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Biobutanol, produced by solventogenic bacteria like Clostridium beijerinckii through acetone, butanol, and ethanol (ABE) fermentation, is a promising green alternative to fossil fuels. However, this ABE fermentation suffers drawbacks, such as a relatively low productivity that limits its industrial deployment. Electro-fermentation (EF) can modify the intracellular ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide (NAD+/NADH), a critical factor for butanol synthesis by C. beijerinckii, thus allowing higher product titers (or productivity) to be reached. RESULTS: The present study focuses on glucose-based ABE-EF at laboratory-scale. Three replicate EF reactors (300 mL) were constructed and used to grow C. beijerinckii. Different cathode materials were electrochemically characterized and tested. The effect of voltage application on process productivity was assessed by performing chronoamperometry tests at constant cathode potential (Ecat), ranging from −0,7 to −1,0 V vs Ag/AgCl. These trials were compared with a benchmark ABE fermentation that was performed without voltage application. Poising Ecat at −0,8 V vs Ag/AgCl resulted in the optimal EF scenario. In these conditions, the specific growth rate of bacteria was 73% higher than the benchmark ABE fermentation, with a productivity of 125,6 mg-butanol/L/h, a process yield of 0,15 g-butanol/g-glucose, and a selectivity for butanol of 87% (34%, 25%, and 11% higher than the benchmark fermentation, respectively). The addition of 0,5 mM neutral red as redox mediator in the broth led to a further improvement of butanol productivity (152,9 mg L h−1), requiring a very low energy consumption of 0,02 kWh/kg-butanol to run the EF process. CONCLUSION: Applying EF enhances the ABE fermentation in terms of butanol production, selectivity, and energy consumption.

Since 2011, unprecedented beaching events of Sargassum seaweed have caused major environmental, health and economic problems in the Caribbean, Gulf of Mexico, northern Brazil and the western coast of Africa. Not only are Sargassum influxes threatening already fragile and often endangered coastal ecosystems, such as coral reefs, mangroves and seagrass beds, they also disrupt the livelihoods of communities, especially those associated with the tourism and fishing sectors. The aim of this study is to develop a plan to turn these “brown tides” into opportunities for sustainable, scalable and efficient harvesting and valorisation approaches that will deliver environmental and socioeconomic benefits to the Dutch Caribbean and other end-users in the region. In this report we have reviewed the state-of-the-art of research on recent Sargassum blooms and influxes in the Caribbean with respect to biology, ecology, origin, distribution, socio-ecological impact and management options, in addition to existing valorisation chains and uses of Sargassum biomass, and environmental and socio-economic impacts of Sargassum valorisation. We conclude that value chains based on valorisation of nearshore Sargassum biomass into biofuel and agricultural products (i.e. fertilizer, animal feed supplement) seem the most promising for the Dutch Caribbean islands, since they will contribute to sustainable energy and food security, while reducing environmental impact of the energy and agricultural sector. Some of the identified management and valorisation strategies could also be applied to other areas that are affected by massive Sargassum influxes, such as the Gulf of Mexico. During 2020 pelagic Sargassum samples have been collected in different locations in the Caribbean region (Bonaire, Mexico, St Maarten) and Florida. These samples have been analysed for major components (including sugars and ash) and for content in iodine and heavy metals. The values obtained in these analysis were compared to values reported in the literature. The results of these analysis are of importance for the definition of possible applications of the Sargassum biomass or its products as component in the food and feed value chains Based on our literature review, a number of knowledge gaps have been identified that are related to the availability of pelagic Sargassum biomass, the environmental impacts of the harvesting, technological challenges, effects of Sargassum leachates to the environment (in case of agricultural uses) and on socio-economic impacts of the current and predicted actions (Chapter 7). These knowledge gaps need to be addressed before (commercial) harvesting and valorisation actions concerning pelagic Sargassum are taken. Therefore, we defined an implementation plan (Section 7.5 and Fig. 1), which addresses these knowledge gaps to set the first steps in the short term towards efficient and sustainable management and valorisation of pelagic Sargassum in the Dutch Caribbean. In the potential value chains defined, prevention of landings of Sargassum on the coasts is preferred to harvesting onshore. This is due to the negative ecological impact of harvesting onshore and the lower quality of beached Sargassum, that decays very rapidly.

