Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.

“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

Investigation of lactic acid bacteria mediated bioprotection with applications in cereal industry. Case-study: malting process

Antifungal compounds produced by Lactic acid bacteria (LAB) metabolites can be natural and reliable alternative for reducing fungal infections pre- and post-harvest with a multitude of additional advantages for cereal-base products. Toxigenic and spoilage fungi are responsible for numerous diseases and economic losses. This thesis includes an overview of the impact fungi have on aspects of the cereal food chain. The applicability of LAB in plant protection and cereal industry is discussed in detail. Specific case studies include Fusarium head blight, and the impact of fungi in the malting and baking industry. The impact of Fusarium culmorum infected raw barley on the final malt quality was part of the investigation. In vitro infected barley grains were fully characterized. The study showed that the germinative energy of infected barley grains decreased by 45% and grains accumulated 199 μg.kg-1 of deoxynivalenol (DON). Barley grains were subsequently malted and fully characterized. Fungal biomass increased during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Infected malt grains revealed extreme structural changes due to proteolytic, (hemi)-cellulolytic and starch degrading activity of the fungi, this led to increased friability and fragmentation. Infected grains also had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt when compared to the control. The protein compositional changes and respective enzymatic activity of infected barley and respective malt were characterized using a wide range of methods. F. culmorum infected barley grains showed an increase in proteolytic activity and protein extractability. Several metabolic proteins decreased and increased at different rates during infection and malting, showing a complex F. culmorum infection interdependence. In vitro F. culmorum infected malt was used to produce lager beer to investigate changes caused by the fungi during the brewing processes and their effect on beer quality attributes. It was found, that the wort containing infected malt had a lower pH, a higher FAN, higher β-glucan and a 45% increase in the purging rate, and led to premature yeast flocculation. The beer produced with infected malt (IB) had also a significantly different amino acid profile. IB flavour characterization revealed a higher concentration of esters, fusel alcohols, fatty acids, ketones, and dimethylsulfide, and in particular, acetaldehyde, when compared to the control. IB had a greater proportion of Strecker aldehydes and Maillard products contributing to an increased beer staling character. IB resulted in a 67% darker colour with a trend to better foam stability. It was also found that 78% of the accumulated mycotoxin deoxynivalenol in the malt was transferred into beer. A LAB cell-freesupernatant (cfs), produced in wort-base substrate, was investigated for its ability to inhibit Fusarium growth during malting. Wort was a suitable substrate for LAB exhibiting antifungal activity. Lactobacillus amylovorus DSM19280 inhibited 104 spores.mL-1 for 7 days, after 120 h of fermentation, while Lactobacillus reuteri R29 inhibited 105 spores.mL-1 for 7 days, after 48 h of fermentation. Both LAB cfs had significant different organic acid profiles. Acid-base antifungal compounds were identified and, phenyllactic, hydroxy-phenyllactic, and benzoic acids were present in higher concentrations when compared to the control. A 3 °P wort substrate inoculated With L. reuteri R29 (cfs) was applied in malting and successfully inhibited Fusarium growth by 23%, and mycotoxin DON by 80%. Malt attributes resulted in highly modified grains, lower pH, higher colouration, and higher extract yield. The implementation of selected LAB producing antifungal compounds can be used successfully in the malting process to reduce mould growth and mycotoxin production.

Biomethane production from grass silage: laboratory assessment to maximise yields

