Second-generation bioethanol of hydrothermally pretreated stover biomass from maize genotypes

Twelve maize genotypes, were agronomically evaluated and their stover hydrothermally pretreated in a temperature range of 210–225 °C to assess the effects of genotype and pretreatment severity on stover recalcitrance toward bioethanol conversion. Maize genotypes exhibited significant variation for biomass yield and all agronomic evaluated, while among all cell wall constituents measured in the unpretreated stover, only ash content showed differences among genotypes. The pretreatment severities assayed impacted most stover compositional traits, and the glucose recovered after enzymatic hydrolysis displayed a similar profile among genotypes with similar genetic background. Harsher pretreatment conditions maximized the potential cellulosic bioethanol production (208–239 L/t), while the mildest maximized the bioethanol from the hemicellulosic hydrolysates (137–175 L/t). Consequently, when both pentose and hexose sugars were considered, the total potential bioethanol produced at the lowest and highest pretreatment temperatures was similar in all genotypes (292–358 L/t), indicating that the lowest temperature (210 °C) was the optimal among all assayed. Importantly, the ranking of genotypes for bioethanol yield (L/ha) closely resembled the ranking for stover yield (t/ha), indicating that breeding for biomass yield would increase the bioethanol production per hectare regardless of the manufacturing process. Similarly, the genetic regulation of corn stover moisture is possible and relevant for efficient energy production as biomass moisture has a potential impact on stover transportation, storage and processing requirements. Overall, these results indicate that local landrace populations are important genetic resources to improve cultivated crops, and that simultaneous breeding for production of grain and stover bioethanol is possible in corn.     

Corn stover ethanol yield as affected by grain yield,Bt trait,and environment

Literature values for glucose release from corn stover are highly variable which would likely result in tremendous variability in bio-refinery ethanol yield from corn stover feedstock. A relatively recent change in United States corn genetics is the inclusion of the Bacillus thuringiensis (Bt) trait, which now accounts for three-fourths of all US planted corn acreage. The objective of this study was to evaluate the effect of corn grain yield, inclusion of the Bt trait, and location environment on corn stover quality for subsequent ethanol conversion. Two hybrid pairs (each having a Bt and non-Bt near-isoline) were analyzed giving a total of 4 hybrids. In 2010 and 2011, field plots were located in Michigan at four latitudinal differing locations in four replicated plots at each location. Stover composition and enzymatic digestibility was analyzed and estimated ethanol yield (g g⁻¹) was calculated based on hydrolyzable glucan and xylan levels. Analysis showed that there were no significant differences in total glucose or xylose levels nor in enzymatically hydrolyzable glucan and xylan concentrations between Bt corn stover and the non-Bt stover isolines. Regression analyses between corn grain yield (Mg ha⁻¹) and corn stover ethanol yield (g g⁻¹) showed an inverse relationship indicative of a photosynthate source-sink relationship. Nevertheless, the quantity of stover produced was found to be more critical than the quality of stover produced in maximizing potential stover ethanol yield on a land area basis.     

The effects of physical and chemical preprocessing on the flowability of corn stover

Continuous and reliable feeding of biomass is essential for successful biofuel production. However, the challenges associated with biomass solids handling are commonly overlooked. In this study, we examine the effects of preprocessing (particle size reduction, moisture content, chemical additives, etc.) on the flow properties of corn stover. Compressibility, flow properties (interparticle friction, cohesion, unconfined yield stress, etc.), and wall friction were examined for five corn stover samples: ground, milled (dry and wet), acid impregnated, and deacetylated. The ground corn stover was found to be the least compressible and most flowable material. The water and acid impregnated stovers had similar compressibilities. Yet, the wet corn stover was less flowable than the acid impregnated sample, which displayed a flow index equivalent to the dry, milled corn stover. The deacetylated stover, on the other hand, was the most compressible and least flowable examined material. However, all of the tested stover samples had internal friction angles >30°, which could present additional feeding and handling challenges. All of the “wetted” materials (water, acid, and deacetylated) displayed reduced flowabilities (excluding the acid impregnated sample), and enhanced compressibilities and wall friction angles, indicating the potential for added handling issues; which was corroborated via theoretical hopper design calculations. All of the “wetted” corn stovers require larger theoretical hopper outlet diameters and steeper hopper walls than the examined “dry” stovers.     

