threshold for a variety of anaerobic bacteria (12.5 mcg/ml), and this was maintained for 5.5 hrs. The lower limit of the MIC of 3.0 mcg/ml was exceeded for a correspondingly longer period. Further pharmacokinetic analysis of the experimental data made it possible to calculate specific medication schedules for the goat. These were established on the basis that serum metronidazole concentrations should be maintained at a level which was bactericidal for the majority of anaerobic bacteria, which included Bacteroides spp., Fusibacterium spp., and Clostridia spp. The recommendation given was that 0.5% w/v metronidazole solution should be administered at a dose rate of 20 mg/kg BWgt and repeated every 4-6 hrs. Using the 40% w/v metronidazole suspension, the dose rate should be 45 mg/kg BWgt and the medication should be repeated every 10-12 hrs. In each case the loading dose was only fractionally greater at 20.3 mg/kg BWgt and 48.5 mg/kg BWgt respectively. The drug concentration in interstitial fluid (tissue cage fluid), gave some indication of the antimicrobial activity in extravascular tissues, a feature which can not be extrapolated from a profile of serum concentrations.
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    Cultivation and community composition analysis of plant-adherent rumen bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Noel, Samantha
    Ruminants have a symbiotic relationship with the complex community of microbes that reside within their rumen. These microbes are able to break down recalcitrant plant material that would otherwise be indigestible by the host. Ruminal bacteria that attach to the ingested plant material are important for the degradation of plant fibre. The number of bacteria cultured from the rumen is estimated to represent only some 10% of the total diversity. This has led to the belief that a large proportion of bacteria in the rumen are unculturable. In this study, liquid media that mimic the physico-chemical composition of the rumen, were used in combination with dilution to a single cell, to obtain >1000 cultures of anaerobic bacteria from the plant-adherent fraction of bovine rumen contents from 20 rumen samples. The phylogenetic affiliation of 828 of these cultures was assessed by comparative analysis of partial 16S rRNA gene sequences. There were 626 unique sequence types (V1-V3 of the 16S rRNA gene), and 200 of these isolates were novel (<96% similarity to a previously-cultured bacterium). The near full-length 16S rRNA gene sequences from 186 selected isolates (representing 801 of the total sequenced isolates) were classified into 14 families including two potentially new families, 77 genera including 59 potentially new genera, and 122 species including 103 potentially new species. The total bacterial communities in the same rumen samples were characterised using FLX-titanium 16S rRNA gene amplicon pyrosequencing of the V1-V3 region of the 16S rRNA gene. These data were then compared with the isolates that had been cultured. The majority of the isolates and amplicon sequences were associated with the phyla Firmicutes and Bacteroidetes. Sequences were grouped into operational taxonomic units (OTUs) at 96% sequence similarity. At this level, 32% of the plant adherent community (i.e., total pyrosequences) and 7.7% of the observed diversity (i.e., unique OTUs) were in OTUs that contained a newly-cultivated isolate. More OTUs (169) contained a sequence from an isolate cultured for the first time in this study compared to the number of OTUs (103) that contained sequences from previously-isolated bacteria. The isolates gained in this study can begin to bridge the gap between the cultured and the uncultured.
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    Interactions between commensal obligate anaerobes and human intestinal cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Manawatu, New Zealand
    (Massey University, 2013) Ulluwishewa, Dulantha
    The human intestinal epithelium is formed by a single layer of epithelial cells which regulates intestinal barrier permeability. Increased permeability can result in the entry of potentially harmful compounds into the body, and is implicated in autoimmune, inflammatory and atopic diseases. The intestinal tract is inhabited by an estimated 1014 microbes and it is increasingly evident that they affect intestinal barrier function. However, over 90% of commensal intestinal bacteria are obligate anaerobes, making it difficult to co-culture them with oxygen-requiring mammalian cells in vitro. To investigate the interactions between obligate anaerobes and epithelial cells that regulate the intestinal barrier, an apical anaerobic model of the human intestinal epithelium, which utilises a dual-environment co-culture chamber, was developed and validated. The chamber allowed for polarised monolayers of the intestinal cell line Caco-2 to be grown such that the apical (luminal) side was exposed to an anaerobic environment, while maintaining an aerobic basal side. The cell viability and barrier function of Caco-2 monolayers was unaffected by culture in the apical anaerobic model for at least 12 hours. Global gene expression analysis predicted upregulation of cell survival and proliferation in Caco-2 cells cultured in the apical anaerobic model, compared to Caco-2 cells grown under conventional conditions, suggesting an adaptation of the Caco-2 cells to a lower supply of oxygen. The apical anaerobic model was used to co-culture the commensal obligate anaerobe Faecalibacterium prausnitzii with Caco-2 cells. The survival of F. prausnitzii was improved in the anaerobic apical environment compared to when cultured in an aerobic atmosphere. Live F. prausnitzii, but not non-viable (UV-killed) F. prausnitzii, were shown to increase permeability across Caco-2 monolayers. Furthermore, global gene expression analysis suggested that live F. prausnitzii cells have more profound effects on Caco-2 cells than non-viable F. prausnitzii, illustrating the importance of maintaining viability of obligate anaerobes in an in vitro co-culture system. The apical anaerobic model can be used to gain insights into the mechanisms of crosstalk between commensal obligate anaerobic bacteria and intestinal cells, and new knowledge generated using this model will assist in the development of strategies to improve intestinal barrier function.
