Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Predicting methane emission of dairy cows using milk composition
Gastelen, Sanne van - \ 2017
Wageningen University. Promotor(en): Wouter Hendriks, co-promotor(en): Jan Dijkstra; Kasper Hettinga. - Wageningen : Wageningen University - ISBN 9789463437097 - 266
dairy cows - dairy cattle - methane production - emission - milk composition - fatty acids - cattle feeding - fermentation - nutrition physiology - animal nutrition - pollution - melkkoeien - melkvee - methaanproductie - emissie - melksamenstelling - vetzuren - rundveevoeding - fermentatie - voedingsfysiologie - diervoeding - verontreiniging

Enteric methane (CH4) is produced as a result of microbial fermentation of feed components in the gastrointestinal tract of ruminant livestock. Methane has no nutritional value for the animal and is predominately released into the environment through eructation and breath. Therefore, CH4 not only represents a greenhouse gas contributing to global warming, but also an energy loss, making enteric CH4 production one of the main targets of greenhouse gas mitigation practices for the dairy industry. Obviously, reduction of CH4 emission could be achieved by simply reducing livestock numbers. However, the global demand for dairy products has been growing rapidly and is expected to further grow in the future. Therefore, it is critical to minimize environmental impact to produce high-quality dairy products. The overall aim of this PhD research was, therefore, to develop a proxy for CH4 emission that can be measured in milk of dairy cows.

There are currently a number of potentially effective dietary CH4 mitigation practices available for the livestock sector. The results of Chapter 3 show that replacing fiber-rich grass silage with starch-rich corn silage in a common forage-based diet for dairy cattle offers an effective strategy to decrease enteric CH4 production without negatively affecting dairy cow performance, although a critical level of starch in the diet seems to be needed. Little is known whether host genetics may influence the CH4 emission response to changes in diet. Therefore, the interaction between host DGAT1 K232A polymorphism with dietary linseed oil supplementation was evaluated in Chapter 7. The results of Chapter 7 indicate that DGAT1 K232A polymorphism is associated with changes in milk composition, milk N efficiency, and diet metabolizability, but does not affect digestibility and enteric CH4 emission, whereas linseed oil reduces CH4 emission independent of the DGAT1 K232A polymorphism.

Accurate and repeatable measurements of CH4 emission from individual dairy cows are required to assess the efficacy of possible mitigation strategies. There are several techniques to estimate or measure enteric CH4 production of dairy cows, including climate respiration chambers, but none of these techniques are suitable for large scale precise and accurate measurements. Therefore, the potential of various metabolites in milk, including milk fatty acids (MFA), as a proxy (i.e., indicators or animal traits that are correlated with enteric CH4 production) for CH4 emission of dairy cows gained interest. Until recently, gas chromatography was the principal method used to determine the MFA profile, but this technique is unsuitable for routine analysis. This has led to the application of Fourier-transform infrared spectroscopy (FTIR) for determination of the MFA profile. Chapter 2 provides an overview of the recent research that relates MFA with CH4 emission, and discusses the opportunities and limitations of using FTIR to estimate, indirectly via MFA or directly, CH4 emission of dairy cattle. The recent literature on the relationship between MFA and CH4 emission gives inconsistent results. Where some studies found a clear and strong relation, other studies consider MFA to be unreliable predictors for CH4 emitted by dairy cows. Even the studies that do find a clear relation between MFA and CH4 emissions do not describe similar prediction models using the same MFA. These discrepancies can be the result of many factors, including dietary composition and lactation stage. Additionally, literature showed that the major advantages of using FTIR to predict CH4 emission include its simplicity and potential practical application on a large scale. Disadvantages include the inability to predict important MFA for the prediction of CH4 emission, and the moderate power of FTIR to directly predict CH4 emission. The latter was also demonstrated in Chapter 9, in which the CH4 prediction potential of MFA was compared with that of FTIR using data from 9 experiments (n = 218 individual cow observations) covering a broad range of roughage-based diets. The results indicate that MFA have a greater potential than FTIR spectra to estimate CH4 emissions, and that both techniques have potential to predict CH4 emission of dairy cows, but also limited current applicability in practice. Much focus has been placed on the relationship between MFA and CH4 emission, but milk also contains other metabolites, such as volatile and non-volatile metabolites. Currently, milk volatile metabolites have been used for tracing animal feeding systems and milk non-volatile metabolites were shown to be related to the health status of cows. In Chapter 4, the relationship between CH4 emission and both volatile and non-volatile metabolites was investigated, using data and milk samples obtained in the study described in Chapter 3. In general, the non-volatile metabolites were more closely related to CH4 emissions than the volatile metabolites. More specifically, the results indicate that CH4 intensity (g/kg fat- and protein-corrected milk; FPCM) may be related to lactose synthesis and energy metabolism in the mammary gland, as reflected by the milk non-volatile metabolites uridine diphosphate-hexose B and citrate. Methane yield (g/kg dry matter intake) on the other hand, may be related to glucogenic nutrient supply, as reflected by the milk non-volatile acetone. Based on the metabolic interpretations of these relationships, it was hypothesized that the addition of both volatile and non-volatile metabolites in a prediction model with only MFA would enhance its predictive power and, thus, leads to a better proxy in milk for enteric CH4 production of dairy cows. This was investigated in Chapter 5, again using data and milk samples described in Chapter 3. The results indicate that MFA alone have moderate to good potential to estimate CH4 emission. Furthermore, including volatile metabolites (CH4 intensity only) and non-volatile metabolites increases the CH4 emission prediction potential.

The work presented in Chapters 3, 4 and 5, was based upon a small range of diets (i.e., four roughage-based diets in which grass silage was replaced partly or fully by corn silage) of one experiment. Therefore, in Chapter 6, the relationship between CH4 emission and the milk metabolome in dairy cattle was further quantified. Data (n = 123 individual cow observations) were used encompassing a large of roughage-based diets, with different qualities and proportions of grass, grass silage and corn silage. The results show that changes in individual milk metabolite concentrations can be related to the ruminal CH4 production pathways. These relationships are most likely the result from changes in dietary composition that affect not only enteric CH4 production, but also the profile of volatile and non-volatile metabolites in milk. Overall, the results indicate that both volatile and non-volatile metabolites in milk might provide useful information and increase our understanding of CH4 emission of dairy cows. However, the development of CH4 prediction models revealed that both volatile and non-volatile metabolites in milk hold little potential to predict CH4 emissions despite the significant relationships found between individual non-volatile metabolites and CH4 emissions. Additionally, combining MFA with milk volatile metabolites and non-volatile metabolites does not improve the CH4 prediction potential relative to MFA alone. Hence, it is concluded that it is not worthwhile to determine the volatile and non-volatile metabolites in milk in order to estimate CH4 emission of dairy cows.