Improved methods for the determination of drying conditions and fraction insoluble solids (FIS) in biomass pretreatment slurry

Accurate and precise chemical characterization of biomass feedstocks and process intermediates is a requirement for successful technical and economic evaluation of biofuel conversion technologies. The uncertainty in primary measurements of the fraction insoluble solid (FIS) content of dilute acid pretreated corn stover slurry is the major contributor to uncertainty in yield calculations for enzymatic hydrolysis of cellulose to glucose. This uncertainty is propagated through process models and impacts modeled fuel costs. The challenge in measuring FIS is obtaining an accurate measurement of insoluble matter in the pretreated materials, while appropriately accounting for all biomass derived components. Three methods were tested to improve this measurement. One used physical separation of liquid and solid phases, and two utilized direct determination of dry matter content in two fractions. We offer a comparison of drying methods. Our results show utilizing a microwave dryer to directly determine dry matter content is the optimal method for determining FIS, based on the low time requirements and the method optimization done using model slurries.     

Improvement of enzymatic saccharification of Populus and switchgrass by combined pretreatment with steam and wet disk milling

To reduce the recalcitrance of lignocellulosic biomass for subsequent biological processing, we pretreated energy crop feedstocks with mild steam treatment (ST; 130 and 150 °C for 60 min) and wet disk milling (WDM). We tested two phylogenetically different, but typical energy crop feedstocks: Populus trichocarpa and switchgrass (Panicum virgatum). WDM after ST facilitated the fibrillation of both types of biomass, resulting in an increase of specific surface area, improved enzymatic saccharification yield, and decrease in cellulose crystallinity. After steam treatment at 150 °C followed by 17 cycles of WDM, enzymatic hydrolysis resulted in almost complete glucan to glucose conversion in both feedstocks.     

Anaerobic digestion of corn silage on a commercial scale: Differential utilization of its chemical constituents and characterization of the solid digestate

The utilization of different chemical constituents of corn silage during industrial-scale anaerobic digestion was determined. Corn silage together with the resulting solid digestate generated during biogas production were collected from an industrial plant during a regular operating period. Moisture, water and ethanol extractives, ash, total nitrogen, starch, cellulose, the monomeric composition of hemicellulose, acid soluble and acid insoluble lignin were measured in both corn silage and corn silage solid digestate. The relative consumption of each component of corn silage during its anaerobic digestion was estimated with reference to acid insoluble lignin. It was assumed that lignin was not digested throughout the process. Starch and large fractions of extractives and acid soluble lignin were digested. In contrast, the digestion of cellulose and particularly hemicellulose were limited (40% and 29% respectively). Of the hemicellulose monomers, xylose was the least digested (20%). The present work shows that the digestate produced by commercial corn-silage anaerobic digestion contains a notable quantity of cell wall polymers. These could potentially be used in biorefinery processes, e.g. ethanol and xylo-oligosaccharide production.     

Compositional differences among upland and lowland switchgrass ecotypes grown as a bioenergy feedstock crop

Feedstock quality mainly depends upon the biomass composition and bioenergy conversion system being used. Higher cellulose and hemicellulose concentrations are desirable for biochemical conversion, whereas higher lignin is favored for thermochemical conversion. The efficiency of these conversion systems is influenced by the presence of high nitrogen and ash concentrations. Switchgrass (Panicum virgatum L.) varieties are classified into two ecotypes based on their habitat preferences, i.e., upland and lowland. The objectives of this study were to quantify the chemical composition of switchgrass varieties as influenced by harvest management, and to determine if ecotypic differences exist among them. A field study was conducted near Ames, IA during 2012 and 2013. Upland (‘Cave-in-Rock’, ‘Trailblazer’ and ‘Blackwell’) and lowland switchgrass varieties (‘Kanlow’ and ‘Alamo’) were grown in a randomized block design with six replications. Six biomass harvests were collected at approximately 2-week intervals each year. In both years, delaying harvest increased cellulose, hemicellulose and lignin concentrations while decreasing nitrogen and ash concentrations in all varieties. On average, Kanlow had the highest cellulose and hemicellulose concentration (354 and 321 g kg⁻¹ DM respectively), and Cave-in-Rock had the highest lignin concentration (33 g kg⁻¹ DM). The lowest nitrogen and ash concentrations were observed in Kanlow (14 and 95 g kg⁻¹ DM respectively). In general, our results indicate that delaying harvest until fall improves feedstock quality, and ecotypic differences do exist between varieties for important feedstock quality traits. These findings also demonstrate potential for developing improved switchgrass cultivars as bioenergy feedstock by intermating lowland and upland ecotypes.     

