Browsing by Subject "Enzym"

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Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.
Function and composition of the soil microbial community in calcareous grassland exposed to elevated atmospheric carbon dioxide
(2003) Ebersberger, Diana; Kandeler, Ellen
Terrestrial ecosystems generally respond to rising atmospheric carbon dioxide (CO2) concentrations with increased net primary productivity and increased water use efficiency. This may change the amount and quality of organic substances entering the soil and fuelling microbial metabolism. Soil microorganisms and their activity might also be affected by increased soil moisture at elevated CO2. This thesis was designed to analyse the response of the soil microbial community in a species-rich calcareous grassland in the Swiss Jura Mountains, which had been exposed to ambient and elevated CO2 concentrations (365 and 600 ppm) for six growing seasons. In the first study, laboratory incubation experiments were conducted to explore the relationship between litter quality under elevated carbon dioxide and enzymes involved in carbon cycling. Naturally senescent, mixed litter from the long-term field experiment was incubated with soil material for 10, 30 and 60 days. Soil samples were then obtained close to the litter layer using a microtome cutting device. Litter and soil samples were analysed for invertase and xylanase activity. The lower litter quality produced under elevated CO2, i.e. wider C/N ratio, yielded lower invertase and xylanase activities of litter. Litter addition stimulated activities in adjacent soil. Invertase activities of adjacent soil were not affected by litter quality, while soil xylanase activity was higher in soil compartments adjacent to litter from elevated CO2 plots. The reduced enzyme activities of litter produced under elevated CO2 can slow decomposition, at least during the initial stages. Since the effects of litter quality on enzyme activities in adjacent soil were small, we conclude that CO2-induced belowground C-inputs (e.g. increased root mass) and altered moisture conditions are more important controls of enzyme activities than altered litter quality. In the second study, functional diversity of the soil microbial community was assessed by analysing N-mineralisation and activities of enzymes of the C-, N-, P- and S-cycle of soil samples taken in spring and summer 1999, in the 6th season of CO2 exposure. In spring, N-mineralisation increased significantly by 30% at elevated CO2, while there was no significant difference between treatments in summer. The response of soil enzymes to CO2 enrichment was also more pronounced in spring, when alkaline phosphatase and urease activities were increased most strongly, by 32% and 21%, respectively. In summer, activity differences between CO2 treatments were greatest in the case of urease and protease (+21% and +17% at elevated CO2). The significant stimulation of N-mineralisation and enzyme activities at elevated CO2 was probably caused by higher soil moisture and/or increased root biomass. In the third study, soil microbial community structure of soil samples taken in spring and summer 1999 was analysed by means of PLFA profiles and 16S rDNA fingerprints obtained by PCR-DGGE. PLFA profiles were not affected by elevated CO2. Ordination analysis of DNA fingerprints revealed a significant relation between CO2 enrichment and variation in DNA fingerprints. This variation must be attributed to low intensity bands because dominant bands did not differ between treatments. Diversity of the bacterial community (number of bands in DNA fingerprints and Shannon indices) was not affected. The observed minute, but significant changes in the structure of the soil bacterial community might be caused by changes in the quality of rhizodeposits at elevated CO2. These could either result from altered rhizodeposition of individual plants or from altered species composition of the calcareous grassland.The 4th part of the thesis compiles data on soil microorganisms, soil fauna, soil structure and nitrogen cycle of calcareous grassland after CO2 exposure for six growing seasons. Microbial biomass, soil basal respiration and the metabolic quotient were not altered significantly. PLFA analysis revealed no significant shift in the ratio of fungi to bacteria. Protozoans, bacterivorous and fungivorous nematodes, acarians, collembolans, and root-feeding nematodes were not affected by elevated CO2. Total nematode numbers averaged slightly lower (-16%) and nematode mass was significantly reduced (by 43%) due to fewer large-diameter nematodes classified as omnivorous and predacious. CO2 exposure resulted in a shift towards smaller aggregate sizes; this was caused by higher soil moisture. Reduced aggregate sizes result in reduced pore neck diameters. This can confine the locomotion of large-diameter nematodes and possibly accounts for their decrease. The CO2 enrichment also affected the nitrogen cycle. N stocks in living plants and surface litter increased, but N in soil organic matter and microorganisms remained unaltered. N mineralisation increased considerably, but microbial N did not differ between treatments, indicating that net N immobilization rates were unaltered.
