Carbohydrate-rich Substrates Research Articles

Presence of lactic acid bacteria in hydrogen production by dark fermentation: competition or synergy.

Dark fermentation in mixed cultures has been extensively studied due to its great potential for sustainable hydrogen production from organic wastes. However, microbial composition, substrate competition, and inhibition by fermentation products can affect hydrogen yield and production rates. Lactic acid bacteria have been identified as the key organisms in this process. On one hand, lactic acid bacteria can efficiently compete for carbohydrate rich substrates, producing lactic acid and secreting bacteriocins that inhibit the growth of hydrogen-producing bacteria, thereby decreasing hydrogen production. On the other hand, due to their metabolic capacity and synergistic interactions with certain hydrogen-producing bacteria, they contribute positively in several ways, for example by providing lactic acid as a substrate for hydrogen generation. Analyzing different perspectives about the role of lactic acid bacteria in hydrogen production by dark fermentation, a literature review was done on this topic. This review article shows a comprehensive view to understand better the role of these bacteria and their influence on the process efficiency, either as competitors or as contributors to hydrogen production by dark fermentation.

Performance of Hermetia illucens reared on pure, mixed, and sequentially mixed organic wastes regionally available in central Argentina

Abstract Black soldier fly larvae (Hermetia illucens L.) are proficient decomposers of organic matter. We compared experiments with local substrates containing: domestic residues (DR), and brewery wastes (BW) in pure form, or mixed with in two different proportions (BW70 or BW30), and two sequential diets in which larvae were fed first DR and then BW (DR/BW) or vice versa (BW/DR). The effects of the diets on life cycle parameters for mass production of H. illucens larvae were evaluated using general linear mixed models, ANOVA and Kruskal-Wallis. The pure DR diet significantly prolonged the developmental period (47.8 days), although the pupal stage was shorter in larvae fed on this diet (13.8 days). Larval survival was similar between treatments (over 85%), but pupal survival tended to be higher in the mixed diets (over 96%). Larval weights differed significantly between treatments, with mixed diets promoting greater weight gains than pure and sequential diets. Larvae fed a DR/BW diet gained more weight than those fed a BW/DR diet. The highest mean larval weights (0.23 g) were observed in the mixed diet BW70, followed by the sequential diet DR/BW (0.22 g). Mixed diet BW70 also resulted in larger wings and bodies in both sexes. Sequential diet BW/DR and pure diet DR resulted in the smallest adult sizes. Sex ratios varied, favouring male emergence (1.8 M:0.2 F) in pure diet DR and in both sequential mixed diets (1.7 M:0.3 F and 1.3 M:0.7 F, respectively). This study highlights the importance of higher total protein plus carbohydrate concentrations in diets for optimal larval rearing, favouring mixed diets BW70. Carbohydrate-rich substrates should be provided earlier in the cycle to benefit larval and adult performance. Locally available waste vegetable or fruit peels, brewery waste, and spent yerba mate proved to be viable substrates for larval rearing, providing economic and environmental benefits for H. illucens rearing.

Sulphite addition during steam pretreatment enhanced both enzyme-mediated cellulose hydrolysis and ethanol production

Sulphite addition during steam pretreatment of softwoods under acidic, neutral and alkaline conditions was assessed to try to minimize lignin condensation. Although pretreatment under neutral/alkaline conditions resulted in effective lignin sulphonation, non-uniform size reduction was observed. In contrast, acidic sulphite steam treatment at 210 °C for 10 min resulted in homogenous particle size reduction and water-insoluble component that was 62% carbohydrate and 33% lignin. This carbohydrate-rich substrate was readily hydrolyzed and fermented which indicated the lack of fermentation inhibitors in the steam-pretreated whole slurry. The use of high solid loading (25% w/v) resulted in a hydrolysis yield of 58% at an enzyme loading of 40 mg protein/g glucan and efficient fermentation (46.6 g/L of ethanol). This indicated that the addition of acidic sulphite at the steam pretreatment of softwoods improved both the enzymatic hydrolysis and fermentation of steam-pretreated whole slurries.Graphical