Overall, in comparison with FTIR, volatile and non-volatile metabolites, the MFA are the most accurate and precise proxy in milk for CH4 emission of dairy cows. However, most of MFA-based models to predict CH4 emission tend to be accurate only for the production system and the environmental conditions under which they were developed. In Chapter 8 it was demonstrated that previously developed MFA-based prediction equations did not predict CH4 emission satisfactory of dairy cows with different DGAT1 genotypes or fed diets with or without linseed oil. Therefore, the greatest shortcoming today of MFA-based CH4 prediction models is their lack of robustness. Additionally, MFA have restricted practical application, meaning that most MFA retained in the current CH4 prediction models cannot be determined routinely because of the use of gas chromatography. The MFA that can be determined with the use of infrared spectroscopy are however no promising predictors for CH4 emission. Furthermore, MFA have only a moderate CH4 prediction potential. This together suggests that it might not be the best option to focus in the future on MFA alone as a proxy for CH4 emission of dairy cows.

The FTIR technique has a low to moderate CH4 prediction potential. However, FTIR has a great potential for practical high throughput application, facilitating repeated measurements of the same cow potentially reducing random noise. Results of this thesis also demonstrated that FTIR spectra do not have the potential to detect differences in CH4 emission between diets which are, in terms of forage level and quality, commonly fed in practice. Moreover, the robustness of FTIR spectra is currently unknown. Hence, it remains to be investigated whether FTIR spectra can predict CH4 emissions from dairy cows housed under different conditions from those under which the FTIR-based prediction equations were developed. It is therefore concluded that the accuracy and precision to predict CH4 emission using FTIR needs to increase, and the capacity of FTIR to evaluate the differences in CH4 emission between dairy cows and different types of diets needs to improve, in order to actually be a valuable proxy for CH4 emission of dairy cows.

Lactic acid fermentation of human excreta for agricultural application
Andreev, Nadejda - \ 2017
Wageningen University. Promotor(en): Piet Lens, co-promotor(en): B. Boincean; M. Ronteltap. - Leiden : CRC Press/Balkema - ISBN 9781138049895 - 207
lactic acid - manure fermentation - fermentation - fertilizers - human faeces - biochar - composting - melkzuur - mestvergisting - fermentatie - kunstmeststoffen - mensenfeces - compostering

Human excreta is a valuable fertilizer for improving soil quality and crop productivity, with a potential to replace or complement the mineral fertilizers. The main challenges related to human excreta regarding agricultural applications are microbial contamination risks, loss of nutrients, and odor issues. Fertilization by lacto-fermented faeces supplemented by biochar has benefits such as improved soil bulk density, nitrate and potassium concentrations as well as the yield and yield components of corn, compared to untreated, simple stored faeces, urine, cattle manure, and unfertilized controls. Even though the mineral fertilizer produced corn with significantly higher height and leaf length, it did not add significantly higher yields than lacto-fermented faeces supplemented by biochar. A faeces treatment process by combined lacto-fermentation with thermophilic composting and biochar supplementation had better reduction of coliforms, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, and higher germination of radish and growth of tomatoes than combined lacto-fermentation with vermicomposting. Urine lacto-fermentation contributed to a pH reduction below 4, a decrease in the ammonium concentration and odor strength, as well as an increase in the germination rates compared to untreated stored urine. The results of this study provide important information that can set the basis for scaling up a sustainable technology for the treatment of source separated human excreta while improving its potential for resource recovery.

Effect Molares® op biogas opbrengst bij co-vergisting
Durksz, Durk - \ 2017
Lelystad : ACRRES - Wageningen UR (Rapport / WPR 738) - 25
bio-energie - co-vergisting - biogas - fermentatie - installatieontwerp - voorbehandeling - malen - gasproductie - bioenergy - co-fermentation - fermentation - plant design - pretreatment - grinding - gas production
Microbial chain elongation based on methanol
Chen, Wei-Shan - \ 2017
Wageningen University. Promotor(en): Cees Buisman; Carolien Kroeze, co-promotor(en): David Strik. - Wageningen : Wageningen University - ISBN 9789463431989 - 201
feedstocks - renewable resources - organic wastes - waste utilization - fermentation - methanol - industriële grondstoffen - vervangbare hulpbronnen - organisch afval - afvalhergebruik - fermentatie

Our society relies heavily on fossil resources to fulfill our energy and commodity demands and this dependence has led to negative economic, environmental and societal consequences. The re-generation rate of fossil resources is much slower than their consumption rate, making these resources a non-renewable feedstock for the supply of energy and goods to our society. Moreover, the rapid consumption of fossil resources releases the carbon sequestrated in the last few million years in a much shorter time span, which contributes to the carbon dioxide (CO2) concentration increase in the atmosphere and potentially global warming. The geographically-uneven distribution of fossil resources also induces social insecurities and political conflicts. An alternative feedstock is necessary for energy and goods supply to our society, and such alternative feedstock should be renewable, economically sustainable, environmentally sound and geographically wide-spread,.

Organic waste is an emerging and promising alternative feedstock. The production of organic waste is inevitable, occurs in large quantities and is geographically wide-spread, especially the so-called “mixed organic waste,” e.g. organic fraction of municipal solid waste (OFMSW) and food processing waste. Mixed organic waste contains a large quantity of carbon materials that can be valorised into energy carriers and commodities. However, the extremely heterogeneous composition and the relatively high water content of mixed organic waste make its valorisation via the current waste management methods (e.g. incineration, composting and anaerobic digestion) less efficient and not economically attractive. Given this context, a novel bioprocess based on a mixed culture fermentation, i.e. microbial chain elongation, was developed to promote the valorisation of mixed organic waste. In microbial chain elongation, the diverse, complex organic matter in mixed organic waste are homogenised via hydrolysis and bacterial acidification into basic building blocks; like short chain fatty acids (SCFAs), CO2 and hydrogen (H2). After the homogenisation, energy-rich co-substrates like ethanol are added to these basic building blocks to synthesise medium chain fatty acids (MCFAs) via a mixed culture fermentation. MCFAs are organic compounds with a higher economic value and a higher energy content. Microbial chain elongation can be operated under a non-sterile condition, which makes it applicable to valorise mixed organic waste where diverse microorganisms exist. Caproate is the most dominant product in the microbial chain elongation of mixed organic waste and ethanol, which can be produced at a high rate and selectivity. Caproate has a higher economic value, a lower solubility in water and an interesting market potential. Thus, caproic acid production from mixed organic waste and ethanol via microbial chain elongation is currently undergoing up-scaling and commercialisation.

