How Can We Advance Integrative Biology Research in Animal Science in 21st Century? Experience at University of Ljubljana from 2002 to 2022.

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health.

Influence of support materials on continuous hydrogen production in anaerobic packed-bed reactor with immobilized hydrogen producing bacteria at acidic conditions.

This study assesses the impact of different support materials (Mutag BioChip™, expanded clay and activated carbon) on microbial hydrogen production in an anaerobic packed-bed reactor (APBR) treating synthetic waste water containing glucose as the main carbon source at low pH value. The APBRs were inoculated with acid pretreated anaerobic sludge and operated at pH value of 4±0.2 and hydraulic retention time (HRT) of 3h. The maximum hydrogen yield of 1.80mol H2/mol glucose was achieved for the APBR packed with Mutag BioChip™ (R1), followed by expanded clay (R2, 1.74mol H2/mol glucose) and activated carbon (R3, 1.46mol H2/mol glucose). It was observed that the investigated support materials influenced the immobilization of hydrogen producing bacteria and consequently hydrogen production performance as well as composition of soluble metabolites. The main metabolic products were acetic acid and butyric acid accompanied with a smaller content of ethanol. The data indicated that in reactors with higher hydrogen yield (R1 and R2), acetate/butyrate (HAc/HBu) ratios were 1.7 and 1.6, respectively, while in the reactor with the lowest hydrogen yield (R3) the obtained HAc/HBu ratio was 4.8. Finally, stable hydrogen and organic acids production throughout the steady-state operation period at low pH values was achieved in all reactors.

Influence of Thermal and Bacterial Pretreatment of Microalgae on Biogas Production in Mesophilic and Thermophilic Conditions.

Microalgae biomass has a great potential in search for new alternative energy sources. They can be used as a substrate for the biogas production in anaerobic digestion. When using microalgae, the efficiency of this process is hampered due to the resistant cell wall. In order to accelerate the hydrolysis of cell wall and increase the efficiency of biogas production we applied two different pretreatments - biological and thermal under mesophilic and thermophilic conditions. During biological pretreatment we incubated microalgae with anaerobic hydrolytic bacteria Pseudobutyrivibrio xylanivorans Mz5T. In thermal pretreatment we incubated microalgae at 90 °C. We also tested a combined thermal and biological pretreatment in which we incubated P. xylanivorans Mz5T with thermally pretreated microalgae. Thermal pretreatment in mesophilic and thermophilic process has increased methane production by 21% and 6%, respectively. Biological pretreatment of microalgae has increased methane production by 13%, but only under thermophilic conditions (pretreatment under mesophilic conditions showed no effect on methane production). Thermal-biological pretreatment increased methane production by 12% under thermophilic conditions and by 6% under mesophilic conditions.

Biogas production from brewery spent grain enhanced by bioaugmentation with hydrolytic anaerobic bacteria.

Lignocellulosic substrates are widely available but not easily applied in biogas production due to their poor anaerobic degradation. The effect of bioaugmentation by anaerobic hydrolytic bacteria on biogas production was determined by the biochemical methane potential assay. Microbial biomass from full scale upflow anaerobic sludge blanket reactor treating brewery wastewater was a source of active microorganisms and brewery spent grain a model lignocellulosic substrate. Ruminococcus flavefaciens 007C, Pseudobutyrivibrio xylanivorans Mz5(T), Fibrobacter succinogenes S85 and Clostridium cellulovorans as pure and mixed cultures were used to enhance the lignocellulose degradation and elevate the biogas production. P. xylanivorans Mz5(T) was the most successful in elevating methane production (+17.8%), followed by the coculture of P. xylanivorans Mz5(T) and F. succinogenes S85 (+6.9%) and the coculture of C. cellulovorans and F. succinogenes S85 (+4.9%). Changes in microbial community structure were detected by fingerprinting techniques.

Identification of GH10 xylanases in strains 2 and Mz5 of Pseudobutyrivibrio xylanivorans.

