Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H 2 O 2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H 2 O 2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H 2 O 2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H 2 O 2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H 2 O 2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H 2 O 2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu + state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H 2 O 2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu + state of LPMOs, which may prevent the formation of uncoupled side reactions.

Biogas plants can contribute to future energy systems’ stability through flexible power generation. To provide power flexibly, a demand-oriented biogas supply is necessary, which may be ensured by applying flexible feeding strategies. In this study, the impacts of applying three different feeding strategies (1x, 3x and 9x feeding per day) on the biogas and methane production and process stability parameters were determined for a biogas plant with a focus on waste treatment. Two feedstocks that differed in (1) high fat and (2) higher carbohydrate content were investigated during semi-continuous fermentation tests. Measurements of the short chain fatty acids concentration, pH value, TVA/TIC ratio and total ammonium and ammonia content along with a molecular biology analysis were conducted to assess the effects on process stability. The results show that flexible biogas production can be obtained without negative impacts on the process performance and that production peaks in biogas and methane can be significantly shifted to another time by changing feeding intervals. Implementing the fermentation tests’ results into a biogas plant simulation model and an assessment of power generation scenarios focusing on peak-time power generation revealed a considerable reduction potential for the needed biogas storage capacity of up to 73.7%.

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Oil from oilseeds can be extracted by mechanical extraction (pressing), aqueous extraction, or by extraction with organic solvents. Although solvent extraction is the most efficient method, organic solvents are a potential hazard to the life and health for workers as well as to the environment, when solvent vapors are released and act as air pollutant with a high ozone forming potential. Pressing is safer, environmentally friendly, and it preserves valuable natural components in the resulting oils. The problems associated with pressing are the high energy consumption and the lower yield on oil extraction, because the applied mechanical force does not completely destroy the structural cell components storing the oil. In seed cells, the oil is contained in the form of lipid bodies (oleosomes) that are surrounded by a phospholipid monolayer with a protein layer on the surface. These lipid bodies are further protected by the seed cell walls consisting mainly of polysaccharides such as pectins, hemicelluloses and cellulose, but also of glycoproteins. The use of hydrolases to degrade these barriers is a promising pretreatment strategy to support mechanical extraction and improve the oil yield. It is advisable to use a combination of enzymes different activities when considering the multicompartment and multicomponent structure of oilseed cells. This article gives an overview on the microstructure and composition of oilseed cells, reviews enzymes capable to destroy oil containing cell compartments, and summarizes the main parameters of enzymatic treatment procedures, such as the composition of the enzyme cocktail, the amount of enzyme and water used, temperature, pH, and the duration. Finally, it analyzes the efficiency of proteolytic, cellulolytic, and pectolytic enzyme pretreatment to increase the yield of mechanically extracted oil from various types of vegetable raw materials with the main focus on oilseeds.

The pretreatment of pumpkin seeds with proteolytic, cellulolytic and pectolytic enzymes and their effect on cell structure and oil yield was studied. Pumkin seed samples were treated with pepsin, papain, Viscozyme L, cellulase or pectinase. The evaluation of cell integrity by immediate hexane extraction (shaking method) showed that all samples treated with different enzymes had a higher oil yield, ranging from 33.2 to 34.1 % of seed weight, than the control samples (32.1 %). The number of disrupted cells was also higher than the control (64.4%), ranging from 67.6 to 69.5 %. The highest percentage of disrupted cells, 71.0 and 71.1 %, was found in samples treated with pepsin+Viscozyme L+pectinase and pepsin+cellulase+pectinase mixtures, respectively. To study the effect of enzymatic pretreatment on the microstructure of pumkin seeds, ultramicrotome slices of pumpkin seeds were treated with individual enzymes and enzyme mixtures. The highest percentage of cell destruction was observed in samples treated with Viscozyme L and the mixture of pepsin+Viscozyme L+pectinase. The yield of pressed oil from pumpkin seeds pretreated with the pepsin+Viscozyme L+pectinase mixture was 7.0 % higher than that of control samples. The quality parameters, chemical composition and antioxidant activity of pressed oils were investigated and the enzymatic pretreatment did not significantly affect the free fatty acid content, peroxide value, fatty acid composition, or phytosterol profiles. The antioxidant activity expressed as DPPH° radical scavenging effect of the enzymatically pretreated pressed oil was 2.7 % higher than that of the sample not pretreated with enzymes.

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