Many studies were done to improve the process of caproate production via microbial chain elongation to make it of industrial interest. The on-going commercialisation of microbial chain elongation also supports the economic feasibility. However, until now, no study addressed the environmental sustainability of microbial chain elongation. Chapter 2 of this thesis took the first attempt in analysing the life-cycle environmental impacts of caproic acid production from organic waste via microbial chain elongation, based on the literature and existing business case. The use of ethanol as a co-substrate (i.e. the electron donor) was shown to be the largest cause the environmental impact. This was found in in all assessed cases and all impact categories studied, and regardless of the feedstocks from which ethanol was produced. An alternative for ethanol as electron donor in microbial chain elongation is, therefore, an effective way to improve the environmental sustainability of microbial chain elongation.

In Chapter 3, we investigated the use of methanol as an alternative electron donor in microbial chain elongation, i.e. methanol chain elongation, for butyrate and caproate production. Methanol chain elongation was previously demonstrated using a pure culture, but never with a mixed culture. To employ organic waste as feedstock, the feasibility of applying methanol chain elongation in an open mixed culture condition needs to be investigated. In Chapter 3, it was demonstrated in a batch incubation that methanol chain elongation could occur with a mixed culture, where butyrate was the dominant product (4.2 g/L). Caproate production via methanol chain elongation was also demonstrated, though only in a low concentration (0.1 g/L). In a continuous reactor operation, continuous butyrate production (1.5 g/ was achieved via microbial chain elongation of acetate and methanol. However, caproate was not observed in the continuous methanol chain elongation. Interestingly, microorganisms that can perform methanol chain elongation were likely present in the inoculum taken from a previous ethanol chain elongation reactor without any methanol supplement.

In Chapter 4, the use of methanol chain elongation to synthesise a novel product, i.e. isobutyrate, was proposed and investigated. Methanol chain elongation was found to continuously produce butyrate as the main metabolite, the accumulation of which was found to trigger isobutyrate formation in several previous methanogenic anaerobic digestion studies. It was, therefore, hypothesised that by elevating the butyrate concentration in the medium, methanol chain elongation might be able to produce isobutyrate as another metabolite. The result showed that isobutyrate could be produced as the main product, up to 6.2 g/L, when using acidified supermarket food waste and methanol as the substrate. A continuous methanol chain elongation using synthetic medium was also performed, which achieved a production rate of 2.0 g/ over five hydraulic retention times. Moreover, the production of isovalerate was also observed. Isobutyrate has a much larger market potential than caproate, though its production relies wholly on fossil-based feedstock. Isobutyrate biosynthesis was demonstrated in previous studies, but was only achieved using metabolically engineered microorganisms as the biocatalyst and glucose as the substrate. Methanol chain elongation, in contrast, could employ derivatives from organic waste as the substrates and a self-regenerating mixed culture biocatalyst for producing isobutyrate. Moreover, methanol chain elongation may be integrated into the current microbial chain elongation production facility without a significant infrastructure retrofit. All these advantages make methanol chain elongation an interesting and promising isobutyrate production process. The relatively large market potential of isobutyrate promotes the application of chain elongation and the use of organic waste for value-added chemical production.

In Chapter 5, isobutyrate production was integrated with the caproate production via microbial chain elongation, by concurrently feeding both methanol and ethanol to a mixed culture. The result from Chapter 3 supports the possibility of coexistence of ethanol and methanol chain elongation microorganisms in the same microbiome. In Chapter 4, the possible concurrence of methanol and ethanol chain elongation was also observed. Based on these observations, we hypothesised that methanol and ethanol chain elongation could be integrated to simultaneously produce caproate and isobutyrate. The result showed that such integration was possible when a stable pH was maintained. When pH was controlled between 6.2 – 6.5 and butyrate was supplied in the medium, caproate and isobutyrate could be produced simultaneously. Additionally, increasing the ethanol feeding rate promoted the chain elongation of butyrate to caproate via ethanol chain elongation. The outcome of this chapter demonstrated the possibility of producing two valuable products in a single reactor with a mixed culture which, coupled with further process improvement, may be of industrial interest.

In Chapter 6, we reflected on the caproate production performance of methanol chain elongation, in comparison with other electron donors used in microbial chain elongation, i.e. ethanol and lactate. Furthermore, we also reflected on the isobutyrate production via methanol chain elongation, in comparison with other emerging products in microbial chain elongation. These reflections could serve as a benchmark for methanol chain elongation as a waste management strategy. Based on this benchmarking, we proposed that methanol chain elongation is a promising bioprocess for isobutyrate production but not for caproate production. A potential strategy for improving the isobutyrate production via methanol chain elongation was proposed and discussed. The outcomes of this thesis may contribute to future application and assessments of microbial chain elongation in waste management. It may fuel discussion on how to further promote microbial chain elongation for a more sustainable waste management.

Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding
Vissers, Anne M. - \ 2017
Wageningen University. Promotor(en): Harry Gruppen; Wouter Hendriks, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789463432023 - 154
phenols - leaves - seaweeds - tannins - beta vulgaris - laminaria - proteins - catechol oxidase - nuclear magnetic resonance spectroscopy - in vitro - mass spectrometry - browning - fermentation - animal feeding - fenolen - bladeren - zeewieren - tanninen - eiwitten - kernmagnetische resonantiespectroscopie - massaspectrometrie - bruinkleuring - fermentatie - diervoedering

Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation.

Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties.
High molecular weight PhT from L. digitata could potentially modify protein properties by non‑covalent interactions. L. digitata contained PhT with subunits mainly connected via C‑O-C linkages, as determined using NMR spectroscopy. Further mass spectrometric analysis revealed the presence of a wide range of oligomers with degrees of polymerisation between 3 and 27. The interaction between PhT and proteins (b-casein and bovine serum albumin) was studied using model systems with different pH values, representing the various environments throughout the ruminants digestive tract. Phlorotannins bound to protein independent of pH, and broadened the pH range of protein precipitation from 0.5 to ~1.5 pH unit around the protein’s pI. At the pH of the abomasum of 2-3, the proteins re-solubilised again, presumably by increase in their net charge. Due to their ability to form water insoluble complexes, PhT could improve ruminal fermentation in vitro in a dose dependent manner, resulting in lower methane production and ammonia (NH3) concentration. The decreased NH3 concentration reflected decreased dietary protein breakdown in the rumen, which is considered a nutritional and environmental benefit.