Auto-selection of microorganisms of sewage sludge used as an inoculum for fermentative hydrogen production from different substrates

When mixed organic waste is used for hydrogen production by dark fermentation, the microbial community which is most adapted to the actual biopolymer composition of the substrate is auto-selected. In this research, six substrates simulating different biopolymers (proteins, fats, carbohydrates) and their mixtures were used to enrich hydrogen-producing bacteria adapted to these substrates from non-pretreated sewage sludge. Phylum Firmicutes dominated in the microbial community (67–100%) regardless of the substrate used, as was shown by high-throughput sequencing. Microbial diversity was low when using carbohydrate-rich substrates and the microbial community was mainly represented by Ruminococcus (26–90%) and Thermoanaerobacterium (6–67%). Dark fermentation of fats and proteins was characterized by higher microbial diversity. Thermoanaerobacterium (21%), Thermobrachium (19%), Tepidiphilus (16%) and Acetomicrobium (14%) dominated when using fats, while Thermobrachium (34%), Acetomicrobium (16%) and Clostridium sensu stricto 7 (12%) dominated when using proteins, as substrate. Different microbial communities and substrates resulted in diverse process performance and metabolic pathways. Dark fermentation of starch achieved the maximum hydrogen yield of 138 mL/g volatile solids with 60.4% hydrogen content in biogas. The dominance of genus Ruminococcus was thought to be responsible for the highest hydrogen production. Minor quantities of methane from proteins and fats were produced by Methanothermobacter and Methanosarcina. Based upon the stable 13C isotope analysis, the hydrogenotrophic pathway was a slightly more predominant methane formation route than the others considered.

Process Design Guided by Life Cycle Assessment to Reduce Greenhouse Gas-Related Environmental Impacts of Food Processing

Food processing can generate large amounts of carbohydrate-rich waste that inevitably has environmental and social impacts. Meanwhile, certain heterotrophic marine microorganisms, including algae and thraustochytrids, have the potential to convert carbohydrate-rich substrates into oil-rich biomass over relatively short time frames. To assess the merits of this apparent synergy, an initial conceptual process was developed based on the use of raw potato processing waste as feed in an algal bioreactor to produce bio-oil for further use within the food industry. A practical flowsheet was established with a conventional 200 kL bioreactor whereby the unit processes were identified, the mass balance developed, and estimates made of the various material and energy demands. These inputs were used to develop a baseline life cycle assessment (LCA) model and to identify opportunities for reducing environmental impacts. With the functional unit (FU) being 1 tonne cooking oil, the baseline configuration had a greenhouse gas (GHG) footprint of 2.4 t CO2-e/FU, which is comparable to conventional process routes. More detailed LCA revealed that electricity for stirring the bioreactor contributed approximately 78% of the total GHG footprint. By adjusting the operating conditions, the most promising scenario produced 0.85 t CO2-e/FU—approximately four times less than the conventional process—and shows the potential advantages of applying LCA as a tool to develop and design a new production process.

A novel d-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar d-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize d-xylose through expression of heterologous d-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new d-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on d-xylose media. The d-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L−1) appeared not to inhibit d-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for d-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

Effects of biochar addition on the anaerobic digestion of carbohydrate-rich, protein-rich, and lipid-rich substrates

ABSTRACTAlthough biochar addition into the anaerobic digestion of food waste (FW) is an efficient means to enhance methane production, the effects of biochar on various FW components remain unclear. Laboratory batch experiments were conducted to investigate the impact of sewage sludge-derived biochar (SSB) supplementation on the anaerobic digestion (AD) of major FW components, including carbohydrate-rich, protein-rich, and lipid-rich substrates. The lag phase of AD with the carbohydrate-rich substrate was 48.6% shorter when SSB was added, and the cumulative methane yield was 4.74 times higher compared to AD without biochar. SSB supplementation also increased the rate of methane production from the lipid-rich substrate. However, the effect of SSB addition on AD of the protein-rich substrate was minor. Analysis of the microbial communities revealed that methanogen growth was enhanced during AD of the carbohydrate-rich and lipid-rich substrates, but not the protein-rich substrate, following SSB supplementation. Also, the most dominant methanogenic genus varied with the substrates. SSB addition promoted the growth of hydrolytic and fermentative bacteria, particularly phylum Bacteroidetes.Implications: Biochar supplementation has been studied to overcome the shortcomings of anaerobic digestion (AD). However, the effects of biochar on different substrates remain unclear. This study compared carbohydrate-rich, protein-rich, and lipid-rich substrates in anaerobic digestion with sewage sludge-derived biochar (SSB). SSB supplementation improved methane generation from all but the protein-rich substrate. The study results imply that the effect of SSB addition on AD varied with the substrate due to the substrates underwent different degradation processes with different microbial communities.

