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Voor het beheer van verharde oppervlakken wordt hier aandacht geschonken aan de betekenis en de voordelen van toepassing van DOB: Duurzaam OnkruidBeheer op verhardingen

Abstract Plant species that expand their range in response to current climate change will encounter soil communities that may hinder, allow or even facilitate plant performance. It has been shown repeatedly for plant species originating from other continents that these plants are less hampered by soil communities from the new than from the original range. However, information about the interactions between intra‐continental range expanders and soil communities is sparse, especially at community level. Here we used a plant–soil feedback experiment approach to examine if the interactions between range expanders and soil communities change during range expansion. We grew communities of range‐expanding and native plant species with soil communities originating from the original and new range of range expanders. In these conditioned soils, we determined the composition of fungi and bacteria by high‐throughput amplicon sequencing of the ITS region and the 16S rRNA gene respectively. Nematode community composition was determined by microscopy‐based morphological identification. Then we tested how these soil communities influence the growth of subsequent communities of range expanders and natives. We found that after the conditioning phase soil bacterial, fungal and nematode communities differed by origin and by conditioning plant communities. Despite differences in bacterial, fungal and nematode communities between original and new range, soil origin did not influence the biomass production of plant communities. Both native and range expanding plant communities produced most above‐ground biomass in soils that were conditioned by plant communities distantly related to them. Synthesis. Communities of range‐expanding plant species shape specific soil communities in both original and new range soil. Plant–soil interactions of range expanders in communities can be similar to the ones of their closely related native plant species.

‘Ik sta al drie jaar met stip op nummer 1 qua energiebesparing’, zegt John Kreuk niet zonder trots. Kreuk is bollenkweker in Callantsoog en doet mee aan het onderzoeksproject State of the Art. Bollenkwekers. Onderzoekers van Praktijkonderzoek Plant en Omgeving, onderdeel van Wageningen UR, en Agentschap NL doen daarin al tien jaar samen onderzoek naar energiebesparing. Resultaat: de energierekeningen in teelt en broei zijn gemiddeld een derde lager dan in 1995.

Worldwide, anthropogenic activities threaten surface water quality by aggravating eutrophication and increasing total nitrogen to total phosphorus (TN:TP) ratios. In hydrologically connected systems, water quality management may benefit from in-ecosystem nutrient retention by preventing nutrient transport to downstream systems. However, nutrient retention may also alter TN:TP ratios with unforeseen consequences for downstream water quality. Here, we aim to increase understanding of how nutrient retention may influence nutrient transport to downstream systems to improve long-term water quality management. We analyzed lake ecosystem state, in-lake nutrient retention, and nutrient transport (ratios) for 3482 Chinese lakes using the lake process-based ecosystem model PCLake+. We compared a low climate change and sustainability-, and a high climate change and economy-focused scenario for 2050 against 2012. In both scenarios, the effect of nutrient input reduction outweighs that of temperature rise, resulting in more lakes with good ecological water quality (i.e., macrophyte-dominated) than in 2012. Generally, the sustainability-focused scenario shows a more promising future for water quality than the economy-focused scenario. Nevertheless, most lakes remain phytoplankton-dominated. The shift to more macrophyte-dominated lakes in 2050 is accompanied by higher nutrient retention fractions and less nutrient transport to downstream waterbodies. In-lake nutrient retention also alters the water's TN:TP ratio, depending on the inflow TN:TP ratio and the ecosystem state. In 2050 higher TN:TP ratios are expected in the outflows of lakes than in 2012, especially for the sustainability-focused scenario with strong TP loading reduction. However, the downstream impact of increased TN:TP ratios depends on actual nutrient loadings and the limiting nutrient in the receiving system. We conclude that nutrient input reductions, improved water quality, higher in-lake nutrient retention fractions, and lower nutrient transport to downstream waterbodies go hand in hand. Therefore, water quality management could benefit even more from nutrient pollution reduction than one would expect at first sight.

The IPCC Guidelines for National Greenhouse Gas Inventories provide default methodologies for estimating emissions of the most important greenhouse gases at a national scale. The methodology for estimating emissions of nitrous oxide (N2O) from agriculture was revised in 1996 by an international working group. Here we summarize this new methodology and apply it to the global data. The new method aims at assessing the full nitrogen cycle and takes into account N2O formation in agricultural fields (direct emissions), animal waste management systems (AWMSs) as well as indirect emissions taking place at remote places after nitrogen is lost from the agricultural fields. Using the IPCC method, we estimated that global agricultural N2O emissions almost doubled between 1960 (3.5 Tg N2O-N) and 1994 (6.2 Tg N2O-N). Direct emissions, animal waste management systems and indirect emissions make about equal contribution to total current emissions.