Optimal use of biogas from waste streams : an assessment of the potential of biogas from digestion in the EU beyond 2020
Kampman, Bettina ; Leguijt, Cor ; Scholten, Thijs ; Tallat-Kelpsaite, Jurga ; Brückmann, Robert ; Maroulis, Georgios ; Lesschen, Jan Peter ; Meesters, Koen ; Sikirica, Natasa ; Elbersen, Berien - \ 2017
Luxembourg : European Commission - 158
biogas - gas production - fermentation - assessment - production possibilities - residual streams - european union - biobased economy - gasproductie - fermentatie - beoordeling - productiemogelijkheden - reststromen - europese unie
As the European Commission is working on the further development and concretisation of the post-2020 climate and energy policies, this study was commissioned to zoom in on the potential role, cost and benefits of biogas, and to assess the key barriers and drivers of biogas deployment in the EU. An important question to address was what policies at both EU and Member State level can best contribute to the effective and efficient growth of biogas deployment in the EU. The study focussed on biogas production by digestion processes of local waste streams, i.e. on biogas production from sewage sludge, landfill gas and from suitable organic waste streams from agriculture, the food industry and households.
Biogas production and digestate utilisation from agricultural residues : deliverable nº: 6.2.1
Corre, W.J. ; Conijn, J.G. - \ 2016
HYSOL project - 39 p.
renewable energy - anaerobic digestion - biogas - crop residues - agricultural wastes - sustainable energy - electricity supplies - innovations - biobased economy - fermentation - digestate - hernieuwbare energie - anaërobe afbraak - oogstresten - agrarische afvalstoffen - duurzame energie - elektriciteitsvoorzieningen - innovaties - fermentatie - digestaat
The HYSOL project aims at hybridisation of concentrated solar power with a gas turbine in order to guarantee a stable and reliable electricity supply, based on renewable energy. The production of fully renewable electricity in a Hybrid Concentrated Solar Power (HCSP) plant includes the use of renewable gas. In task 6.2 of the HYSOL project research into the possibilities of sustainable biogas production from agricultural residues by anaerobic digestion has been performed. In this report results are described of part of this research focussing on potential biogas production and digestate production and utilisation from animal manure and crop residues.
Anaerobic digestion of cellulose and hemicellulose in the presence of humic acids
Azman, Samet - \ 2016
Wageningen University. Promotor(en): Fons Stams; Grietje Zeeman, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462579613 - 189
humic acids - hydrolysis - anaerobic digestion - cellulose - hemicelluloses - biomass - renewable energy - energy recovery - biogas - fermentation - bioprocess engineering - humuszuren - hydrolyse - anaërobe afbraak - hemicellulosen - biomassa - hernieuwbare energie - energieterugwinning - fermentatie - bioproceskunde

Research on the hydrolysis step of the AD became more important with the increased use of recalcitrant waste products such as manure, sewage sludge and agricultural biomass for biogas production. Hydrolysis is often the rate limiting step of the overall AD. Hydrolysis enhancement is one of the required steps to optimise biogas production. Despite the progress to overcome the limitations of hydrolysis, inhibition of hydrolysis is still poorly researched. Humic acid-like molecules (HA) are one of the inhibitors of the anaerobic hydrolysis and their effect on the overall AD process is generally overlooked.

In this thesis, the HA inhibition on anaerobic digestion of cellulosic material and mitigation strategies, using cation and enzyme addition, to overcome the inhibition were investigated. In addition, the microbial community dynamics during AD in the presence and absence of HA were examined. In this scope, in Chapter 2, we reviewed the literature and pinpointed the urgent need for comprehensive studies on the role of hydrolytic microorganisms and environmental factors that effects their abundance within biogas plants. Consequently, the hydrolysis mechanism and involved hydrolytic enzymes were discussed. The overall discussion showed that a holistic approach, including microbiological and engineering studies should be chosen to disclose the role of hydrolytic microbes within biogas reactors. In Chapter 3 and, Chapter 4 the effect of HA on anaerobic cellulose hydrolysis and methanogenesis, in batch wise incubations is reported, respectively. Our results showed that pulse addition of 5 g L-1 HA caused a 50 % decrease in hydrolysis rate of anaerobic cellulose degradation (Chapter 3). Moreover, VFA accumulation was observed in the presence of HA during the anaerobic cellulose degradation, which indicated the possible inhibition of HA on methanogenesis. Based on the results of Chapter 3, pure cultures of methanogens and a mixed culture were tested to study the vulnerability of methanogenesis to HA inhibition. Hydrogenotrophic methanogenesis in pure cultures was inhibited by more than 75% in the presence of 1 g L-1 HA whereas, acetoclastic methanogenesis by Methanosaeta concilii was only slightly affected by HA up to 3 g L-1. When methanogenic granular sludge was incubated with HA, the specific methanogenic activity tests showed less inhibition, when compared to the pure cultures of methanogens. HA inhibition was also observed during long-term CSTR operation at an HRT of 20 days, 35°C and a mixture of cellulose and xylan as a subtrate (Chapter 6). 8 g L-1 HA inhibited the hydrolysis efficiency of the cellulose and xylan digestion by 40 % and concomitantly reduced the methane yields.

Mitigation of the HA inhibition is required to increase the hydrolysis efficiency and methane yields of cellulosic biomass digestion. Therefore, two different strategies were tested for their potential use as mitigation agents, viz. addition of cations such as, calcium magnesium and iron (Chapter 3 and Chapter 6) and addition of hydrolytic enzymes (Chapter 6). Addition of magnesium, calcium and iron salts mitigated the HA inhibition and hydrolysis efficiencies reached up to 75, 65 and 72%, respectively, compared to the control groups in the batch wise incubations (Chapter 3). However, in long term CSTR operations, calcium addition did not show a positive effect on hydrolysis inhibition. On the other hand, enzyme addition helped to reverse the negative effect of HA.

The microbial communities involved in AD were also studied. Chapter 5 and Chapter 6 dealt with microbial community analyses with 16S rRNA next generation sequencing. In Chapter 5, five replicate reactors were monitored during the start-up period. Transient feeding strategy was used to acclimatise anaerobic sludge to efficient cellulose and xylan degradation. During the experiment, Bacteriodales, Clostridiales and Anaerolineales became dominant bacterial populations while, Methanobacteriaceae and Methanospirillaceae were the dominant archaeal populations within the reactors. In Chapter 6, the microbial population dynamics in the presence and absence of HA were monitored. Microbiological analyses showed that the abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA.