Upgraded biofuel from residue biomass by Thermo-Catalytic Reforming and hydrodeoxygenation

Research is focused on the utilisation of waste or residue biomass for bioenergy conversion. A promising conversion technology for the production of liquid biofuels from residue biomass is a process called Thermo-Catalytic Reforming (TCR^®​) which is a combination of prior thermal treatment of the biomass at mild temperatures (intermediate pyrolysis) followed by a second catalytic treatment step at elevated temperatures (reforming). This article focuses on the conversion of TCR^® liquids from digestate as a feedstock for subsequent hydrocarbon production. The generated bio-oil showed a lower heating value of 34.0 MJ kg⁻¹ with an oxygen content of 7.0% and a water content of 2.2%. The bio-oil was hydrodeoxygenated using an industrial NiMo–Al₂O₃ catalyst at temperatures of 503 K–643 K and a pressure of 14 MPa. The hydrodeoxygenated bio-oil reached a lower heating value of 42.3 MJ kg⁻¹ with an oxygen content below 0.8 mg kg⁻¹ and water content of 30 ppm. Product yields and catalyst life give confidence that upgrading of the TCR^®​ bio-oil offers a suitable option to meet the high standards of common fuels.     

Changes in feedstock quality in willow chip piles created in winter from a commercial scale harvest

Storage and handling are important facets of biomass logistics because there are associated costs and biomass properties can change significantly as material proceeds through the supply chain. Thus, this aspect of biomass supply systems requires continued study. Shrub willow chips were harvested, and used to create six piles that each contained between 10 and 22 Mg (fresh biomass). Material was monitored for several months in temporary storage to assess changes in biomass quality (moisture, ash and energy content). Internal pile temperatures increased due to biological activity and conditions within a pile quickly differentiated based on location (shell, core, top, and side). Mean moisture content increased from 42 to 47% (mass fraction) between harvest and delivery of the chips, but ranged between 37 and over 60% over the next three months depending on pile location with the shell generally drier than the core. Mean ash content increased 1 to 2% points (mass fraction) between harvesting, reloading and delivery to the trial location, but became more variable during storage. Higher heating values (HHV) were stable between 18.6 and 19.0 MJ kg⁻¹ over the six months, but lower heating values (LHV) ranged between 8.6 and 11.7 MJ kg⁻¹ and mirrored changes in moisture content. There was minimal change in chip quality over two months, but quality became more variable over longer time periods. This period could be extended, and negative effects on chip quality could be mitigated, by improving storage methods, blending different types of chips, or employing pretreatments.     

Effects of delayed winter harvest on biomass yield and quality of napiergrass and energycane

Napiergrass (Cenchrus purpureus (Schumach.) Morrone) and energycane (Saccharum hyb.) are perennial grasses that are well-suited for biomass production in the southeastern USA. The purpose of this study was to determine the effects of delayed winter harvest on biomass yield and quality of these grasses. The study was conducted on two adjacent sites near Midville, GA. Each site used a split-plot design with four replications, with species as the main plot, and harvest times (December, January, or February) as sub-plots. Dry matter (DM) yields were measured by mechanical harvesting, and a sample of biomass was taken from each harvest for determination of ethanol production by simultaneous saccharification and fermentation (SSF). Biomass moisture, N, P, K, and ash mass fractions were also measured. Energycane DM yields were stable from December (46.8 Mg ha⁻¹) to January (42.9 Mg ha⁻¹), but then declined (36.8 Mg ha⁻¹), while napiergrass yields declined sharply from December (47.0 Mg ha⁻¹) to January (35.0 Mg ha⁻¹). Napiergrass moisture mass fraction was reduced by an average of 18% in February harvests compared to December. Mass fractions of N, K, and ash tended to decrease with later harvesting, but sometimes increased due to changes in biomass composition. Delaying harvest of napiergrass from December to January reduced N removal by an average of 144 kg ha⁻¹, while delaying harvest of energycane to February reduced N removal by an average of 54 kg ha⁻¹. In SSF, later-harvested energycane produced less ethanol per unit of DM while napiergrass was less affected by harvest date.     