Genes encoding glycosyl hydrolase family 11 (GH11) xylanases and xylanases have been identified from Pseudobutyrivibrio xylanivorans. In contrast, little is known about the diversity and distribution of the GH10 xylanase in strains of P. xylanivorans. Xylanase and associated activities of P. xylanivorans have been characterized in detail in the type strain, Mz5. The aim of the present study was to identify GH10 xylanase genes in strains 2 and Mz5 of P. xylanivorans. In addition, we evaluated degradation and utilization of xylan by P. xylanivorans 2 isolated from rumen of Creole goats. After a 12-h culture, P. xylanivorans 2 was able to utilize up to 53% of the total pentose content present in birchwood xylan (BWX) and to utilize up to 62% of a ethanol-acetic acid-soluble fraction prepared from BWX. This is the first report describing the presence of GH10 xylanase-encoding genes in P. xylanivorans. Strain 2 and Mz5 contained xylanases which were related to GH10 xylanase of Butyrivibrio sp. Identifying xylanase-encoding genes and activity of these enzymes are a step toward understanding possible functional role of P. xylanivorans in the rumen ecosystem and contribute to providing an improved choice of enzymes for improving fiber digestion in ruminant animals, agricultural biomass utilization for biofuel production, and other industries.

The effects of engineered nanoparticles on the cellular structure and growth of Saccharomyces cerevisiae.

In order to study the effects of nanoparticles (NPs) with different physicochemical properties on cellular viability and structure, Saccharomyces cerevisiae were exposed to different concentrations of TiO2-NPs (1-3 nm), ZnO-NPs (<100 nm), CuO-NPs (<50 nm), their bulk forms, Ag-NPs (10 nm) and single-walled carbon nanotubes (SWCNTs). The GreenScreen assay was used to measure cyto- and genotoxicity, and transmission electron microscopy (TEM) used to assess ultrastructure. CuO-NPs were highly cytotoxic, reducing the cell density by 80% at 9 cm(2)/ml, and inducing lipid droplet formation. Cells exposed to Ag-NPs (19 cm(2)/ml) and TiO2-NPs (147 cm(2)/ml) contained dark deposits in intracellular vacuoles, the cell wall and vesicles, and reduced cell density (40 and 30%, respectively). ZnO-NPs (8 cm(2)/ml) caused an increase in the size of intracellular vacuoles, despite not being cytotoxic. SWCNTs did not cause cytotoxicity or significant alterations in ultrastructure, despite high oxidative potential. Two genotoxicity assays, GreenScreen and the comet assay, produced different results and the authors discuss the reasons for this discrepancy. Classical assays of toxicity may not be the most suitable for studying the effects of NPs in cellular systems, and the simultaneous assessment of other measures of the state of cells, such as TEM are highly recommended.

Expression of cellulosome components and type IV pili within the extracellular proteome of Ruminococcus flavefaciens 007.

BACKGROUND: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose. METHODOLOGY/PRINCIPAL FINDINGS: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel. CONCLUSIONS/SIGNIFICANCE: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

Experimental evidence of false-positive Comet test results due to TiO2 particle--assay interactions.

We have studied the genotoxicity of TiO2 particles with a Comet assay on a unicellular organism, Tetrahymena thermophila. Exposure to bulk- or nano-TiO2 of free cells, cells embedded in gel or nuclei embedded in gel, all resulted in a positive Comet assay result but this outcome could not be confirmed by cytotoxicity measures such as lipid peroxidation, elevated reactive oxygen species or cell membrane composition. Published reports state that in the absence of cytotoxicity, nano- and bulk-TiO2 genotoxicity do not occur directly, and a possible explanation of our Comet assay results is that they are false positives resulting from post festum exposure interactions between particles and DNA. We suggest that before Comet assay is used for nanoparticle genotoxicity testing, evidence for the possibility of post festum exposure interactions should be considered. The acellular Comet test described in this report can be used for this purpose.