In conclusion, this thesis confirms that HA inhibit the hydrolysis and methanogenesis within both batch incubations and CSTR systems. Microbial populations were also affected by HA. Therefore, hydrolytic enzyme addition can be an option to mitigate HA inhibition and enhance hydrolysis and methanogenesis during conversion of biomass to biogas.

Controlling the self-assembly of protein polymers via heterodimer-forming modules
Domeradzka, Natalia Eliza - \ 2016
Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - massaspectrometrie - microscopie - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie

Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.

The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.

In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.

Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.

A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.

Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.

In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.

Methodology for estimating emissions from agriculture in the Netherlands. : Calculations of CH4, NH3, N2O, NOx, PM10, PM2.5 and CO2 with the National Emission Model for Agriculture (NEMA)
Vonk, J. ; Bannink, A. ; Bruggen, C. van; Groenestein, C.M. ; Huijsmans, J.F.M. ; Kolk, J.W.H. van der; Luesink, H.H. ; Oude Voshaar, S.V. ; Sluis, S.M. ; Velthof, G.L. - \ 2016
Wageningen : Statutory Research Tasks Unit for Nature & the Environment (WOt-technical report 53) - 164 p.
air pollutants, greenhouse gases, livestock, crops, animal housing, manure storage, manure application, inorganic fertilizer, enteric fermentation, manure management, agricultural soils, liming, NIR, CRF, IIR, NFR - landbouw - gewassen - landbouwgronden - vee - huisvesting, dieren - dierlijke meststoffen - rundveemest - mestverwerking - begrazing - broeikasgassen - luchtverontreinigende stoffen - emissie - ammoniakemissie - kooldioxide - methaan - anorganische meststoffen - fermentatie - bekalking - nederland - compost - rioolslib - teelt - oogstresten - rijp worden - agriculture - crops - agricultural soils - livestock - animal housing - animal manures - cattle manure - manure treatment - grazing - greenhouse gases - air pollutants - emission - ammonia emission - carbon dioxide - methane - inorganic fertilizers - fermentation - liming - netherlands - composts - sewage sludge - cultivation - crop residues - ripening
The National Emission Model for Agriculture (NEMA) is used to calculate emissions to air from agricultural activities in the Netherlands on a national scale. Emissions of ammonia (NH3) and other N-compounds (NOx and N2O) from animal housing, manure storage, manure application and grazing are assessed using a Total Ammoniacal Nitrogen (TAN) flow model. Furthermore, emissions from application of inorganic N-fertilizer, compost and sewage sludge, cultivation of organic soils, crop residues, and ripening of crops are calculated. NEMA is also used to estimate emissions of methane (CH4) from enteric fermentation and manure management, particulate matter (PM) from manure management and agricultural soils, and carbon dioxide
(CO2) from liming. Emissions are calculated in accordance with international guidance criteria and reported in an annual Informative Inventory Report (IIR; for air pollutants) and National Inventory Report (NIR; for greenhouse gases). This methodology report describes the outline and backgrounds of the emission
calculations with NEMA
In vitro fermentation and immunomodulating characteristics of dietary fibres
Rösch, C. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462577954 - 130 p.
dietary fibres - degradation - enzymes - immunomodulatory properties - cytokines - glycosides - fermentation - voedingsvezels - degradatie - enzymen - immunomodulerende eigenschappen - cytokinen - glycosiden - fermentatie


Dietary fibres are a diverse group of substances, indigestible by human digestive enzymes, but (partially) fermentable in the human large intestine by the resident microbiota. Many health beneficial effects of fibres such as lowering blood cholesterol levels or increasing stool bulk have been reported. For some fibres, immunomodulating properties have been shown. Other studies investigate the degradation fate of fibres by the bacteria. In this PhD thesis BMDCs from TLR2/4 knock out mice were validated to be unresponsive to naturally present contaminants like LPS and proved to be a good tool to analyse the immune response of dietary fibres. A variety of 44 fibres, was tested on these immune cells and all fibres were found to modulated the immune system differently. Also, different immunomodulating properties of an oat and barley β-glucan having rather similar chemical structures, were found. The insoluble fraction of the β-glucans induced highest amounts of cytokines. As a consequence, sample preparation such as drying, dispersing and heating were shown to affect the immunomodulatory properties. The in vitro fermentation characteristics of barley β-glucan and sugar beet pectin and the immunomodulatory properties of their degradation products on BMDCs were compared and shown to be substrate and degradation product specific. This study showed, that glycosidic degradation products of both fibres induced higher amounts of cytokines than their intact polysaccharide. An in vitro batch fermentation of soluble, indigestible maltodextrins by human faecal inocula was monitored and the activity of carbohydrate degrading enzymes, produced by the microbiota, was analysed. Results revealed that the maltodextrin was only slowly and incompletely fermented, despite the high potential of microbial enzymes present to degrade typical starch linkages.

Overall, this thesis showed that dietary fibres interact and influence the immune system dependent on their individual chemical fine structure. Additionally, an evaluation of the health impact of dietary fibres can only be complete when also glycosidic fermentation products are considered.

Development of a lactic acid production process using lignocellulosic biomass as feedstock
Pol, E.C. van der - \ 2016
Wageningen University. Promotor(en): Gerrit Eggink, co-promotor(en): Ruud Weusthuis. - Wageningen : s.n. - ISBN 9789462576735 - 167 p.
lignocellulose - biomass - biobased chemicals - sugarcane bagasse - lactic acid - sugars - byproducts - inhibitors - pretreatment - bacillus coagulans - furfural - saccharification - fermentation - quantitative techniques - production processes - biomassa - chemicaliën uit biologische grondstoffen - suikerrietbagasse - melkzuur - suikers - bijproducten - remmers - voorbehandeling - versuikering - fermentatie - kwantitatieve technieken - productieprocessen

The availability of crude oil is finite. Therefore, an alternative feedstock has to be found for the production of fuels and plastics. Lignocellulose is such an alternative feedstock. It is present in large quantities in agricultural waste material such as sugarcane bagasse.

In this PhD thesis, lignocellulose is chemically and enzymatically pretreated to depolymerise sugars present in this structure. The released sugar monomers are fermented by micro-organisms to lactic acid, which is a precursor for the bioplastic PLA. In this thesis, it is shown that it is possible to produce lactic acid from lignocellulosic biomass with high yields and high productivities.