On-farm biogas production is typically associated with forage maize as the biomass source. Digesters are designed and operated with the focus of optimising the conditions for this feedstock. Thus, such systems may not be ideally suited to the digestion of grass. Ireland has ca. 3.85 million ha of grassland. Annual excess grass, surplus to livestock requirements, could potentially fuel an anaerobic digestion industry. Biomethane associated with biomass from 1.1 % of grassland in Ireland, could potentially generate over 10 % renewable energy supply in transport. This study aims to identify and optimise technologies for the production of biomethane from grass silage. Mono-digestion of grass silage and co-digestion with slurry, as would occur on Irish farms, is investigated in laboratory trials. Grass silage was shown to have 7 times greater methane potential than dairy slurry on a fresh weight basis (107 m3 t-1 v 16 m3 t-1). However, comprehensive trace element profiles indicated that cobalt, iron and nickel are deficient in mono-digestion of grass silage at a high organic loading rate (OLR) of 4.0 kg VS m-3 d-1. The addition of a slurry co-substrate was beneficial due to its wealth of essential trace elements. To stimulate hydrolysis of high lignocellulose grass silage, particle size reduction (physical) and rumen fluid addition (biological) were investigated. In a continuous trial, digestion of grass silage of <1 cm particle size achieved a specific methane yield of 371 L CH4 kg-1 VS when coupled with rumen fluid addition. The concept of demand driven biogas was also examined in a two-phase digestion system (leaching with UASB). When demand for electricity is low it is recommended to disconnect the UASB from the system and recirculate rumen fluid to increase volatile fatty acid (VFA) and soluble chemical oxygen demand (SCOD) production whilst minimising volatile solids (VS) destruction. At times of high demand for electricity, connection of the UASB increases the destruction of volatiles and associated biogas production. The above experiments are intended to assess a range of biogas production options from grass silage with a specific focus on maximising methane yields and provide a guideline for feasible design and operation of on-farm digesters in Ireland.

Functional application of lactic acid bacteria exopolysaccharide in complex food systems

Lactic acid bacteria expolysaccharides (LAB-EPS), in particular those formed from sucrose have the potential to improve food and beverage rheology and enhance their sensory properties potentially replacing or reducing expensive hydrocolloids currently used as improvers in food and beverage industries. Addition of sucrose not only enables EPS formation but also affects organic acid formation, thus influencing the sensory properties of the resulting food/beverage products. The first part of the study the organoleptic modulation of barley malt derived wort fermented using in situ produced bacterial polysaccharides has been investigated. Weisella cibaria MG1 was capable to produce exopolysaccharides during sucrosesupplemented barley malt derived wort fermentation. Even though the strain dominated the (sucrose-supplemented) wort fermentation, it was found to produce EPS (14.4 g l-1) with lower efficiency than in SucMRS (34.6 g l-1). Higher maltose concentration in wort led to the increased formation of oligosaccharide (OS) at the expense of EPS. Additionally, small amounts of organic acids were formed and ethanol remained below 0.5% (v/v). W. cibaria MG1 fermented worts supplemented with 5 or 10% sucrose displayed a shear-thinning behaviour indicating the formation of polymers. This report showed how novel and nutritious LAB fermented wort-base beverage with prospects for further advancements can be formulated using tailored microbial cultures. In the next step, the impact of exopolysaccharide-producing Weissella cibaria MG1 on the ability to improve rheological properties of fermented plant-based milk substitute plant based soy and quinoa grain was evaluated. W. cibaria MG1 grew well in soy milk, exceeding a cell count of log 8 cfu/g within 6 h of fermentation. The presence of W. cibaria MG1 led to a decrease in gelation and fermentation time. EPS isolated from soy yoghurts supplemented with sucrose were higher in molecular weight (1.1 x 108 g/mol vs 6.6 x 107 g/mol), and resulted in reduced gel stiffness (190 ± 2.89 Pa vs 244 ± 15.9 Pa). Soy yoghurts showed typical biopolymer gels structure and the network structure changed to larger pores and less cross-linking in the presence of sucrose and increasing molecular weight of the EPS. In situ investigation of Weissella cibaria MG1 producing EPS on quinoa-based milk was performed. The production of quinoa milk, starting from wholemeal quinoa flour, was optimised to maximise EPS production. On doing that, enzymatic destructuration of protein and carbohydrate components of quinoa milk was successfully achieved applying alpha-amylase and proteases treatments. Fermented wholemeal quinoa milk using Weissella cibaria MG1 showed high viable cell counts (>109 cfu/mL), a pH of 5.16, and significantly higher water holding capacity (WHC, 100 %), viscosity (> 0. 5 Pa s) and exopolysaccharide (EPS) amount (40 mg/L) than the chemically acidified control. High EPS (dextran) concentration in quinoa milk caused earlier aggregation because more EPS occupy more space, and the chenopodin were forced to interact with each other. Direct observation of microstructure in fermented quinoa milk indicated that the network structures of EPS-protein could improve the texture of fermented quinoa milk. Overall, Weissella cibaria MG1 showed favorable technology properties and great potential for further possible application in the development of high viscosity fermented quinoa milk. The last part of the study investigate the ex-situ LAB-EPS (dextran) application compared to other hydrocolloids as a novel food ingredient to compensate for low protein in biscuit and wholemeal wheat flour. Three hydrocolloids, xanthan gum, dextran and hydroxypropyl methylcellulose, were incorporated into bread recipes based on high-protein flours, low-protein flours and coarse wholemeal flour. Hydrocolloid levels of 0–5 % (flour basis) were used in bread recipes to test the water absorption. The quality parameters of dough (farinograph, extensograph, rheofermentometre) and bread (specific volume, crumb structure and staling profile) were determined. Results showed that xanthan had negative impact on the dough and bread quality characteristics. HPMC and dextran generally improved dough and bread quality and showed dosage dependence. Volume of low-protein flour breads were significantly improved by incorporation of 0.5 % of the latter two hydrocolloids. However, dextran outperformed HPMC regarding initial bread hardness and staling shelf life regardless the flour applied in the formulation.