In 2015/2016, the total municipal solid waste (MSW) collected by local authority in the U.K. was 26 million tonnes and over 57% is still put into landfill or incinerated. MSW is a promising feedstock for bio-butanol production as it has a high lignocellulosic fibre content such as paper, wood, and food waste, about 50 wt% of a typical MSW stream. The study evaluates acetone, butanol, ethanol and hydrogen production from autoclaved municipal solid waste feedstock. Life cycle assessment is undertaken to evaluate the acetone, butanol, ethanol and hydrogen production process, considering cogeneration of heat and power from residual biogenic waste based on experimental data and process modelling. Acetone, butanol, and ethanol product yield can be achieved at 12.2 kg butanol, 1.5 kg ethanol, 5.7 kg acetone, and 0.9 kg hydrogen per tonne MSW. The product yield is relatively low compared to other lignocellulosic feedstocks primarily because of the lower hydrolysis yield (38% for glucose) achieved in this study; however, hydrolysis yields could be improved in future optimisation work. The conversion shows a net primary energy demand of −1.11 MJ/MJ liquid biofuels (butanol and ethanol) and net greenhouse gas emission of −12.57 g CO2eq/MJ liquid biofuels, achieving a greenhouse gas reduction of 115% compared to gasoline comparator.

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.

In this study, the removal of IBE from aqueous solutions by gas stripping has been characterized. The effect of one or more components in the solution on the kinetics of the separation has been studied, both at 37 °C and at 70 °C. Gas stripping has been applied to batch, repeated batch and continuous cultures of Clostridium beijerinckii grown on a glucose/xylose mixed sugar substrate mimicking lignocellulosic hydrolysates, with the aim of finding optimal conditions for a stable IBE-producing culture with high productivity. An innovative repeated-batch process has been demonstrated in which the gas-stripping is performed at 70 °C, resulting in a prolonged stable IBE culture.

Biomass fermentation to Iso-propanol, Butanol and Ethanol (IBE) is particularly important as IBE is a common building block in the development of biorefineries and IBE-producing bacteria are robust industrial organisms, capable to utilize the sugars of the lignocellulosic biomass. Research is focused on increasing fermentation yields and the reduction of energy that is required to separate the volatile products. The paper addresses both of these challenges combining experimental innovations with a systems engineering approach. IBE is recovered from a gas-stripped fermenter whose potential for adsorption is researched and integrated with downstream options for separation. Design and integration is assisted using a systems approach that relies on mathematical models that regress and extrapolate experimental data for scale-up calculations. Process integration involves synthesis challenges to define biorefinery portfolios and systems integration to combine fermentation, stripping, adsorption, and distillation. The analysis considers 4 alternative biorefinery cases and presents results with significant savings in energy use and costs (up to 87% savings reported) after the application of energy integration to the IBE plant. Scenarios are analysed economically and confirm benefits in the use of adsorption and viable production yields.

Four dilute-acid pretreated and hydrolysed lignocellulosic raw materials were evaluated as substrates for fermentative hydrogen production by Caldicellulosiruptor saccharolyticus. Their fermentability was ranked in the order: barley straw > wheat straw > corn stalk > corn cob. The content of 5-hydroxymethylfurfural (HMF) in medium with hydrolysates prepared from corn cob (1.0 g/L) and corn stalk (0.8 g/L), respectively, reached levels likely to be toxic for growth of C. saccharolyticus. HMF was absent in wheat and barley straw hydrolysates. Furfural concentrations in media with wheat straw, barley straw and corn stalk hydrolysates were low (0.2-0.3 g/L), while it was higher in corn cob hydrolysates (0.6 g/L). (Less)