Microbial community structure and function is shaped by microhabitat characteristics in soil
(2016) Ditterich, Franziska; Kandeler, Ellen
Soil microorganisms play a key role in degradation processes in soil, such as organic matter decomposition and degradation of xenobiotics. Microbial growth and activity and therefore degradation processes are influenced by different ecological factors, such as substrate availability, pH and temperature. During soil development different microhabitats are formed which differ in their physiochemical properties. There is some evidence that mineral composition is a driver for specific microbial colonization. Thereby, the heterogeneity of soils with differences in mineral composition and substrate availability can lead to a spatial distribution of soil microorganisms. At the soil-litter interface, a biogeochemical hot spot in soil, the abundance and activity of soil microorganisms increases due to high substrate availability, and degradation processes such as pesticide degradation are enhanced. This thesis aimed to clarify the influence of habitat properties on the structure and function of the microbial community in soil. In particular, focus was on mineral-microbe interactions that result from the mineral composition and substrate availability in an artificial soils system. Furthermore this thesis was designed to increase our understanding of the bacterial and fungal roles in pesticide degradation at the soil-litter interface using 4-chloro-2-methylphenoxyacetic acid (MCPA) as a model xenobiotic. These two aspects of the thesis were examined in three studies. The first study focused on the succession of microbial communities and enzyme activities in an artificial soils system with varying mineral composition and substrate availability over a period of 18 months. In the second study a microcosm experiment was used to study the bacterial pathway of MCPA degradation at the soil-litter interface. Over a period of 27 days the succession of bacterial degraders was followed. The third study focused on the degradation of MCPA in soil by nonspecific fungal enzymes, through the addition of fungal laccases as well as litter during 42 days of incubation. Both studies indicated the involvement of fungi in MCPA degradation and the importance of the ecological behavior of different degraders as a function of substrate availability. Results of the first study indicated that the microbial community was affected by mineral properties under high substrate availability and by the availability of beneficial nutrients at the end of incubation when substrate had become limited. The measured enzyme activities provided clear evidence that microbial community structure was driven by nutrient limitation during incubation. In the presence of easily available organic substrates at the beginning of the experiment, the soil microbial community was dominated by copiotrophic bacteria (e.g. Betaproteobacteria), whereas under substrate limitation at the end of incubation, more recalcitrant compounds became important to oligotrophic bacteria (e.g. Acidobacteria), which then became dominant. The results of the second study indicated that the contribution of the potential degraders to degradation of MCPA differed, and this was also seen in the succession of specific bacterial MCPA degraders. Added litter stimulated MCPA degradation due to the availability of litter-derived carbon and induced a two-phase response of fungi. This was seen in the development of pioneer and late stage fungal communities. Both fungal communities were probably involved in MCPA degradation. Therefore, the third study focused on the fungal pathway. These results indicated that the fungal laccases used had no direct influence on degradation and were as efficient as litter in providing additional nutrient sources, increasing MCPA degradation by bacteria and fungi. The observed differences between litter and enzyme addition underscored the observation that the enzyme effect was short-lived and that substrate quality is an important factor in degradation processes. In conclusion, this thesis demonstrated that soil microbial communities and therefore degradation processes are driven by mineral composition as well as substrate availability and quality. In addition, this thesis extends our understanding of degradation processes such as the degradation of xenobiotics, with MCPA as model compound, in soil. The combined insights from all three studies suggest that the use of a simple system such as the artificial soil system can increase our understanding of complex mechanisms such as degradation of pesticides.
Microbial consortia as inoculants for improvedcrop performance
(2020) Bradácová, Klára; Neumann, Günter
The use of microbial consortia products (MCP) based on combinations of different strains of plant growth-promoting microorganisms (PGPM) and frequently also on non-microbial bio-stimulants (BS) with complementary beneficial properties, is discussed as a strategy to increase the efficiency and the flexibility of BS-based crop production strategies under variable environmental conditions. Moreover, MCP application aims at the restoration of plant-beneficial, soil biological processes disturbed by soil degradation and intensive use of agro-chemicals. This PhD thesis was initiated to characterize the modes of action and the potential advantages of a representative commercial MCP formulation over selected single strain PGPM inoculants, with documented effects on plant growth promotion and pathogen suppression. In total, nine pot and field experiments were conducted with three crops (maize, spring wheat, tomato) on seven different soils with three organic and inorganic fertilization regimes. Only in one out of nine experiments conducted in this thesis, clear evidence for superior MCP performance was detectable in a drip-irrigated tomato field experiment conducted under the challenging environmental conditions of the Negev desert in Israel (Bradáčová et al., 2019c). This finding demonstrates that MCP inoculants can exhibit an advantage over single strain inoculants but not as a general feature. Selective interactions with the type and dosage of the selected fertilizers, as well as avoidance of inhibitory effects on root growth during MCP rhizosphere establishment, have been identified as critical factors. A further characterization of the conditions, promoting beneficial plant-MCP interactions is mandatory for a more targeted and reproducible MCP application.