Reduction of salt in yeasted wheat bread: impact on bread quality and solutions using sourdough fermented by functional lactic acid bacteria strains

The dietary intake of sodium chloride has increased considerably over the last few decades due to changes in the human diet. This higher intake has been linked to a number of diseases including hypertension and other cardiovascular diseases. Numerous international health agencies, as well as the food industry, have now recommended a salt intake level of 5-6 g daily, approximately half of the average current daily intake level. Cereal products, and in particular bread, are a major source of salt in the Western diet. Therefore, any reduction in the level of salt in bread could have a major impact on global health. However, salt is a critical ingredient in bread production, and its reduction can have a deleterious effect on the production process as well as on the final bread quality characteristics such as shelf-life, bread volume and sensory characteristics, all deviating from the bakers’ and consumers’ expectations. This work addresses the feasibility of NaCl reduction in wheat bread focusing on options to compensate NaCl with the use of functional sourdoughs. Three strains were used for the application of low-salt bread; L. amylovorus DSM19280, W. cibaria MG1 and L. reuteri FF2hh2. The multifunctional strain L. reuteri FF2hh2 was tested the first time and its application could be demonstrated successfully. The functionalities were based on the production of exopolysaccharides as well as the production of antifungal compounds. While the exopolysaccharides, mainly high molecular dextrans, positively influenced mainly bread loaf volume, crumb structure and staling rate, the strains producing antifungal compounds prolonged the microbial shelf life significantly and compensated the lack of salt. The impact on the sensory characteristics of bread were evaluated by descriptive sensory evaluation. The increase in surface area as well as the presence of organic acids impacted significantly on the flavour profile of the sourdough bread samples. The flavour attribute “salt” could be enhanced by sourdough addition and increased the salty perception. Furthermore, a trained sensory panel evaluated for the first time the impact of yeast activity, based on different salt and yeast concentrations, on the volatile aroma profile of bread crumb samples. The analytical measurements using high resolution gas chromatography and proton-transfer-reaction mass spectrometry (PTR-MS) resulted in significantly different results based on different yeast activities. Nevertheless, the extent of the result could not be recognised by the sensory panel analysing the odour profile of the bread crumb samples. Hence, the consumer cannot recognised low-salt bread by its odour. The use of sourdough is a natural option to overcome the broad range of technological issues caused by salt reduction and also a more popular alternative compared to existing chemical salt replacers.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.