Exposure to Al2O3 nanoparticles changes the fatty acid profile of the anaerobe Ruminococcus flavefaciens.

One of the main mechanisms of nanoparticle toxicity is known to be the generation of reactive oxygen species (ROS) which primarily damage cell membranes. However, very limited data on membrane effects in anaerobic environments (where ROS could not be the cause of membrane damage) are available. In the following study, rumen anaerobe Ruminococcus flavefaciens 007C was used as a bacterial model to assess the potential effects of Al(2)O(3) and TiO(2) nanoparticles on membranes in an anaerobic environment. Fatty acid profiles of cultures after exposure to Al(2)O(3) or TiO(2) nanoparticles were analyzed and compared with the profiles of non-exposed cultures or cultures exposed to bulk materials. Analysis revealed dose-effect changes in membrane composition exclusively when cells were exposed to Al(2)O(3) nanoparticles in a concentration range of 3-5 g/L, but were not present in cultures exposed to bulk material. On the other hand, the tested concentrations of nano-TiO(2) did not significantly affect the membrane profile of the exposed bacterium. The results suggest the possibility that Al(2)O(3) induces changes in bacterial membranes by direct physical interaction, which was supported by TEM image analysis.

Expression patterns of Ruminococcus flavefaciens 007S cellulases as revealed by zymogram approach.

The expression of Ruminococcus flavefaciens 007S cellulases in different incubation time points (growth stages) and their substrate inducibility were analyzed by comparing the zymogram expression profiles of cultures grown on insoluble cellulose (Avicel) with cellobiose-grown cultures. The molecular weights of the enzymes were compared to (putative) cellulases encoded in the R. flavefaciens FD-1 genome.

Membrane Changes Associated with Exposure of Pseudomonas putida to Selected Environmental Pollutants and their Possible Roles in Toxicity.

A bacterial model system (Pseudomonas putida DSM 50026) was used in this research to assess potential effect of five selected chemically diverse environmental pollutants on cell membranes. Long chain fatty acid profiles of cultures exposed to environmentally relevant concentrations of atrazine (ATR), metolachlor (MET), pentachlorobiphenyl (PCB), hexachlorobenzene (HCB) and fluoranthene (FL), were analyzed and compared to non-exposed cultures. To assess sensitivity of membrane-based responses, the impact of each toxicant on culture growth was also followed spectrophotometrically. Results revealed changes in fatty acid profiles when cells were exposed to PCB, HCB and FL in concentrations below the inhibitory levels. Moreover, the observed membrane responses were similar to the ones previously associated with adaptation to some membrane-active compounds. On the other hand, exposure of cells to any of the two herbicides, ATR or MET, did not induce any significant changes in fatty acid profiles. However, when combined with a commonly used fertilizer compound, NH4NO3 growth impairment was observed. Synergistic effect of the two herbicides with NH4NO3 might be a consequence of changes in fatty acid profile increasing membrane fluidity, likely induced by NH4+ ions.

Exposure to CuO nanoparticles changes the fatty acid composition of protozoa Tetrahymena thermophila.

In the current study, the toxicity mechanism of nanosized CuO (nCuO) to the freshwater ciliated protozoa Tetrahymena thermophila was studied. Changes in fatty acid profile, lipid peroxidation metabolites and reactive oxygen species (ROS) were measured. Bulk CuO and CuSO(4) served as controls for size and solubility and 3,5-dichorophenol (3,5-DCP) as a control for a chemical known to directly affect the membrane composition. Exposure to all copper compounds induced the generation of ROS, whereas nCuO was most potent. The latter effect was not solely explained by solubilized Cu-ions and was apparently particle-related. 24 h exposure of protozoa to 80 mg/L of nCuO (EC50) significantly decreased the proportion of two major unsaturated fatty acids (UFA) (C18:3 cis-6,9,12, C18:2 cis-9,12), while it increased the relative amount of two saturated fatty acids (SFA) (C18:0, C16:0). Analogous effect was not observed when protozoa were exposed to equitoxic suspensions of bulk CuO, Cu-ions or 3,5-DCP. As changes in the UFA:SFA upon exposure of protozoa to nCuO were not detected at 2 h exposure and no simultaneous dose- or time-dependent lipid peroxidation occurred, it is likely that one of the adaptation mechanisms of protozoa to nCuO was lowering membrane fluidity by the inhibition of de novo synthesis of fatty acid desaturases. This is the first study of the effects of nanoparticles on the membrane fatty acid composition.