Hydrogen was produced from carrot pulp hydrolysate, untreated carrot pulp and (mixtures of) glucose and fructose by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana in pH-controlled bioreactors. Carrot pulp hydrolysate was obtained after enzymatic hydrolysis of the polysaccharide fraction in carrot pulp. The main sugars in the hydrolysate were glucose, fructose, and sucrose. In fermentations with glucose hydrogen yields and productivities were similar for both strains. With fructose the hydrogen yield of C. saccharolyticus was reduced which might be related to uptake of glucose and fructose by different types of transport systems. With T. neapolitana the fructose consumption rate and consequently the hydrogen productivity were low. The hydrogen yields of both thermophiles were 2.7–2.8 mol H2/mol hexose with 10 g/L sugars from carrot pulp hydrolysate. With 20 g/L sugars the yield of T. neapolitana was 2.4 mol H2/mol hexose while the yield of C. saccharolyticus was reduced to 1.3 mol H2/mol hexose due to high lactate production in the stationary growth phase. C. saccharolyticus was able to grow on carrot pulp and utilized soluble sugars and, after adaptation, pectin and some (hemi)cellulose. No growth was observed with T. neapolitana when using carrot pulp in agitated fermentations. Enzymatic hydrolysis of the polysaccharide fraction prior to fermentation increased the hydrogen yield with almost 10% to 2.3 g/kg of hydrolyzed carrot pulp.

Hydrogen can be produced through dark anaerobic fermentation using carbohydrate-rich biomass, and through photofermentation using the organic acids produced from dark fermentation. Sugar beet is an ideal energy crop for fermentative production of hydrogen in the EU due to its environmental profile and its potential availability in the area. In this work, various aspects of cultivating sugar beet in the EU for biohydrogen were highlighted, with special focus on The Netherlands and Greece. Moreover, fermentation of sugar beet juice with Caldicellulosiruptor saccharolyticus at sucrose concentration 10 g/l was performed, and was found comparable to the fermentation on pure sucrose except that the hydrogen production was 10% higher on sugar beet juice. A conservative estimate of the annual hydrogen potential in the EU was made (300 × 106 kg hydrogen), considering the utilization of sugar beet pulp in hydrogen production.

Production of hydrogen by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana was studied in serum flasks and in pH-controlled bioreactors with glucose, and hydrolyzed and untreated potato steam peels (PSP) as carbon sources. Two types of PSP hydrolysates were used: one in which the starch in the PSP was liquefied with alpha-amylase, and one in which the liquefied starch was further hydrolyzed to glucose by amyloglucosidase. When the PSP hydrolysates or untreated PSP were added at circa 10–14 g/L of glucose units, both strains grew well and produced hydrogen with reasonable to high molar yields (2.4–3.8 moles H2/mole glucose units), and no significant production of lactate. The hydrogen production rates and yields were similar with untreated PSP, hydrolyzed PSP, and pure glucose, showing that C. saccharolyticus and T. neapolitana are well equipped for the utilization of starch. When the concentrations of the substrates were increased, growth and hydrogen production of both strains were hampered. At substrate concentrations of circa 30–40 g/L of glucose units, the molar hydrogen yield of C. saccharolyticus was severely reduced due to the formation of high amounts of lactate, while T. neapolitana was unable to grow at all. The results showed that PSP and PSP hydrolysates are very suitable substrates for efficient fermentative hydrogen production at moderate substrate loadings.

In Petunia hybrida flowers, both pollination and stigma wounding induced a transient increase in ethylene pro­ duction and hastened corolla senescence. Ethylene production by different flower parts was measured in situ using laser photoacoustic (LPA) spectroscopy. In pollinated flowers, ethylene was exclusively produced by the stigma/style region whereas wounding of the stigma induced ethylene production both by the stigma/style region and by the remaining flower parts. In aminoethoxyvinylglycine (AVG)-treated flowers, sub­ sequent treatment of the unwounded stigma with 1«aminocyclopropane-1-carboxylic acid (ACC) induced ethylene production exclusively by the stigma/style region whereas treatment of a previously wounded stigma with ACC induced a simultaneous increase in ethylene production by the stigma/style region and the remaining flower parts. These results suggest that following stigma wounding, either ACC or ethylene is involved in inter-organ communication. Following pol­ lination, the signal is apparently not directly related to ethylene.