Structure and fermentation of natural and manufactured lactose-based oligosaccharides
Difilippo, E. - \ 2016
Wageningen University. Promotor(en): Harry Gruppen; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576155 - 128 p.
milks - lactose - oligosaccharides - ingestion - bioactive compounds - isolation - characterization - fermentation - colostrum - food analysis - melksoorten - oligosacchariden - inname - bioactieve verbindingen - isolatie - karakterisering - fermentatie - voedselanalyse

At early stages of life, infant immature intestine is not fully developed, exposing the new-born to potential diseases. Compounds that can exert beneficial actions on the infant intestine are bioactive lactose-based oligosaccharides (LBOs). The natural source of LBOs is mother milk. When human milk is lacking, dietary supplementation with infant formula fortified with manufactured LBOs, such as galacto-oligosaccharides (GOS), is pursued. GOS have been shown to have several properties in common with HMOs. LBOs composition and intestinal fate is extensively described for humans, whereas they are hardly investigated for domestic animal. In this PhD thesis, composition of LBOs in equine and porcine colostrum were described and new structures were elucidated. The analysis were performed mainly using liquid chromatography and capillary electrophoresis techniques. High inter- and intra-individual variation were found for oligosaccharides present in equine and porcine milk. In vivo fermentation fate of porcine milk oligosaccharides (PMOs) was also described analysing PMOs as found in fecal samples of piglets. The results were correlated to existing literature on HMOs. Dietary oligosaccharides are partially present systemically, as suggested from HMO studies. GOS and PMOs in blood, urine and fecal samples from an in vivo feeding trial on piglet were described. Intact dietary oligosaccharides including GOS and milk oligosaccharides from the piglet diet were found in piglet blood and urine samples. All dietary oligosaccharides were fermented/absorbed in vivo, not being detectable in the piglet fecal samples. On the other hand, GOS in vitro fermentation by piglet inoculum delineate a unique fermentation profile regarding GOS size consumption compared to GOS in vitro fermentation by human fecal inoculum. Similar degradation profile regarding GOS linkage types was observed for GOS fermentation by piglet and human inocula.

Metabolic engineering of Escherichia coli for itaconate production
Vuoristo, K.S. - \ 2016
Wageningen University. Promotor(en): Gerrit Eggink; Johan Sanders, co-promotor(en): Ruud Weusthuis. - Wageningen : Wageningen University - ISBN 9789462576001 - 162 p.
fermentation - escherichia coli - aspergillus niger - biobased chemistry - bioengineering - acids - organic acids - glutamates - tca - production - chemicals - fermentatie - chemie op basis van biologische grondstoffen - zuren - organische zuren - glutamaten - productie - chemicaliën

Interest in sustainable development together with limited amounts of fossil resources have increased the demand for production of chemicals and fuels from renewable resources. The market potential for bio-based products is growing and a transition from petrochemicals to biomass-based chemicals is ongoing. Itaconic acid is a C5-dicarboxylic acid which can be produced by microbial conversion processes. It can be easily polymerized and is an appealing building block for the chemical industry with many potential applications. However, biobased chemicals have to compete with their petrochemical counterparts, and yield and productivity of the microbial processes are therefore of the utmost importance. Traditionally itaconic acid is produced using the ascomycete Aspergillus terreus. This process is not competitive with petrochemical processes due to high production costs caused by low yields, and difficult and expensive product recovery. Maximizing product yield is important to lower production costs. This thesis looked at ways to reach theoretical maximum yield in a recombinant production host, Escherichia coli.

Chapter 2 describes the construction of an itaconate biosynthesis pathway in E. coli. The key enzyme of microbial itaconate production is cis-Aconitate decarboxylase (CadA) that converts the citric acid cycle intermediate cis-aconitate into itaconate. We focused on optimizing heterologous expression of cadA from Aspergillus terreus in E. coli. Initially this resulted in low CadA activities and production of trace amounts of itaconate. CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium and this resulted in enhanced itaconate titres. The itaconate titre was further increased in aerobic bioreactor cultures by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting genes encoding phosphate acetyltransferase and lactate dehydrogenase. The maximum itaconate yield from glucose obtained in this study was only 0.09 mol/mol, due to high flux of carbon to by-products such as acetate and pyruvate. Pyruvate is a precursor molecule for itaconate biosynthesis and its accumulation suggested that the activity of CadA might be one of the rate limiting steps. It was concluded that further optimization of cadA expression, and reduction of acetate formation should be achieved to obtain higher itaconate yield.

As sufficient cis-aconitate decarboxylase activity is crucial for itaconate production, in chapter 3 ways to increase the activity of CadA were investigated. A recently characterized cis-aconitate decarboxylase of mammalian origin was therefore expressed in E.coli. The novel cis-aconitate decarboxylase from Mus musculus encoded by immunoresponsive gene 1 (irg1) produced comparable amounts of itaconate as CadA from A. terreus. In addition, the effects of codon optimization and harmonization on enzymatic activities of heterologously expressed cadA and irg1 were studied. Codon harmonization increased the activity of CadA in cell free extracts, but this did not result in higher itaconate production in bioreactor cultures. This suggests that other factors such as itaconate transport may limit the production.

In chapter 4, proof of principle for an anaerobic fermentation process for the production of itaconic acid was obtained by using the mixed acid fermentation pathway of E. coli. Itaconic acid production was redox balanced by co-producing succinate or ethanol with H2 and CO2. Expression of cadA together with citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum resulted in 0.66 mM (1.2 % Cmol) itaconate under anaerobic conditions. Unexpectedly, strains started to produce significant amounts of glutamate when the itaconate pathway was introduced. As glutamate production depends on the availability of nitrogen in the medium, a nitrogen-limited medium was tested to diminish glutamate production. This enhanced the production of itaconate to up to 2.9 mM (5.4 % C mol %). Here, anaerobic production of itaconate from glucose was reported for the first time. The observed itaconate yields and productivities were still modest. Eliminating the pathways to major by-products like glutamate, succinate, and acetate, and enhancing the pathway between pyruvate and itaconate is crucial to obtain a cost-competitive anaerobic itaconic acid process production.

To investigate how itaconate production can be improved, the insights from the previous chapters together with existing scientific literature were combined with our pathway design proposals in chapter 5. The tricarboxylic acid (TCA) cycle is an important source of precursors for biobased chemicals. The opinion article takes a closer look at the metabolic engineering of TCA cycle for the production of chemicals high yield. For most TCA cycle products the maximum pathway yield is much lower than the theoretical maximum yield. For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt. A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved: the conversion of acetyl-CoA and glyoxylate to malate in the glyoxylate shunt and the conversion of 2-oxoglutarate into succinyl-CoA in the TCA cycle. This way, the pathway yield for products originating from the oxidative TCA cycle branch such as citrate, itaconate and L-glutamate becomes identical to the theoretical maximum. Future research should focus on implementing these solutions in suitable production hosts, and increasing the ATP yield of the production pathways. This will minimize the oxygen requirement of the process, or even allow for anaerobic operation, and should lead to reduced operational costs and maximal product yields.