Ester production by microorganisms is of great importance to biotechnological processes. These volatile compounds impart pleasant, fruity aromas to beer, wine, and other fermented products. Yeast in particular are well known for their ability to produce volatile esters. The genetic and enzymatic intricacies of ester production have been the focus of many studies, mainly in S. cerevisiae. Despite this, ester synthesis in yeast is not fully understood. This holds particularly true for yeasts that produce high amounts of ethyl acetate from sugars, such as Kluyveromyces marxianus and Wickerhamomyces anomalus. As was introduced in Chapter 1, this ability has been described more than a century ago, but the enzymatic mechanisms behind the synthesis were unclear. Circumstantial evidence suggested that an alcohol acetyltransferase (AAT) was responsible for the ethyl acetate formation. Ethyl acetate is a versatile commodity chemical that is currently produced in unsustainable processes. Understanding the enzymes responsible for bulk ethyl acetate synthesis in yeast could enable rational design of novel production strains. This, in turn could facilitate the development of biobased ethyl acetate production processes. This thesis has made several important breakthroughs in the field of ester production in yeast, particularly ethyl acetate. The most significant of these was the discovery of the elusive enzyme responsible for bulk ethyl acetate synthesis in yeast. The identification of the ethyl acetate-producing enzyme in W. anomalus is described in Chapter 2. The purified enzyme showed AAT activity with ethanol and acetyl-CoA and was therefore named Ethanol acetyltransferase 1 (Eat1). Production of Eat1 in Escherichia coli enabled efficient ethyl acetate production in E. coli. The enzyme could thus potentially be used to develop new biobased ethyl acetate production processes. However, Eat1 was also able to function as a thioesterase and esterase active against acetyl-CoA and ethyl acetate, respectively. It was observed that the presence of ethanol was able to repress the hydrolytic activities, at which point the AAT activity was dominant. How ethanol can control the activities of Eat1 is unclear. It highlights that sufficient ethanol concentrations must be present to produce ethyl acetate with Eat1. It is also shown that Eat1 homologs are present in other bulk ethyl acetate-producing yeasts. These homologs are only distantly related to known AATs. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family. The discovery of this novel enzyme family was the cornerstone of the research presented in this thesis. The identification enabled further studies on the physiology of Eat1 and bulk ethyl acetate production in the native yeasts in Chapter 3. The cellular location of Eat1 in Kluyveromyces lactis was determined. The enzyme localised to the mitochondria of the yeast. This observation opposed the literature consensus which assumed that bulk ethyl acetate synthesis occurred in the yeast cytosol. Cytosolic acetyl-CoA flux in yeast is low and would presumably not support the synthesis of high amounts of ethyl acetate. The localisation of Eat1 in the mitochondria could better explain how bulk ethyl acetate synthesis occurs. The current hypothesis suggests that bulk ethyl acetate is an overflow product of yeast under iron limited conditions. Under these conditions, the entry of acetyl-CoA into the TCA cycle is impaired and acetyl-CoA accumulates. Eat1 is then proposed to relieve the accumulation by forming ethyl acetate. The TCA cycle and the major flux of acetyl-CoA in yeast are located in the mitochondria, where Eat1 is also located. It is thereby established that bulk ethyl acetate is a mitochondrial product of certain yeasts. Chapter 4 describes the engineering of efficient ethyl acetate production in E. coli using Eat1 as the catalyst. Unlike yeast, the metabolism of E. coli can support the synthesis of ethyl acetate under anaerobic conditions. This removes the need for aeration, which is costly on a large scale. Establishing the anaerobic ethyl acetate pathway was faced with several bottlenecks, and removing them was the main theme of this chapter. The pathway towards ethyl acetate could be improved by disrupting by-product formation and optimising the expression levels of eat1. Further improvements could be made by removing the N-terminal mitochondrial localisation sequence of Eat1. These sequences typically destabilise proteins unless they are removed. These approaches did improve ethyl acetate formation by Eat1 to the point where ethanol levels were no longer sufficient to repress the hydrolytic activities of Eat1. To prevent ester hydrolysis the volatility of ethyl acetate was used to strip it from the fermenter. The result was ethyl acetate production at 63.4 % of the pathway maximum. We then looked beyond ethyl acetate as a bulk chemical in Chapter 5 and investigated the role Eat1 has in general ester formation by yeasts, particularly S. cerevisiae. The formation of esters is industrially relevant also in S. cerevisiae as these compounds contribute to the aroma of fermented foods. S. cerevisiae naturally produces a variety of alcohols and thus provided a convenient platform to compare Eat1 homologs from different yeast. Expression of various eat1 genes resulted in an increase of various in acetate and propionate ester levels. By disrupting the S. cerevisiae eat1 gene the inverse effect was observed. Eat1 therefore seems to contribute to acetate ester synthesis in S. cerevisiae as well. In this chapter, a S. cerevisiae strain was generated where all known AAT genes were disrupted. Opposite to the expectations, ester production persisted in this strain, showing that even more ester-producing mechanisms exist. Chapter 6 focuses on the complex field of ester production as bulk chemicals, from the fundamentals of microbial ester production. Much research has been devoted to understanding the ester-producing processes in microorganisms. This is a daunting task as the structures and functions of esters in microorganisms are in many cases unrelated. Most esters are produced via the AAT reaction. With the exception of Eat1, which was identified in this thesis, other AATs have been studied for a long time. Still, relatively little is known about their structure and catalytic mechanisms, likely because there are no crystal structures available yet. Nevertheless, they have been applied extensively in metabolic engineering of ester production. The AAT reaction is simple. As long as a suitable alcohol and acyl-CoA are provided, the AAT will catalyse the ester formation. The real challenge of engineering ester formation is in the efficient supply of alcohols and acyl-CoA substrates. Some remarkable success has been made in recent years which is outlined in the review. In many cases, esters are less toxic and more readily extractable than the alcohols and acids they are composed of. They could therefore serve as a platform compound. In summary, this thesis contributed significantly to the knowledge of ester synthesis in yeast. The progress made on the production of ethyl acetate specifically can be used to develop new biotechnological, more sustainable production processes for this versatile compound. At the same time, much remains to be discovered.