Neue Cytochrom P450 Enzyme des Sesquiterpenlacton Stoffwechsels der Sonnenblume (Helianthus annuus L.)
(2016) Frey, Maximilian; Spring, Otmar
In the present work additional steps towards the elucidation of the biosynthetic pathway of H. annuus sesquiterpene lactones (STL) were achieved. Firstly candidate sequences were retrieved from a transcriptome database by filtering according to expression pattern and similarity to P450 enzymes known to participate in STL biosynthetic pathways. Open reading frames (ORFs) were obtained using 3´-and 5´-RACE-PCR. Previously described and newly identified candidate genes were then transformed in yeast vectors and expressed in combination with different substrate vectors. A high throughput micro approach was developed that allowed the expression and analysis of many yeast strains at the same time. For the transient expression in N. benthamiana the genes of known and putative enzymes were introduced via Agrobacterium mediated transformation. Using the in planta expression system the complete STL pathway of sunflower to costunolide was reconstructed de novo in a step-by-step approach. Previously described Michael-addition reactions of α-methylene-γ-lactone type STL to the thiol group of cysteine or glutathione in tobacco expression systems could be observed for all STL investigated. Chemically synthesized STL adducts were used as reference for the identification of in planta produced STL adducts. Enzyme characterization was conducted in two different in vivo expression systems, in yeast (Saccharomyces cervisiae) and tobacco (Nicotiana benthamiana). For the investigated biosynthetic pathway, differences between these two expression systems were discussed. Candidate gene M4 showed an unexpected product in yeast (farnesyl-δ-lactone) and led in combination with HaG8H to the production of costunolide. In the plant expression system, germacrene A acid was converted to costunolide by M4 in the absence of HaG8H. In both cases, M4 was involved in the synthesis of costunolide and should therefore be assigned Helianthus annuus costunolide synthase the underlying reaction mechanism should however be investigated more thoroughly. Helianthus annuus costunolide 14-hydroxylase HaC14H (candidate M33) was characterized in yeast and tobacco. A classification into subfamily CYP71CB, together with Tp8878 the Tanacetum parthenium costunolide/parthenolide 3β-hydroxylase is proposed. It was shown that in planta the main product of HaG8H exists most likely as inunolide, which would be the entry point for the biosynthesis of 8-epixanthatine and tomentosine. Candidate S2 from Ikezawa et al. (2011) was found to convert 8β-hydroxy-germacrene A acid to 8β-hydroxy-costunolide (eupatolide) in tobacco, but not in yeast, producing several byproducts. The name Helianthus annuus eupatolide synthase HaES is proposed accordingly. HaES has 47 % amino acid identity to the parthenolide synthase from T. parthenium (TpPTS). A classification into a new CYP71 subfamily is proposed. Two alternative metabolic routes led to 8β-hydroxy-costunolide in the expression studies in tobacco, the underlying mechanisms are discussed. Enzymes involved in STL biosynthesis were expressed in inner tissues of young Helianthus annuus plants; the induction of expression of STL biosynthesis enzymes in leaf primordia correlates with the development of capitate glandular trichomes (CGT) and STL synthesis. HaC14H was found in a chromosomal region in proximity to several P450 enzyme candidates, that share the same subfamily and the expression in capitate glandular trichomes (CGT). Therefore involvement of these enzymes in later steps of the biosynthesis of the elaborate STL structures found in CGT is likely.