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    The occurrence of chromatiaceae in waste treatment lagoons and their utilisation to treat fellmongery effluent : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1979) McFarlane, Paul Northcote
    A study of the occurrence of Chromatiaceae in waste treatment lagoons was made. To determine the important factors leading to their dominance, an investigation of the effect of various environmental parameters on the growth of a Chromatium species was made. Chromatium minutissimum was isolated and identified from an anaerobic lagoon treating meatworks effluent. An experimental design was used to screen the effects of temperature, pH, sulphide and acetate concentrations and light intensity on the batch growth of this bacterium in pure culture. Empirical models were developed which described the maximum population and the exponential growth rate as a function of these variables. Comparison of these models with lagoon data indicated that they provided a conservative estimate of the exponential growth rate and maximum population under lagoon conditions and that, under the range of environmental conditions expected in New Zealand, the hydraulic retention time is of major importance in limiting the development of this phototrophic bacterium in lagoons. The developed models may possibly be used to characterise the growth of other Chromatiaceae. To study the growth of the Chromatiaceae in mixed culture various lagoon samples were incubated in daylight. A succession from anaerobic non-phototrophic bacteria to phototrophic bacteria to algae was observed in these batch cultures. Thus, in addition to low hydraulic retention times preventing the growth of the Chromatiaceae, competition from the algae precludes their dominance at longer retention times. Seven lagoon systems in which the Chromatiaceae were known to occur were then investigated. The lagoons studied ranged from facultative to anaerobic. The wastes treated varied from domestic sewage to strong industrial and agricultural effluents. A succession from non-phototrophic anaerobes to Chromatiaceae to algae was observed in many instances and a three stage succession theory was formulated. This theory was used to explain the occurrence of the Chromatiaceae in all the lagoon systems studied and it may be used to design lagoons in which the dominance of the Chromatiaceae is favoured or prevented. The study of the lagoon systems indicated the potential of the Chromatiaceae for treating effluents containing reduced sulphur compounds. In N.Z., fellmongery effluent is the most important sulphide-bearing effluent. Experiments were therefore performed to develop criteria for the design of anaerobic lagoons using the Chromatiaceae to treat fellmongery effluent. Experiments were conducted to determine the effects of temperature and sulphide concentration on the performance of .088 m3 laboratory lagoons, in which Thiocapsa roseopersicina was dominant, treating a synthetic fellmongery effluent. Temperatures from 10°C to 25°C and influent sulphide concentrations of 200 mg/l to 1,500 mg/l were studied. Good treatment was obtained under a wide range of conditions although inhibition of growth occurred at influent sulphide concentrations of approximately 900 mg/l. Concentrated fellmongery effluents may therefore be treated by these lagoons. COD removals varied from 66.1% - 87.1% and sulphide removals from 89.5% - 98.4%. Design equations which described the performance of the laboratory lagoons were developed. To confirm the accuracy of these equations, pilot scale experiments were conducted on a 5.74 m3 lagoon system treating actual fellmongery effluent. A good degree of treatment was again achieved and the laboratory-developed equations provided a good estimate of the pilot-scale effluent over the range of conditions studied. Suitable criteria have therefore been developed for the design of anaerobic lagoons using the Chromatiaceae to treat fellmongery effluent.
