Archaeal and Bacterial Content in a Two-Stage Anaerobic System for Efficient Energy Production from Agricultural Wastes.

Anaerobic digestion (AD) is a microbially-driven process enabling energy production. Microorganisms are the core of anaerobic digesters and play an important role in the succession of hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. The diversity of participating microbial communities can provide new information on digester performance for biomass valorization and biofuel production. In this study anaerobic systems were used, operating under mesophilic conditions that realized biodegradation processes of waste wheat straw pretreated with NaOH-a renewable source for hydrogen and methane production. These processes could be managed and optimized for hydrogen and methane separately but combining them in a two-stage system can lead to higher yields and a positive energy balance. The aim of the study was to depict a process of biohydrogen production from lignocellulosic waste followed by a second one leading to the production of biomethane. Archaeal and bacterial consortia in a two-stage system operating with wheat straw were identified for the first time and the role of the most important representatives was elucidated. The mixed cultures were identified by the molecular-biological methods of metagenomics. The results showed that biohydrogen generation is most probably due to the presence of Proteiniphilum saccharofermentans, which was 28.2% to 45.4% of the microbial community in the first and the second bioreactor, respectively. Archaeal representatives belonging to Methanobacterium formicicum (0.71% of the community), Methanosarcina spelaei (0.03%), Methanothrix soehngenii (0.012%), and Methanobacterium beijingense (0.01%) were proven in the methane-generating reactor. The correlation between substrate degradation and biogas accumulation was calculated, together with the profile of fatty acids as intermediates produced during the processes. The hydrogen concentration in the biogas reached 14.43%, and the Methane concentration was 69%. Calculations of the energy yield during the two-stage process showed 1195.89 kWh·t-1 compared to a 361.62 kWh·t-1 cumulative yield of energy carrier for a one-stage process.

Simultaneous Biodegradation of Phenol and n-Hexadecane by Cryogel Immobilized Biosurfactant Producing Strain Rhodococcus wratislawiensis BN38.

The capability of the biosurfactant-producing strain Rhodococcus wratislawiensis BN38 to mineralize both aromatic and aliphatic xenobiotics was proved. During semicontinuous cultivation 11 g/l phenol was completely degraded within 22 cycles by Rhodococcus free cells. Immobilization in a cryogel matrix was performed for the first time to enhance the biodegradation at multiple use. A stable simultaneous hydrocarbon biodegradation was achieved until the total depletion of 20 g/l phenol and 20 g/l n-hexadecane (40 cycles). The alkanotrophic strain R. wratislawiensis BN38 preferably degraded hexadecane rather than phenol. SEM revealed well preserved cells entrapped in the heterogeneous super-macroporous structure of the cryogel which allowed unhindered mass transfer of xenobiotics. The immobilized strain can be used in real conditions for the treatment of contaminated industrial waste water.

Impact of Waste Cooking Oils Addition on Thermophilic Dry Co-Digestion of Wheat Straw and Horse Manure for Renewable Energy Production in Two Stages.

Anaerobic co-digestion of waste wheat straw and horse manure in two steps was revealed as a promising option for renewable energy production in the form of hydrogen and methane. Addition of waste cooking oils, disposal of which could cause damage to health or the environment, as a third substrate for digestion, is suggested as an approach not only to help handle the increasing volume of food waste worldwide but also to improve process performance. In the present study, waste cooking oil, in a concentration of 5%, appeared to be a positive modulator of anaerobic digestion with the production of hydrogen and did not lead to inhibition of the hydrolysis phase. The overall efficiency of the two-stage anaerobic digestion of the mixture, which contains mainly lignocellulose waste, is positively dependent on thermochemical pretreatment with the alkali reagent (Ca(OH)2), but elevated temperature (55 °C) and cooking oil addition revealed the opportunity to omit the pre-treatment step. Nevertheless, the overall energy production was lower due to the methane production step. However, the addition of waste cooking oils to the process in which lig-nocellulose is not pretreated (V3) led to an increase in the methane production and energy yield compared to V1. The anaerobic digestion of lignocellulosic waste is a complex process and comprises successive degradation pathways and syntrophic microbial associations' activities, so the division in two reactors ensured suitable conditions for the microorganisms residing in each of them. In this study, along with the production of hydrogen and methane and the separation of the hydrolysis and methanogenesis stages, utilization of agriculture- and kitchen-generated wastes was realized in the context of waste-to-energy sustainable production methods.