Periodic peristalsis enhancing the high solids enzymatic hydrolysis performance of steam exploded corn stover biomass

Enzymatic hydrolysis beyond 15% solid loading offers many advantages such as increased sugar and ethanol concentrations and decreased capital cost. However, difficult mixing and handling limited its industrialized application. A novel intensification method, periodic peristalsis, had been exploited to improve the high solids enzymatic hydrolysis performance of steam exploded corn stover (SECS). The optimal steam explosion conditions were 200 °C and 8 min, under which glucan and xylan recovery was 94.3% and 64.8%, respectively. Glucan and xylan conversions in periodic peristalsis enzymatic hydrolysis (PPEH) were 28.0–38.5% and 25.0–36.0% higher than those in static state enzymatic hydrolysis with solid loading increasing from 1% to 30%, respectively, while they were 1.0–11.2% and 3.0–9.2% higher than those in incubator shaker enzymatic hydrolysis (ISEH). Glucan and xylan conversion in PPEH at 21% solid loading reached 71.2% and 70.3%, respectively. Periodic peristalsis also facilitated fed-batch enzymatic hydrolysis of which SECS was added completely before transition point. Results presented that PPEH shortened the transition point time from solid state to slurry state, decreased the viscosity of hydrolysis mixture, and reduced the denaturation effect of enzymes compared with ISEH, and hence improve the high solids enzymatic hydrolysis efficiency.     

Improving methane production from wheat straw by digestate liquor recirculation in continuous stirred tank processes

Wheat straw is an abundant, cheap substrate that can be used for methane production. However, the nutrient content in straw is inadequate for methane fermentation. In this study, recycling digestate liquor was implemented in single-stage continuous stirred tank processes for enrichment of the nutrient content of straw with the aim of improving the methane production. The VS-based organic loading rate was set at 2 g/(L d) and the solid retention time at 40 days. When wheat straw alone was used as the substrate, the methane yields achieved with digestate liquor recycling was on average 240 ml CH₄/g VS giving a 21% improvement over the processes without recycling. However, over time, the processes suffered from declining methane yields and poor stability evidenced by low pH. To maintain process stability, wheat straw was co-digested with sewage sludge or supplemented with macronutrients (nitrogen and phosphorous). As a result, the processes with digestate liquor recycling could be operated stably, achieving methane yields ranging from 288 to 296 ml CH₄/g VS. Besides, the processes could not be operated sturdily with supplementation of macronutrients without digestate liquor recycling. The highest methane yield (296 ± 16 ml CH₄/g VS) was achieved by co-digestion with sewage sludge plus recycling of digestate liquor after filtration (retention of nutrients and microorganisms). This was comparable to the maximum expected methane yield of 293 ± 13 ml CH₄/g VS achieved in batch test. The present study therefore demonstrated that digestate liquor recycling could lead to a decreased dilution of vital nutrients from the reactors thereby rendering high process performance and stability.     

Growth,yield, fiber content and lodging resistance in eight varieties of Cenchrus purpureus (Schumach.) Morrone intended as energy crop

Growth, biomass yield, fiber content and lodging resistance were studied, during a six month growth period, for eight varieties of Cenchrus purpureus, intended as energy crop, in Veracruz, Mexico. Then, only yield at day 182 was assessed for two additional years. The varieties were: CT115 (CT), African Cane (AC), Taiwan (TAI), King Grass (KG), Vruckwona (VRU), Roxo (RX), OM22 (OM) and Cameroon (CAM). Local weather is warm and sub-humid, historical data for monthly average temperature and annual rainfall were 25.8 °C and 1142 mm, respectively. Height, diameter and light interception were measured monthly from day 65–185. At day 185, biomass yield and tiller density were measured. Number of lying tillers was counted to estimate lodging resistance. Cellulose and hemicellulose content were estimated in leaf and stem. No differences were found for dry matter yield or stem yield at day 185 in the first year. Regarding the next two years, TAI yielded above CT, OM or ROX. Average dry matter yield was higher in the second year than in the establishment cycle (38.6 vs 21.1 Mg ha⁻¹), but decreased in the third year (32.2 Mg ha⁻¹). In both stem and whole plant, AC and KG showed higher hemicellulose content than RX, OM or CT; while AC and VRU had higher cellulose than RX in stem, or than CT in the whole plant. Furthermore, varieties AC, KG, VRU and TAI were resistant to lodging and had a higher fiber content, so they are recommended as energetic crops.     