In chapter 6 the implications of the overall results of this thesis for the current research status of itaconate production are presented. Solutions to optimize itaconate production strains and production process were proposed.

Fermented foods : Products of science and craftsmanship
Smid, E.J. - \ 2015
Wageningen : Wageningen University, Wageningen UR - ISBN 9789462573710 - 20 p.
fermentation - microorganisms - food microbiology - fermented foods - fermentation products - public speeches - fermentatie - micro-organismen - voedselmicrobiologie - gefermenteerde voedingsmiddelen - fermentatieproducten - openbare redes
Inaugural lecture upon taking up the post of Personal Professor of Food Microbiology at Wageningen University on 11 June 2015. 'Microbial cultures deliver the typical characteristics of fermented food products through their metabolic activity, by secretion of enzymes in the food matrix and by their particular growth and survival behaviour. Knowledge of the structural complexity and population dynamics provides explanations for compositional stability and overall performance of such cultures. Especially, insight into microbial interactions delivers new design rules for robust composite starter cultures with predictable and desirable industrial performance.'
Replacing lactose from calf milk replacers : effects on digestion and post-absorptive metabolism
Gilbert, M.S. - \ 2015
Wageningen University. Promotor(en): Wouter Hendriks, co-promotor(en): Walter Gerrits; Henk Schols. - Wageningen : Wageningen University - ISBN 9789462576032 - 171
vleeskalveren - lactose - kunstmelk - polysacchariden - glucose - fructose - glycerol - zetmeelvertering - metabolisme - fermentatie - kalvervoeding - diervoeding - voedingsfysiologie - veal calves - filled milk - polysaccharides - starch digestion - metabolism - fermentation - calf feeding - animal nutrition - nutrition physiology

Summary PhD thesis Myrthe S. Gilbert

Replacing lactose from calf milk replacers – Effects on digestion and post-absorptive metabolism

Veal calves are fed milk replacer (MR) and solid feed. The largest part of the energy provided to veal calves originates from the MR. Calf MR contains 40 to 50% lactose, originating from whey, a by-product from cheese production. High and strongly fluctuating dairy prices are a major economic incentive to replace lactose from the calf MR by alternative energy sources. The objective of this thesis was to study the effects of replacing lactose from calf MR on nutrient digestion and fermentation and post-absorptive metabolism.

In Chapter 2 and 3, four starch products (SP) were evaluated for replacing lactose. The four SP differed in size and branching, and consequently required different ratios of starch-degrading enzymes for their complete hydrolysis to glucose. Gelatinized starch required α-amylase and (iso)maltase; maltodextrin required (iso)maltase and α-amylase; maltodextrin with α-1,6-branching required isomaltase, maltase and α-amylase and maltose required maltase. In Chapter 2, adaptation to these SP was assessed during 14 weeks, using a within-animal titration study. Forty male Holstein-Friesian calves (n = 8 per treatment) were assigned to either a lactose control MR or one of four titration strategies, each testing the stepwise exchange of lactose for one of the SP. For control calves, fecal dry matter (DM) content and fecal pH did not change over time. The response in fecal DM content and fecal pH in time did not differ between SP treatments and decreased linearly with 0.57% and 0.32 per week, respectively, where one week corresponded to an increase in SP inclusion of 3%. This indicates that the capacity for starch digestion was already exceeded at low inclusion levels, resulting in SP fermentation. All SP required maltase to achieve complete hydrolysis to glucose and it was, therefore, suggested that maltase is the rate-limiting enzyme in starch digestion in milk-fed calves.

Following the titration, a fixed inclusion level of 18% of the SP in the MR was applied. Effects on starch-degrading enzyme activity, nutrient disappearance, SP fermentation and jugular glucose appearance were measured (Chapter 3). Lactase activity in the brush border was high in the proximal small intestine of all calves, resulting in a high apparent ileal disappearance of lactose (≥ 99% of intake). Maltase and isomaltase activities in the brush border were not increased for any of the SP treatments. Luminal α-amylase activity was lower in the proximal small intestine but greater in the distal small intestine of SP-fed calves compared to control calves. This amylase activity in the distal small intestine of SP-fed calves might have been of microbial origin. Apparent SP disappearance did not differ between SP treatments. The difference between apparent ileal (62%) and total tract (99%) SP disappearance indicated substantial SP fermentation in the large intestine (37% of intake). In addition, total tract SP fermentation was quantified using fecal 13C excretion which originated from the naturally 13C-enriched corn SP. Total tract SP fermentation averaged 89% of intake, regardless of SP treatment. MR leaking into the reticulorumen was measured as the recovery of Cr in the reticulorumen at slaughter after feeding MR pulse-dosed with Cr 4h prior to slaughter. MR leaking into the reticulorumen averaged 11% for SP-fed calves. By difference, this leaves 41% of the SP intake fermented in the small intestine. This coincided with increased fecal nitrogen (N) and DM losses for SP-fed calves. However, apparent total tract crude fat disappearance tended to increase when replacing lactose with SP. The substantial SP fermentation indicates that only 10% of the SP intake was enzymatically hydrolyzed and absorbed as glucose. This was in agreement with the marginal increase in 13C enrichment in peripheral plasma glucose after feeding naturally 13C-enriched gelatinized starch and maltose, compared to a clear increase after feeding naturally 13C-enriched lactose to control calves. It was concluded that fermentation, rather than enzymatic digestion, is the main reason for small intestinal starch disappearance in milk-fed calves. The expected decrease in growth performance with such extensive SP fermentation is partially compensated by the greater crude fat digestion and possibly by a reduced urinary glucose excretion when replacing lactose with SP.