Anaerobic digestion of brewery spent grain in a semi-continuous bioreactor: inhibition by phenolic degradation products.

In this study anaerobic digestion of selected lignocellulosic substrate, namely brewery spent grain (BSG), was studied. In order to facilitate anaerobic digestion several types of pretreatment methods were tested such as: mechanical, chemical (alkali and acid) and thermo-chemical. The anaerobic digestion experiments were carried out in a semi-continuous stirred bioreactors with the organic loading rates between 2.9 and 3.9 kgCOD m-3 d-1 (1.9 and 2.5 kgVSS m-3 d-1 respectively) and corresponding hydraulic retention times of 33-39 days. Biogas production and composition, pH, COD, TSS and VSS, short chain fatty acids and phenolic compounds were measured. A significant inhibition of biogas production occurred, depending on the type of substrate pretreatment. There are indications that p-cresol is responsible for process inhibition when its concentration in the reaction mixture exceeds critical value between 115 and 240 mg L-1. Anaerobic digestion of chemically pretreated BSG (acid and alkali) and untreated-raw BSG was inhibited between the days 56 and 63 of the experiment, followed by thermo-chemically pretreated BSG on day 112 and mechanically pretreated BSG on day 126. Analyses of the substrates showed no phenolic compounds either in raw-untreated BSG or pretreated substrates, therefore the recorded p-cresol is an intermediate degradation product, responsible for process inhibition.

General Microbial Community Flexibility in Biochemical Methane Potential Assay is Highly Correlated to Initial Biogas Production Rates.

Degradation of brewery spent grain as a novel test substrate was explored in routine biochemical methane potential assays (BMP) using three different inocula. Significant differences in the initial biogas production rates from spent grain, methane yield coefficients and final spent grain degradation were observed between inocula. Initial and developed communities degrading novel substrate showed significant differences in archaeal community fingerprints. Differences were observed irrespective of substrate identity (no substrate, glucose, spent grain) providing evidence of a significant general influence of BMP incubation on the microbial phylotypes. A linear relationship between microbial community flexibility in BMP assay and corresponding initial biogas production rates was identified as a novel parameter to diagnose anaerobic processes, particularly under dynamic conditions like start-up.

Cellulosomes - promising supramolecular machines of anaerobic cellulolytic microorganisms.

Cellulose is the main structural component of plant cell wall and thus the most abundant carbohydrate in nature. However, extracting the energy from this abundant source is limited by its recalcitrant nature. The hydrolysis of plant cell wall requires synergystic action of different enzymes, including multiple cellulases, hemicellulases, pectinases, etc. Meanwhile aerobic cellulolytic microorganisms release large quantities of synergistically acting free enzymes in their environment, most anaerobic microorganisms evolved more efficient strategies to optimize the process of plant cell wall degradation, for example production of extracellular multi-enzyme complexes (cellulosomes). Cellulosomes consist of at least one core structural protein, named scaffoldin, which firmly binds numerous enzymatic subunits, and usually also plays a major role in substrate binding. Although the general structure of cellulosomes seems universal, differences in number and identity of complex components do exist among microorganisms. The article surveys the current knowledge about cellulosomes, focusing on three best investigated cellulolytic clostridia, one representative of ruminal bacteria and novel findings concerning anaerobic fungi. Efforts in construction of artificially engineered cellulosomal systems (designer cellulosomes) as well as their biotechnological potential are also discussed.