Pathways of C and N turnover in soil under elevated atmospheric CO2
(2008) Dorodnikov, Maxim; Fangmeier, Andreas
In the present thesis the C and N transformations in soil as influenced by indirect effect of elevated atmospheric CO2, soil physical structure and land use change were studied in four laboratory experiments using stable-C and N isotopes, as well as soil microbiological techniques. To test the interrelations between chemical and biological characteristics of soil organic matter (SOM) as affected by land use change and elevated atmospheric CO2 an approach for SOM partitioning based on its thermal stability was chosen. In the first experiment C isotopic composition of soils subjected to C3-C4 vegetation change (grassland to Miscanthus x gigantheus, respectively) was used for the estimation of C turnover in SOM pools. In the 2nd (Free Air CO2 Enrichment ? FACE ? Hohenheim) and 3rd (FACE Braunschweig) experiments CO2 applied for FACE was strongly depleted in 13C and thus provided an opportunity to study C turnover in SOM based on its δ13C value. Simultaneous use of 15N labeled fertilizers allowed N turnover to be studied (in the 2nd experiment). We hypothesized that the biological availability of SOM pools expressed as the mean residence time (MRT) of C or N is inversely proportional to their thermal stability. Soil samples were analysed by thermogravimetry coupled with differential scanning calorimetry (TG-DSC). According to differential weight losses between 20 and 1000 °C (dTG) and energy release or consumption (DSC), SOM pools (4 to 5 depending on experiment) with increasing thermal stability were distinguished. Soil samples were heated up to the respective temperature and the remaining soil was analyzed for δ13C and δ15N by IRMS. For all three experiments the separation of SOM based on its thermal stability was not sufficient to reveal pools with contrasting turnover rates of C and N. A possible explanation for the inability of thermal oxidation for isolating SOM pools of contrasting turnover times is that the fractionation of SOM pools according to their thermal stability is close to chemical separation. In turn, it was found that chemical separations of SOM failed to isolate the SOM pools of different turnover time because different biochemical plant components (cellulose, lignin) are decomposed in a wide temperature range. Individual components of plant residues may be directly incorporated into, or even mixed with the thermal stable SOM pools and will so mask low turnover rates of these pools. To evaluate the interactions between availability of SOM for decomposition by soil microbial biomass (biological characteristic) under elevated atmospheric CO2 and protection of SOM due to the occlusion within aggregates of different sizes (physical property, responsible for SOM sequestration) we measured the activity of microbial biomass (indicated by enzyme activities) and growth strategies of soil microorganisms (fast- vs. slow growing organisms) in isolated macro- and microaggregates. The contribution of fast (r-strategists) and slowly growing microorganisms (K-strategists) in microbial communities was estimated by the kinetics of the CO2 emission from bulk soil and aggregates amended with glucose and nutrients (Substrate Induced Growth Respiration method). Although Corg and total Cmic were unaffected by elevated CO2, maximal specific growth rates were significantly higher under elevated than ambient CO2 for bulk soil, small macroaggregates, and microaggregates. Thus, we conclude that elevated atmospheric CO2 stimulated the r-selected microorganisms. Such an increase in r-selected microorganisms could increase C turnover in terrestrial ecosystems in a future elevated atmospheric CO2 environment. The activities of β-glucosidase, phosphatase and sulphatase were unaffected in bulk soil and in aggregate-size classes by elevated CO2, however, significant changes were observed in potential enzyme production after substrate amendment. After adding glucose, enzyme activities under elevated CO2 were 1.2-1.9-fold higher than under ambient CO2. This indicates an increased activity of microorganisms, which leads to accelerated C turnover in soil under elevated CO2. Significantly higher chitinase activity in bulk soil and in large macroaggregates under elevated CO2 revealed an increased contribution of fungi to turnover processes. At the same time, less chitinase activity in microaggregates underlined microaggregate stability and the difficulties for fungi hyphae penetrating them. We conclude that quantitative and qualitative changes of C input by plants into the soil at elevated CO2 affect microbial community functioning, but not its total content. Future studies should therefore focus more on the changes of functions and activities, but less on the pools. In conclusion, elevated CO2 concentrations in the atmosphere along with soil physical structure have a pronounced effect on qualitative but not quantitative changes in C and N transformations in soil under agricultural ecosystem. The physical parameters of soil such as aggregation correlate more with biological availability of SOM than the chemical properties of soil organic materials. The increase of soil microbial activity under elevated CO2 detected especially in soil microaggregates, which are supposed to be responsible for SOM preservation, prejudice sequestration of C in agroecosystems affected by elevated atmospheric CO2.