Ethylene (C2H4) is a gaseous plant hormone produced by higher and lower (green) plants and, when grown on appropriate substrates, also by fungi, yeasts and bacteria. Ethylene is involved in many developmental processes in plants and is biologically active in trace amounts (10 – 100 nl/I of air) that may be present in the outside air due to industrial air pollution(1). Fruit ripening and flower senescence especially, in a variety of commercially important crops, are dramatically stimulated by ethylene. Following characterization of the genes coding for the key enzymes in ethylene biosynthesis, i.e. ACC synthase and ACC oxidase, it has become clear that their expression is regulated in a complex manner involving developmental, hormonal and tissue-specific factors. This was recently very elegantly demonstrated for the expression of ACC oxidase genes in developing petunia flowers by Tang et al.(2). The spatial and temporal expression patterns, especially in the reproductive organs, suggest a hitherto unknown role for ethylene in reproductive processes such as the self-incompatible response and the secretion of cellular exudate by the stigma and nectary.

Summary The plant hormone ethylene regulates senescence in carnation flowers. The final step in the biosynthesis of ethylene i.e. the conversion of ACC to ethylene is catalysed by the enzyme ACC oxidase. ACC oxidase was extracted from carnation petals and its characteristics were determined. The enzyme appears to be a soluble enzyme and requires Fe 2+ and ascorbate for maximal activity. CO 2 increases the reaction velocity and the apparent K m for ACC from 30 µM to 425 µM. Both Co 2+ and the ACC analog α-aminoisobutyric acid inhibit the ACC oxidase activity. The change in in vivo ACC oxidase activity in senescing carnation flowers is accompanied by a parallel change in ACC oxidase mRNA abundance and in vitro ACC oxidase activity. Optimal conditions for extraction and determination of authentic carnation petal ACC oxidase activity in vitro are described.

Carnation (Dianthus caryophyllus L. cv. White Sim) petal discs were radiolabelled with (32p)ortho- phosphate and the lipids were extracted and analysed by thin-layer chromatography and autoradiography. Phos- pholipids were identified by co-migration with standards using thin-layer chromatography with different solvent systems. Results showed that (32p)orthophosphate was rapidly incorporated into the minor lipids phosphatidic acid (PtdOH), phosphatidylinositol monophosphate (PtdInsP) and phosphatidylinositol bisphosphate (Ptd- InsP2), and relatively slowly into the structural lipids phosphatidylcholine, -ethanolamine, -glycerol and -inos- itol. Pulse-chase experiments revealed that the label was rapidly lost from PtdOH, PtdInsP and PtdInsP2 while the structural lipids remained radiolabelled. The amount of PtdInsP and PtdInsP2 was found to constitute 0.45% and 0.013%, respectively, of the total phospholipids, on a mo- lar basis. Together these results show that the turnover of the chemically low-abundant polyphosphoinositides is relatively high compared with the major structural phos- pholipids. Phosphatidylinositol monophosphate was fur- ther characterized by showing that it incorporates myo- (3H)inositol and that its major fatty-acid constituents are palmitic acid and linoleic acid. Furthermore, we present evidence for the presence of both phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate iso- mers. The significance of these results is discussed with respect to plant phosphoinositide signal transduction.

The objectives and methodology of the EU-funded research project HYVOLUTION devoted to hydrogen production from biomass are reviewed. The main scientific objective of this project is the development of a novel two-stage bioprocess employing thermophilic and phototrophic bacteria, for the cost-effective production of pure hydrogen from multiple biomass feedstocks in small-scale, cost-effective industries. Results are summarised of the work on pretreatment technologies for optimal biodegradation of energy crops and bio-residues, conditions for maximum efficiency in conversion of fermentable biomass to hydrogen and CO2, concepts of dedicated installations for optimal gas cleaning and gas quality protocols, as well as innovative system integration aimed at minimizing energy demand and maximizing product output. The main technological objective is the construction of prototype modules of the plant which, when assembled, form the basis of a blueprint for the whole chain for converting biomass to pure hydrogen. A brief outline is presented of the progress made towards developing reactors for thermophilic hydrogen production, reactors for photoheterotrophic hydrogen production and equipment for optimal gas cleaning procedures. (Less)

In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatinealpha-chaconinealpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.