Examining trophic-level nematode community structure and nitrogen mineralization to assess local effective microorganisms’ role in nitrogen availability of swine effluent to forage crops

Culturing and using effective microorganisms from locally derived microorganisms has become a common practice employed by farmers throughout Central America as well as parts of South America and Southeast Asia to control foul odors and flies around compost piles and livestock facilities. These local, effective microorganisms (LEM) are applied in a concentrated solution fermented from carbohydrate-rich substrates inoculated with locally-sourced, actively decomposing leaf litter. Despite its growing prevalence among small-scale farmers in some parts of the world, there is little published research that explores the beneficial or detrimental effects of changing the biome of animal-based nutrients and the soils to which LEM are applied. The objectives of this study were to evaluate the effect of combining LEM with swine effluent to fertilize annual ryegrass (Lolium multiflorum) on plant-available nitrogen, nematode community structure, and forage productivity. Forage performance was good across all of the treatments – with relative feed quality (RFQ) values above 200 and 100 in the March and May harvests respectively. In the first, second and third sampling dates, soils from 0 to 5 cm depth showed more nitrogen mineralized in LEM-treated soils than in control soils (p = 0.178, p < 0.0001 and p = 0.027) respectively. After nutrient applications, soil-nematode community structure (0–10 cm) at the trophic group level changed over time. Most changes were similar between treatments, however, plant parasitic nematode populations were found to be significantly higher in LEM-treated plots in all but one of the sampling dates. LEM is gaining popularity as a natural amendment and although we have shown that it does increase N availability and changes trophic-level nematode community structure, more research is needed to optimize the potential of these benefits.

Effect of cellobiose supplementation and dietary soluble fibre content on in vitro caecal fermentation of carbohydrate-rich substrates in rabbits

ABSTRACTThe in vitro caecal fermentation of five substrates low in starch and protein content [d-(+)-glucose (GLU), d-cellobiose (CEL), sugar beet pectin (PEC), sugar beet pulp (SBP) and wheat straw (WS)] was investigated using soft faeces from rabbits receiving different levels of cellobiose and soluble fibre as inoculum. A total of 24 rabbits were supplemented 3 levels of cellobiose in the drinking water (0.0, 7.5, 15.0 g/l) and fed two experimental diets containing either low soluble fibre (LSF) or high soluble fibre (HSF) levels (84.0 and 130 g/kg dry matter). All substrates were subjected to a two-step pepsin/pancreatin in vitro pre-digestion, and the whole residue was used as substrate for the in vitro incubations. Gas production was measured until 144 h, and volatile fatty acid (VFA) production was determined at 24 h incubation. Experimental treatments did not affect SBP fermentation and had only a subtle influence on fermentation of WS and GLU. In contrast, cellobiose supplementation × donors’ diet interactions were detected for most gas production parameters for CEL. Both the fractional gas production (k) and maximal gas production rates were linearly increased (p ≤ 0.042) and the initial delay in the onset of gas production (Lag) linearly decreased (p < 0.001) by cellobiose supplementation with the HSF inoculum, with no differences between the 7.5 and 15.0 doses. In contrast, with the LSF inoculum cellobiose supplementation only affected k values, which were quadratically increased (p = 0.043) and had maximal values for the 7.5 dose. A quadratic effect (p ≤ 0.018) of cellobiose supplementation was observed for total VFA production at 24 h when CEL and PEC were fermented, obtaining the maximal VFA production for the 7.5 dose of cellobiose. Total VFA production for CEL was greater with LSF than with HSF inoculum (20.7 vs. 12.9 mmol/l; p = 0.014), but the opposite was found for WS (3.97 vs. 6.21 mmol/l; p = 0.005). The use of LSF inoculum for CEL fermentation sharply reduced acetate (p = 0.001) and increased butyrate proportions (p ≤ 0.001) compared with the HSF inoculum. A positive relationship between total VFA caecal concentrations in rabbits receiving the same experimental treatments and in vitro values was only observed when WS was used as substrate (r = 0.90; p = 0.015; n = 6). The results suggest that experimental factors influenced the fermentative activity of caecal digesta, but the observed response differed with the incubated substrate, being the CEL the most affected.