Glucose, fructose and glycerol do not require enzymatic hydrolysis and can be absorbed directly from the small intestine. However, these lactose replacers might differentially affect glucose and insulin metabolism and with that energy partitioning. The effects of partly replacing lactose with glucose, fructose or glycerol on energy and N partitioning and glucose homeostasis and insulin sensitivity were, therefore, studied in Chapter 4 and 5. Forty male Holstein-Friesian calves either received a lactose control MR or a MR in which one third of the lactose was replaced with glucose, fructose or glycerol (n = 10 per treatment). Energy and N retention were not affected by MR composition. Fructose absorption from the small intestine was incomplete resulting in fructose fermentation. This resulted in fecal losses of DM, energy and N and the lowest numerical energy and N retention for fructose-fed calves. Postprandial plasma concentrations of glucose exceeded the renal threshold for glucose in glucose-fed calves and control calves, which resulted in urinary glucose excretion. Glycerol was likely excreted with the urine of glycerol-fed calves. Oxidation of glucose, fructose and glycerol was quantified by feeding a single dose of [U-13C]glucose, [U-13C]fructose or [U-13C]glycerol with the MR and subsequently measuring 13CO2 production. Oxidation of lactose replacers did not differ between lactose replacers and averaged 72% of intake. However, the time at which the maximum rate of oxidation was reached was delayed for fructose-fed compared to glucose-fed and glycerol-fed calves, indicating that fructose was converted into other substrates before being oxidized. Conversion of fructose and glycerol into glucose was confirmed by an increase in 13C enrichment of peripheral plasma glucose after feeding [U-13C]fructose and [U-13C]glycerol, respectively. Insulin sensitivity did not differ between MR treatments, but was already low at the start of the experiment at 15 weeks of age and remained low throughout the experiment. It was concluded that glucose and glycerol can replace one third of the lactose from the calf MR, but that inclusion of fructose should be lower to prevent incomplete absorption from the small intestine.

In literature and the studies in this thesis, high inter-individual variation in growth performance was found in veal calves. The experiment described in Chapter 6 was, therefore, designed to assess the predictability of later life growth performance by charactering calves in early life. In addition, it was examined whether the ability of calves to cope with MR in which lactose is partially replaced by alternative energy sources can be predicted. From 2 to 11 weeks of age, male Holstein-Friesian calves were fed a lactose control MR and solid feed according to a practical feeding scheme and were characterized individually using targeted challenges related to feeding motivation, digestion, post-absorptive metabolism, immunology, behavior and stress. Based on the results in Chapter 4, a combination of glucose, fructose and glycerol in a 2:1:2 ratio was used to replace half of the lactose from the MR (GFG). From 11 to 27 weeks of age, calves received a lactose control MR or the GFG MR (n = 65 per treatment). Growth performance from 11 to 27 weeks of age tended to be lower for GFG-fed than for control calves (-25 g/d). Measurements in early life explained 12% of the variation in growth performance in later life. However, this was mainly related to variation in solid feed refusals. When growth performance was adjusted to equal solid feed intake, only 4% of the variation in standardized growth performance in later life, reflecting feed efficiency, could be explained by early life measurements. This indicates that > 95% of the variation in feed efficiency in later life could not be explained by early life characterization. It is hypothesized that variation in health status explains substantial variation in feed efficiency in veal calves. Significant relations between fasting plasma glucose concentrations, fecal dry matter and fecal pH in early life and feed efficiency in later life depended on MR composition. These measurements are, therefore, potential tools for screening calves in early life on their ability to cope with a MR in which half of the lactose is replaced by glucose, fructose and glycerol (in a 2:1:2 ratio).

The studies reported in this thesis demonstrate that glycerol, glucose and a combination of glucose, fructose and glycerol in a 2:1:2 ratio are promising lactose replacers. The effects of replacing lactose by other carbohydrate or energy sources described in this thesis are required to evaluate the potential of lactose replacers for inclusion in calf milk replacers and provide input for feed evaluation for calves and ruminants.

Microben maken mede de wijn : regionale verschillen in gistpopulaties beïnvloeden het aromaprofiel van wijn
Maanen, G. van; Vermeulen, T. - \ 2015
Bionieuws 2015 (2015)15. - ISSN 0924-7734 - p. 1 - 1.
wijnen - smaak - gisten - microbiologie - fermentatie - wijnbouw - wijngaarden - wines - taste - yeasts - microbiology - fermentation - viticulture - vineyards
Wijn dankt zijn karakter niet alleen aan lokale omstandigheden in klimaat, bodem en teelt, maar ook aan aanwezige gisten, claimen Nieuw-Zeelandse microbiologen.
Mestvergisting Fermtech systems en Ecobag
Kasper, G.J. ; Kemperman, A.J.M. - \ 2015
Wageningen : Wageningen UR Livestock Research (Rapport / Wageningen UR Livestock Research 874) - 45
mestvergisting - melkveehouderij - biogas - voedingsstoffen - dierlijke meststoffen - systeeminnovatie - bioraffinage - duurzaamheid (sustainability) - fermentatie - biobased economy - manure fermentation - dairy farming - nutrients - animal manures - system innovation - biorefinery - sustainability - fermentation
Een deel van de veehouders wil de energetische waarde van mest benutten om in hun energieverbruik te voorzien. Vergisting is een methode om energie uit mest te halen. Doel is het vergisten van alle soorten dierlijke mest met systemen die een rendabele business case realiseren in de keten ‘(verse) mest tot en met verwaarden van digestaat of mineralenterugwinning m.b.v. mestraffinage’. Hiertoe zijn twee innovatieve technieken van vergisten onderzocht, die op pilotschaal zijn getest. Daarna zal bij opschaling het innovatieve vergistingsproces van grote betekenis kunnen zijn voor het mede oplossen van het Nederlandse mestprobleem. Het doel van dit project in engere zin beperkt zich tot het monitoren van alleen de innovatieve vergistingstechnieken van twee systemen met melkveemest op boerderijschaal.
Zoektocht naar fermentatieprocessen voor producten met minder zout
Smid, E.J. - \ 2015
Wageningen UR
voedselmicrobiologie - fermentatie - houdbaarheid (kwaliteit) - voedseltechnologie - zout - smaak - voeding en gezondheid - food microbiology - fermentation - keeping quality - food technology - salt - taste - nutrition and health
De fermentatie van verse plantaardige of dierlijke producten geeft extra smaak aan voedsel en verlengt de houdbaarheid. Het proces vergt echter vaak veel zout en daarvan krijgt de westerling al veel te veel binnen. In zijn inaugurele rede bij de aanvaarding van het persoonlijk hoogleraarschap Levensmiddelenmicrobiologie aan Wageningen University gaat prof. Eddy Smid op zoek naar alternatieve, ‘natriumarme’ fermentatieprocessen die de gezondheid ten goede komen.
Role of anti-competitor toxins in the origin and maintenance of diversity in Saccharomyces yeast microbial populations
Pieczynska, M.D. - \ 2015
Wageningen University. Promotor(en): Bas Zwaan, co-promotor(en): Arjan de Visser. - Wageningen : Wageningen University - ISBN 9789462573093 - 123
gisten - rna-virussen - toxinen - symbiose - toxiciteit - co-evolutie - fenotypen - fermentatie - yeasts - rna viruses - toxins - symbiosis - toxicity - coevolution - phenotypes - fermentation


Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution.

In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains).

Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4).

Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry.

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