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    Factors influencing the rate and stability of the anaerobic digestive process : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1986) Mawson, Andrew John
    Three factors affecting the rate and stability of the methane fermentation of a readily-hydrolysable feedstock were investigated. The aim of this work was to develop improved processes and control strategies to facilitate economic treatment of industrial wastes by anaerobic digestion. A comparison was made between the performance of a continuously-fed digester and semi-continuous digesters slug fed every second day. A semi-synthetic medium with glucose as the major carbon and energy source was used and seed material was transferred between the digesters, which were operated under similar loading conditions. The continuous digester repeatedly failed even when operated at dilution and loading rates much lower than the maximum values commonly reported. In contrast, the semi-continuous units provided satisfactory performance and could be easily and rapidly recovered from retarded operation. Failure of the continuous digesters was characterised by a steady fall in volatile suspended solids concentration followed by a rapid accumulation of acetate, and was attributed to a deficiency in the medium of one or more essential nutrients. These were thought to be provided in the semi-continuous digester by lysis of acidogenic bacteria or luxury uptake from the medium. Degradation of acetic and propionic acids was investigated in batch culture. Increasing the concentration of either acid from low levels decreased the rate of utilisation of the acid, but the proposed inhibitory role of un-ionised acids was not conclusively supported. Increasing the initial acetate concentration above 1000 to 1500 mg.l-1 significantly reduced the rate of degradation of propionate added at 500 mg.l-1. When acetate was added at 2000 mg.l-1 the rate of propionate utilisation was approximately half of that when acetate was present at 500 mg.l-1 or lower. In batch culture experiments, addition of up to 3.2 mM cysteine-hydrochloride or sodium sulphide, or 4.4. mM sodium thioglycollate did not inhibit total gas production from samples drawn from the continuous digester. However thy rate of methane production in effluent samples from a semi-continuous digester was inhibited by 25 % to 30 % by addition of 3.2 mM cysteine or sulphide. Inhibition was attributed to the sulphide ion. Sodium thioglycollate did not inhibit methane production from acetate but propionate degradation was markedly reduced, with increasing inhibition noted with increasing incubation time. The work adds to a considerable body of investigation into the factors influencing anaerobic digestion and the unresolved problem of process stability in long-term operation of conventional stirred tank digesters has again been highlighted. Indicators and possible causes of process failure have been suggested and further development of these should assist in the continuing increase in the rate of treatment while ensuring acceptable working margins of safety for the process.
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    Casein whey as booster for anaerobic co-digestion of primary sludge : a thesis presented in partial fulfilment of the requirements for the degree of Master of Engineering in Environmental Engineering
    (Massey University, 2012) Güttler, Johanna
    Spare capacity found in many municipal primary sludge digesters could be used to improve the biogas production through the addition of other organic waste. This work investigates the potential of casein whey as an additional substrate. The amount of whey required for maximum biogas production and stable reactor performance was tested, along with the use of cow manure as an additional substrate to enhance reactor stability. Bench-scale continuously stirred tank reactors were operated at 38 °C with an initial hydraulic retention time of 20 days. Biogas production was recorded daily and compared to a control reactor. To assess reactor stability, pH, alkalinity, chemical oxygen demand (COD) and volatile fatty acid concentration were measured. To manage seasonal production, whey (W) was stored at ambient temperature prior to utilisation. This caused 74 % of the lactose to ferment to mainly L-lactate, accompanied by a pH drop from initially 4.5 to 3.6 and decreased COD. While fresh whey co-digested with primary sludge (PS) did not improve the biogas production, stored whey utilised at the ratio 10:3 (PS:W) improved the biogas production to 150 % of the control. Cow manure (CM) co-digested with primary sludge and fresh whey at the ratio 10:7:1 (PS:W:CM) improved the biogas production by up to 200 % after slow acclimatisation to the whey. The addition of cow manure to primary sludge and stored whey did not improve the biogas production beyond the 150 % achieved without cow manure. Investigation into why cow manure improved biogas production in primary sludge and whey co-digestion established that fungi found in cow manure could play an important role in the hydrolysis of complex material and therefore the biogas production. Improved biogas production from fresh whey was only achieved when cow manure was provided. It appeared that additional lactic acid bacteria supplied by cow manure was required to ferment the high lactose concentration in fresh whey. This work has shown how the seasonal availability of whey can be effectively used to improve the biogas production from municipal sludge digestion. During peak milk production fresh whey could be co-digested with primary sludge and cow manure at the ratio 10:5:1 (PS:W:CM) achieving 178 % biogas production. If cow manure is difficult to obtain, the ratio 10:3:0.1 is recommended, achieving 138 % biogas production. When the availability of fresh whey decreases, stored whey at the ratio 10:3 (PS:W) is recommended without cow manure, producing 150 % biogas compared to primary sludge alone. Utilising whey as a viable substrate would improve productivity of municipal sludge digesters as well as alleviating environmental issues associated with whey disposal.