Effect of aerobic pre-treatment on hydrogen and methane production in a two-stage anaerobic digestion process using food waste with different compositions

Aerobic pre-treatment was applied prior to two-stage anaerobic digestion process. Three different food wastes samples, namely carbohydrate rich, protein rich and lipid rich, were prepared as substrates. Effect of aerobic pre-treatment on hydrogen and methane production was studied. Pre-aeration of substrates showed no positive impact on hydrogen production in the first stage. All three categories of pre-aerated food wastes produced less hydrogen compared to samples without pre-aeration. In the second stage, methane production increased for aerated protein rich and carbohydrate rich samples. In addition, the lag phase for carbohydrate rich substrate was shorter for aerated samples. Aerated protein rich substrate yielded the best results among substrates for methane production, with a cumulative production of approximately 351ml/gVS. With regard to non-aerated substrates, lipid rich was the best substrate for CH4 production (263ml/gVS). Pre-aerated P substrate was the best in terms of total energy generation which amounted to 9.64kJ/gVS. This study revealed aerobic pre-treatment to be a promising option for use in achieving enhanced substrate conversion efficiencies and CH4 production in a two-stage AD process, particularly when the substrate contains high amounts of proteins.

Nutrition mediates the expression of cultivar–farmer conflict in a fungus-growing ant

Attine ants evolved farming 55-60 My before humans. Although evolutionarily derived leafcutter ants achieved industrial-scale farming, extant species from basal attine genera continue to farm loosely domesticated fungal cultivars capable of pursuing independent reproductive interests. We used feeding experiments with the basal attine Mycocepurus smithii to test whether reproductive allocation conflicts between farmers and cultivars constrain crop yield, possibly explaining why their mutualism has remained limited in scale and productivity. Stoichiometric and geometric framework approaches showed that carbohydrate-rich substrates maximize growth of both edible hyphae and inedible mushrooms, but that modest protein provisioning can suppress mushroom formation. Worker foraging was consistent with maximizing long-term cultivar performance: ant farmers could neither increase carbohydrate provisioning without cultivars allocating the excess toward mushroom production, nor increase protein provisioning without compromising somatic cultivar growth. Our results confirm that phylogenetically basal attine farming has been very successful over evolutionary time, but that unresolved host-symbiont conflict may have precluded these wild-type symbioses from rising to ecological dominance. That status was achieved by the evolutionarily derived leafcutter ants following full domestication of a coevolving cultivar 30-35 Mya after the first attine ants committed to farming.

Selective short-chain carboxylates production: A review of control mechanisms to direct mixed culture fermentations

ABSTRACTAnaerobic digestion of organic residual streams can be directed to produce carboxylates such as acetate, propionate, and n-butyrate, which can be either directly used in industry or converted into other valuable compounds. This paper reviews the studies working with mixed culture conversion of organic substrates toward carboxylates. It draws connections between standard fermentation parameters and the carboxylate product concentrations and composition. The use of more concentrated carbohydrate-rich substrates, at longer residence times and at neutral pH ranges, stimulates total acid production. When increasing pH to the neutral range, acetate and propionate fractions are elevated. High propionate concentrations and fractions are infrequently reported and mainly appear on high-protein-containing feedstock. High n-butyrate fraction >70% is usually found when pH < 6, at longer retention times or organic loading rates, under CO2 atmosphere or on substrates with high lactate concentrations. The review concludes with prospects for further developments related to the carboxylate platform.