Conditioning of the membrane fatty acid profile of Escherichia coli during periodic temperature cycling.

The membrane fatty acid composition of Escherichia coli becomes conditioned during periodic temperature cycling between 37 and 8 degrees C. After several cycles of temperature change, the bacteria become locked into a low-temperature physiology. Even after a prolonged incubation at high temperature the membrane fatty acid composition of conditioned cells was similar to that of cold-stressed cells.

Bioassays for evaluating the water-extractable genotoxic and toxic potential of soils polluted by metal smelters.

Physicochemical analyses of polluted soils are limited in their ability to determine all hazardous compounds, their bioavailability, and their combined effects on living organisms. Bioassays, on the other hand, can evaluate environmental quality more accurately. This study assesses the genotoxic potential of water extracts from soil polluted with metals (Pb, Cd, and Zn) by the former lead smelter in zerjav, Slovenia using comet assay with Tetrahymena thermophila and human hepatoma cells (HepG2). In addition, the toxicity of soil samples and their extracts was evaluated using Vibrio fischeri and delayed fluorescence of Lemna minor. Chemical analyses of metals using atomic absorption spectrophotometry (AAS) was performed for comparison. Measurements of the total metal concentrations showed that four of five plots near the former lead smelter were highly contaminated with Pb, Cd, and Zn, but the amount of metals in water/soil extracts was low at all the sampling plots. Genotoxicity was demonstrated using T. thermophila for the majority of the extracts, and HepG2 cells for only some of the extracts. Whereas V. fischeri indicated a gradual decrease in soil toxicity with greater distance from the smelter, the toxicity of extracts did not correlate with proximity. Low concentrations of metals in water extracts stimulated L. minor growth. The results indicate that comet assay with T. thermophila and HepG2 cells and the BSPT with V. fischeri are suitable protocols for screening the genotoxic and toxic potential of water/soil extracts by comet assay, whereas chemical analyses of total metal concentrations in soil do not solely suffice for evaluating metal pollution in the environment. Biological assays are thus crucial for risk assessment.

Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay.

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO(2)) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.

Butyrivibrio hungatei sp. nov. and Pseudobutyrivibrio xylanivorans sp. nov., butyrate-producing bacteria from the rumen.

Two novel Gram-negative, anaerobic, non-spore-forming, butyrate-producing bacterial species, strains Mz 5T and JK 615T, were isolated from the rumen fluid of cow and sheep. Both strains were curved rods that were motile by means of single polar or subpolar flagellum and common in the rumen microbial ecosystem. Strain Mz 5T produced high xylanase, proteinase, pectin hydrolase and DNase activities; 1,4-beta-endoglucanase was also detected in the culture medium. The bacterium utilized a wide range of carbohydrates. Glucose was fermented to formate, butyrate, lactate, succinate and ethanol. The DNA G + C content was 42.1 mol%. The complete 16S rDNA sequence was obtained and phylogenetic relationships were determined. Strain Mz 5T and related isolates were located in clostridial cluster XIVa and were closely related to Pseudobutyrivibrio ruminis, Butyrivibrio crossotus, Roseburia cecicola and Eubacterium rectale. The name proposed for this novel bacterium is Pseudobutyrivibrio xylanivorans; the type strain is Mz 5T (=DSM 14809T =ATCC BAA-455T). Strain JK 615T produced no fibrolytic activity, but utilized a wide range of carbohydrates. Glucose was fermented to formate, acetate, butyrate and ethanol. The DNA G + C content was 44-8 mol%. The complete 16S rDNA sequence was obtained and phylogenetic relationships were determined. Strain JK 615T was located in clostridial cluster XIVa and was closely related to Clostridium proteoclasticum, Butyrivibrio fibrisolvens and Eubacterium halii. The name proposed for this novel bacterium is Butyrivibrio hungatei; the type strain is JK 615T (=DSM 14810T =ATCC BAA-456T).