Cob biomass supply for combined heat and power and biofuel in the north central USA

Corn (Zea mays L.) cobs are being evaluated as a potential bioenergy feedstock for combined heat and power generation (CHP) and conversion into a biofuel. The objective of this study was to determine corn cob availability in north central United States (Minnesota, North Dakota, and South Dakota) using existing corn grain ethanol plants as a proxy for possible future co-located cellulosic ethanol plants. Cob production estimates averaged 6.04 Tg and 8.87 Tg using a 40 km radius area and 80 km radius area, respectively, from existing corn grain ethanol plants. The use of CHP from cobs reduces overall GHG emissions by 60%–65% from existing dry mill ethanol plants. An integrated biorefinery further reduces corn grain ethanol GHG emissions with estimated ranges from 13.9 g CO₂ equiv MJ⁻¹ to 17.4 g CO₂ equiv MJ⁻¹. Significant radius area overlap (53% overlap for 40 km radius and 86% overlap for 80 km radius) exists for cob availability between current corn grain ethanol plants in this region suggesting possible cob supply constraints for a mature biofuel industry. A multi-feedstock approach will likely be required to meet multiple end user renewable energy requirements for the north central United States. Economic and feedstock logistics models need to account for possible supply constraints under a mature biofuel industry.     

Combined ethanol and methane production from switchgrass (Panicum virgatum L.) impregnated with lime prior to steam explosion

Pretreatments are crucial to achieve efficient conversion of lignocellulosic biomass to soluble sugars. In this light, switchgrass was subjected to 13 pretreatments including steam explosion alone (195 °C for 5, 10 and 15 min) and after impregnation with the following catalysts: Ca(OH)₂ at low (0.4%) and high (0.7%) concentration; Ca(OH)₂ at high concentration and higher temperature (205 °C for 5, 10 and 15 min); H₂SO₄ (0.2% at 195 °C for 10 min) as reference acid catalyst before steam explosion. Enzymatic hydrolysis was carried out to assess pretreatment efficiency in both solid and liquid fraction. Thereafter, in selected pretreatments the solid fraction was subjected to simultaneous saccharification and fermentation (SSF), while the liquid fraction underwent anaerobic digestion (AD). Lignin removal was lowest (12%) and highest (35%) with steam alone and 0.7% lime, respectively. In general, higher cellulose degradation and lower hemicellulose hydrolysis were observed in this study compared to others, depending on lower biomass hydration during steam explosion. Mild lime addition (0.4% at 195 °C) enhanced ethanol in SSF (+28% than steam alone), while H₂SO₄ boosted methane in AD (+110%). However, methane represented a lesser component in combined energy yield (ethanol, methane and energy content of residual solid). Mild lime addition was also shown less aggressive and secured more residual solid after SSF, resulting in higher energy yield per unit raw biomass. Decreased water consumption, avoidance of toxic compounds in downstream effluents, and post process recovery of Ca(OH)₂ as CaCO₃ represent further advantages of pretreatments involving mild lime addition before steam explosion.     

Assessing sustainable forest biomass potential and bioenergy implications for the northern Lake States region,USA

Forestlands in the United States have tremendous potential for providing feedstocks necessary to meet emerging renewable energy standards. The Lake States region is one area recognized for its high potential of supplying forest-derived biomass; however, the long-term availability of roundwood harvests and associated residues from this region has not been fully explored. Better distribution and temporal availability estimates are needed to formulate emerging state policies regarding renewable energy development. We used a novel predictive methodology to quantify sustainable biomass availability and likely harvest levels over a 100-year period in the Lake States region. USDA Forest Inventory and Analysis estimates of timberland were combined with published growth and yield models, and historic harvest data using the Forest Age Class Change Simulator (FACCS) to generate availability estimates. Monte-Carlo simulation was used to develop probability distributions of biomass harvests and to incorporate the uncertainty of future harvest levels. Our results indicate that 11.27–15.71 Mt y⁻¹ dry roundwood could be sustainably harvested from the Lake States. Assuming 65% collection rate, 1.87–2.62 Mt y⁻¹ residue could be removed, which if substituted for coal would generate 2.12–2.99 GW h of electricity on equivalent energy basis while reducing GHG (CO₂e) emission by 1.91–2.69 Mt annually. In addition to promoting energy security and reducing GHG emissions, forest residues for energy may create additional revenues and employment opportunities in a region historically dependent on forest-based industries.     