Physiological characteristics of the extreme thermophile Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell factory.

Global concerns about climate changes and their association with the use of fossil fuels have accelerated research on biological fuel production. Biological hydrogen production from hemicellulose-containing waste is considered one of the promising avenues. A major economical issue for such a process, however, is the low substrate conversion efficiency. Interestingly, the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus can produce hydrogen from carbohydrate-rich substrates at yields close to the theoretical maximum of the dark fermentation process (i.e., 4 mol H2/mol hexose). The organism is able to ferment an array of mono-, di- and polysaccharides, and is relatively tolerant to high partial hydrogen pressures, making it a promising candidate for exploitation in a biohydrogen process. The behaviour of this Gram-positive bacterium bears all hallmarks of being adapted to an environment sparse in free sugars, which is further reflected in its low volumetric hydrogen productivity and low osmotolerance. These two properties need to be improved by at least a factor of 10 and 5, respectively, for a cost-effective industrial process. In this review, the physiological characteristics of C. saccharolyticus are analyzed in view of the requirements for an efficient hydrogen cell factory. A special emphasis is put on the tight regulation of hydrogen production in C. saccharolyticus by both redox and energy metabolism. Suggestions for strategies to overcome the current challenges facing the potential use of the organism in hydrogen production are also discussed.

The potential for hydrogen-enriched biogas production from crops: Scenarios in the UK

There is increasing international interest in developing low carbon technologies to provide hydrogen renewably. Hydrogen can be produced through dark anaerobic fermentation using carbohydrate-rich substrates, and methane can be produced in a methanogenic second stage. The suitability of a range of crops for hydrogen and methane production in the UK is examined, using selection criteria including yield, harvest window and composition of the crops. The annual potential for hydrogen and methane production is calculated using the selected crops, taking into account the energy required to grow and harvest the biomass and run the process. The fermentable energy crops fodder beet, forage maize, sugar beet and rye grass were identified as the most suitable substrates for this farm-scale process. A conservative estimate of the amount of agricultural land in the UK excluding permanent grassland not already used for food production or energy crops (currently unused set-aside land) has been made (294,960 ha). If this was used to grow a rotation of the selected crops, 9.6 TW h net energy could be produced per year. This equates to electrical power for 2.2 million homes in the UK annually and a reduction of CO2 emissions by over 2.3 million tones per annum in the UK.

Self-heating of anaerobic digesters using energy crops

With the increasing application of energy crops in agricultural biogas plants and increasing digester volumes, the phenomenon of self-heating in anaerobic digesters appeared in some cases. Until now this development was just known from aerobic systems. To obtain an idea of the thermodynamics inside an anaerobic digester, a detailed analysis of all heat fluxes in a full-scale agricultural biogas plant was carried out. Several experiments were realised to quantify the influences of different internal and external energy sources. To estimate the impact of self-heating in anaerobic systems, data of other full-scale agricultural biogas plants in Austria were collected. Alternatives to the cooling of the digesters are discussed based on individual experiences of several plants. A connection between carbohydrate-rich substrates, especially with high starch contents, and the self-heating could be shown. From the results it can be assumed that the anaerobic digestion of most energy crops is exothermic, which is in contrast to the current thermodynamic belief.

Biohydrogen Production in Continuous‐Flow Reactor Using Mixed Microbial Culture

The goal of the proposed project was to develop an anaerobic fermentation process that converts negative-value organic wastes into hydrogen-rich gas in a continuous-flow reactor under different operating conditions, such as hydraulic retention time (HRT), heat treatment, pH, and substrates. A series of batch tests were also conducted in parallel to the continuous study to evaluate the hydrogen conversion efficiency of two different organic substrates, namely sucrose and starch. A heat shock (at 90 degrees C for 15 minutes) was applied to the sludge in an external heating chamber known as a sludge activation chamber, as a method to impose a selection pressure to eliminate non-spore-forming, hydrogen-consuming bacteria and to activate spore germination. The experimental results showed that the heat activation of biomass enhanced hydrogen production by selecting for hydrogen-producing, spore-forming bacteria. The batch feeding at a shorter HRT of 20 hours (or higher organic loading rate) favored hydrogen production, whereas, at a longer HRT of 30 hours, methane was detected in the gas phase. The major organic acids of hydrogen fermentation were acetate, butyrate, and propionate. Up to 23.1% of influent chemical oxygen demand was consumed in biomass synthesis. Batch tests showed that the hydrogen-production potential of starch was lower than sucrose, and better conversion efficiency from starch was obtained at a lower pH of 4.5. However, addition of sucrose to starch improved the overall hydrogen-production potential and hydrogen-production rate. This study showed that sustainable biohydrogen production from carbohydrate-rich substrates is possible through heat activation of settled sludge.