Spatial and temporal variations of microorganisms in grassland soils : influences of land-use intensity, plants and soil properties
(2019) Boeddinghaus, Runa S.; Kandeler, Ellen
Grassland ecosystems provide a wide range of services to human societies (Allan et al., 2015) and plants and soil microorganisms have been identified as key drivers of ecosystem functioning (Soliveres et al., 2016). Therefore, understanding soil microbial distributions and processes in agricultural grassland soils is crucial for characterizing these ecosystems and for predicting how they may shift in a changing environment. Yet we are only beginning to understand these complex ecosystems, which account for about 26% of the world’s terrestrial surface (FAOSTATS, 2018), making it especially urgent to gain better insights into the effects of land-use intensity on soil microbial properties and plant-microbe interactions. This thesis was conducted to evaluate the impact land-use intensity has on soil microbial biogeography of grasslands with respect to both spatial patterns and temporal changes in soil microbial abundance, function (in terms of enzyme activities), and community composition. It also investigated the relationships between plants and the spatial and temporal distributions of soil microorganisms. Thereby both, land-use intensity effects and plant-microbe interactions, were assessed in light of ecological niche and neutral theory. This thesis is based on three observational studies conducted on from one to 150 continuously farmed, un-manipulated grassland sites in three regions of Germany within the Biodiversity Exploratories project (DFG priority program 1374). The first study assessed the effects of land-use intensity and physico-chemical soil properties on the spatial biogeography of soil microbial abundance and function in 18 grasslands sites from two of the three regions, sampled at one time point. The second study analyzed spatial and temporal distributions of alpha- and beta-diversity of arbuscular mycorrhizal fungi in a low land-use intensity grassland with six sampling time points across one season. The third study investigated both legacy and short-term change effects of land-use intensity, soil physico-chemical properties, plant functional traits, and plant biomass properties on temporal changes in soil microbial abundance, function, and community composition in 150 grassland sites across three regions, with particular regard to direct and indirect land-use intensity effects. Although the three studies used different approaches and assessed different soil microbial properties, general patterns were detectable. Abiotic soil properties, namely pH, nitrogen content, texture, and bulk density played fundamental roles for spatial and temporal microbial biogeography. Since these factors were specific and unique for each investigated site, they formed the background based on which other processes occurred. In addition to abiotic soil properties, impacts of land-use intensity and plants were detected, though to various degrees in the three studies. Land-use intensity played a much smaller role than anticipated in the first and third study. No influence on the spatial distribution of soil microbial abundance and function could be detected in the first study. In the third study, short-term changes in and legacy effects of land-use intensity played a minor role with respect to short-term changes in soil microbial abundance, function, and community composition. Where detected, changes in land-use intensity had a direct and negative effect on soil microbial properties in structural equation modelling; i.e., increases in land-use intensity reduced, e.g., soil microbial enzyme activities, while legacy effects of land-use intensity were shown to act both directly and indirectly on soil microbial properties. Thereby indirect legacy effects were mediated via plant functional traits. Only one of the three studies detected minor plant diversity effects on soil microbial properties. Instead, functional properties of the plant communities, i.e., plant functional traits, biomass, and nutritional quality, were significantly related to spatial and temporal distributions of soil microorganisms. Finally, the findings of the three studies suggest that processes related to niche and neutral theory both drive spatial and temporal patterns of soil microbial properties at the investigated plot scale (up to 50 m × 50 m). This thesis concluded that in order to gain deeper insights into the complex functions and processes occurring in grassland ecosystems, a multidisciplinary approach investigating fundamental physico-chemical site characteristics, microbial soil properties, and plants is necessary. The results of the thesis suggest that focus be turned to functional properties of plant and microbial communities, as they are closely intermingled, provide more detailed insights into plant-microbe interactions, and are able to reflect effects of human impacts on grassland soils better than diversity measures.
The effect of enzyme additives on the anaerobic digestion of energy crops
(2014) Brulé, Mathieu; Jungbluth, Thomas
The mechanisms governing the efficiency of commercial fiber-degrading enzyme additives at improving the anaerobic digestion of energy crops were investigated. Standard batch digestion trials (BMP-assays) were performed using the Hohenheim Biogas Test (HBT) on maize straw, maize corn, and rye silage with different inocula. These BMP-assays showed no significant effect of enzyme additives (including commercial cellulase, xylanase, pectinase, laccase) on the methane production rate. However, batch digestion trials performed on grass silage under suboptimal conditions with inoculum of weak bacterial activity revealed significant increases of methane production up to 40%. In another experiment semi continuous acidogenic fermentation was performed in laboratory digesters with maize silage and water added for dilution at OLR 4 kg VS/(m3 × d), HRT 5 days, with the medium kept in the pH range 5 5.5 through quicklime addition. Enzyme additives at a dosage of 10 g/kg substrate VS significantly increased VFA release (+10%) as well as gas production, including H2 production (+20%). The results show that the efficiency of enzyme additives in anaerobic digestion depends on substrate (fibre length and composition) and process parameters (retention time, loading rate, pH, efficiency of bacterial substrate degradation).