Effect of doping pretreated corn stover conditions on yield of bioethanol in immobilized cell systems

The surface characteristics of immobilized yeast before and after adding CO₂-laser pretreated corn stover (LPCS) substrates were investigated using bioethanol production. Response surface methodology (RSM), based on the Box–Behnken design (BBD) for experiments, was used to optimize the doping condition. An optimum experimental condition was obtained at pH 4.5, 2.08% yeast concentration, and 0.20% LPCS substrates. Under this condition, doping LPCS increased the yield of bioethanol from 53% to 84%, which matched the predicted value. After doping LPCS, the results of inverted microscope (IM) and atomic force microscopy (AFM) illustrated that the immobilized gel beads changed from rod-like in shape with a smooth surface to a larger rod-like ultrastructure with a rougher surface. The yield was relatively stable within 28 d, with a downward trend subsequently appearing.     

Enhancement of lipid productivity in green microalgae Chlorella sp. via fast neutron irradiation

Microalgae is becoming a promising candidate for biofuel production and energetic lipid production, which urges the need to develop efficient methods for improvement of the lipid production. In this study, the neutron irradiation was employed to improve lipid production under different dosage irradiation. Effects of different dosages (No.1:8.14 × 10⁹, No.2:5.64 × 10⁸, No.4:9.42 × 10⁷ n/cm²) were compared by investigating associated algae cell growth, total lipid production, metabolization and enzyme activity. Dosages No.2 and No.4 improved the lipid content without negative influence on the growing, whereas irradiations shortened the period of biomass accumulation. The neutron irradiation in No.2 accelerated the synthesis of lipid from the carbohydrate transition process. In addition, the neutron irradiation not only increased the total lipid production by 20% but also speeded up the reaction rate to reach the maximal total lipid production in 28 days. Our results can provide a better understanding of the lipid production in microalgae Chlorella sp. by neutron irradiation, which are critical for evaluation of neutron irradiation based technologies for the sustainable and renewable biofuel production.     

GIS-based assessment of sustainable crop residue potentials in European regions

In this paper a novel model based on a geographic information system (GIS) is presented for the assessment of sustainable crop residue potentials. The approach is applied to analyse the amount and the spatial distribution (1 km × 1 km grid cells) of cereal straw, root crop and oil plant residues for five European regions, considering spatially differentiated environmental sustainability issues, i.e. organic carbon content in topsoil, soil erodibility, and protected areas. The maximum sustainable residue potential varies strongly between the regions and residue types. In the scenarios Basis and Restrict, it accounts for 45–59% and 24–48% of the theoretical potential respectively without considering competing uses. Among the crop residues, cereal straw shows the highest energy potential in all regions under investigation. In terms of wet mass it accounts for 3.7 Mio. t_wet/a in North Rhine-Westphalia, 1.6 Mio. t_wet/a in Île-the-France, 1.2 Mio. t_wet/a in Wallonia, 0.9 Mio. t_wet/a in West Midlands, and 0.3 Mio. t_wet/a in South Netherlands (scenario Basis). Our survey shows that spatially differentiated potential estimations and the inclusion of crop residues other than cereal straw are urgently needed to improve the present rough estimations for crop residues which can be used in a sustainable way. The rather high spatial resolution of our analyses particularly allows for the support of regional stakeholders and prospective investors when it comes to questions of regional availability of biomass resources, transport distances to biomass conversion plants, and identification of suitable plant sites and sizes, respectively.     

Anaerobic digestion of maize and cellulose under thermophilic and mesophilic conditions – A comparative study

The aim of this study was to advance in understanding of digestion process of energy crops. Cellulose and maize silage were fermented in batch mode at mesophilic (38 °C) and thermophilic (55 °C) conditions and corresponding organic loads of 5.5 ± 0.2 kgVS/m³, 11.2 ± 0.3 kgVS/m³ and 16.7 ± 0.4 kgVS/m³.For both substrates more stable and faster digestion took place at 38 °C. Due to complex structure maize degradation was characterized by varying digestion rate and longer total digestion time resulting form breakdown of hard-degradable fractions. The digestion retard at increased OLRs of cellulose and lower degradation level obtained for all cellulose series confirm a higher overloading potential for systems dealing with single-component-substrates but also the enhanced sensitivity of such systems to any inconvenient digestion conditions.Based on observed patterns of volatile fatty acids and oxidation-reduction potential, different fermentation mechanisms can be concluded for cellulose and maize, but also for different temperature modes. Conversion of maize at highly reductive conditions with increased concentrations of butyric acid was accompanied by much higher activity of hydrogenotrophic methanogens than for cellulose digestion.Two factors showed a strong potential to influence test results: an insufficient VS content of inoculum, which caused reduced biogas yields, and a high natural biodiversity of maize silage, resulting in higher biogas yields than calculated based on the maize composition.