Rhamnolipid Surfactants: An Update on the General Aspects of These Remarkable Biomolecules

Pseudomonas strains are able to biosynthesize rhamnose-containing surfactants also known as rhamnolipids. These surface-active compounds are reviewed with respect to chemical structure, properties, biosynthesis, and physiological role, focusing on their production and the use of low-cost substrates such as wastes from food industries as alternative carbon sources. The use of inexpensive raw materials such as agroindustrial wastes is an attractive strategy to reduce the production costs associated with biosurfactant production and, at same time, contribute to the reduction of environmental impact generated by the discard of residues, and the treatment costs. Carbohydrate-rich substrates generated low rhamnolipid levels, whereas oils and lipid-rich wastes have shown excellent potential as alternative carbon sources.

Microbial activities of faeces from unweaned and adult pigs, in relation to selected fermentable carbohydrates

AbstractA comparison of in vitro microbial activity was made between inocula from faeces of adult and unweaned pigs, using a range of carbohydrate-rich substrates. The substrates tested were classified into groups (fibre-rich, grains, gums, pectin, saccharides, storage carbohydrates, and miscellaneous). Proximate and fibre analyses were determined for all substrates tested. Fermentation kinetics were measured according to the cumulative gas production technique. End-point products such as gas, volatile fatty acids and ammonia were also determined. The faeces from the adult pigs (ATF) were collected from four castrated finisher pigs (Dutch Landrace×Great Yorkshire). These were offered a diet based on highly digestible maize starch and fish meal, so as to be as free as possible of fermentable carbohydrates. The unweaned piglet faeces were collected from 19 unweaned piglets (male and female) which were 27 to 30 days old. The parameters were tested for significance by an analysis of variance using the Tukey's studentized range test of multiple comparisons. There were significant differences in the fermentation patterns both due to source of substrate and inoculum. It would appear that the metabolic activity of the microflora does differ significantly between adult and weanling pigs, though this varied for the different carbohydrates tested. Suggestions are made as to how to select potential carbohydrate ingredients for an in vivo experiment, based on their in vitro fermentation characteristics.

Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species

The genetic variability within 600 bp of DNA sequence from the ribosomal internal transcribed spacers (ITS 1 and ITS 2) and the 5.8 S rRNA gene was examined in 52 strains belonging to 29 terverticillate taxa in Penicillium subg. Penicillium from diverse sources and locations. The sequenced region is extremely conserved within the terverticillate penicillia, with only 29 positions differing in one or more taxa. The highest degree of ITS variability was seen among the species close to Penicillium roqueforti ( P. roqueforti, P. carneum and P. paneum ) whereas the ITS variability was very low between closely related taxa, e.g. among the taxa near P. aurantiogriseum. The relationships among the terverticillate penicillia, and related teleomorphs in Eupenicillium , were analysed from bootstrapped ITS sequence data sets using the neighbour-joining method. The terverticillate penicillia form a well supported clade with Eupenicillium crustaceum. Sequence analysis generally confirmed the overall taxonomy in Penicillium subg. Penicillium but relationships between all the terverticillate taxa could not be clearly established due to the low degree of ITS variability. Nevertheless, clades of Penicillium species sharing environmental characteristics did emerge, e.g. species growing on protein and lipid rich substrates (e.g. P. crustosum and P. commune ), species prevalent in dry habitats (e.g. P. chrysogenum and P. nalgiovense ), and those prevalent on carbohydrate rich substrates (e.g. P. aurantiogriseum and P. freii ).