Biosurfactant production by Pseudomonas aeruginosa BN10 cells entrapped in cryogels.

Production of a rhamnolipid biosurfactant by cells of Pseudomonas aeruginosa strain BN10 immobilized into poly(ethylene oxide) (PEO) and polyacrylamide (PAAm) cryogels was investigated under semicontinuous shake flask conditions and compared to biosurfactant secretion by free cells. The biosurfactant synthesis was followed over 9 cycles of operation of the immobilized system, each cycle comprising 7 days at ambient temperature and neutral pH. Type and quantity of the carrier were optimized for the rhamnolipid production. The highest rhamnolipid yield of 4.6 g l(-1) was obtained in the 6th cycle for the immobilized system with 3 g PEO compared to 4.2 g l(-1) obtained for the free cells, thus immobilization provided physiological stability of the cells. Scanning electron microscopy revealed preservation of the cell shape and regular distribution of the cells under the matrix surface. The polymer matrices possessed chemical and biological stability and very good physico-mechanical characteristics which are a prerequisite for a high life span of these materials for the production of rhamnolipids.

Phenol biodegradation by fungal cells immobilized in sol-gel hybrids.

The capability of cells of the fungus Aspergillus awamori, either free or immobilized in hybrid sol-gel material cells, for phenol biodegradation was demonstrated. Phenol was present in the reaction mixture as the sole carbon and energy source, and its decomposition was followed in repeated batch degradation experiments. Atomic force microscopy provided information on the development of self-organizing structures in the materials synthesized by the sol-gel method. Phenol biodegradation was mediated only by the fungal cells, and no absorption by the hybrid matrix was observed. Ten cycles of phenol biodegradation using the immobilized cells system were conducted during which up to 2000 mg l(-1) phenol was completely decomposed. Immobilized cells degraded phenol at 8.33 mg h(-1), twice as fast as free cells. The good performance of the immobilized fungal cell system is promising for the development of an efficient technology for treating phenol-containing waste waters.

Metagenomic Analysis of Bacterial, Archaeal and Fungal Diversity in Two-Stage Anaerobic Biodegradation for Production of Hydrogen and Methane from Corn Steep Liquor.

The main purpose of this study was to identify the microbial communities (bacterial, archaeal and fungal) in a two-stage system of anaerobic bioreactors for the production of hydrogen and methane from the waste substrate-corn steep liquor. Wastes from the food industry are valuable resources with potential in biotechnological production because of their high organic matter contents. In addition, the production of hydrogen and methane, volatile fatty acids, reducing sugars and cellulose content was monitored. Two-stage anaerobic biodegradation processes were performed by microbial populations in the first hydrogen generating bioreactor (working volume of 3 dm3) and in the second methane-generating reactor (working volume of 15 dm3). Cumulative hydrogen yield reached 2000 cm3 or 670 cm3/L a day, while the methane production reached a maximum quantity of 3300 cm3 or 220 cm3/L a day. Microbial consortia in anaerobic digestion systems play an essential role for process optimization and biofuel production enhancement. The obtained results showed the possibility of conducting two separate processes-the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis)-as two stages of anaerobic digestion to favor energy production under controlled conditions with corn steep liquor. The diversity of microorganisms as main participants in the processes in the bioreactors of the two-stage system was followed using metagenome sequencing and bioinformatics analysis. The obtained metagenomic data showed that the most abundant phylum in both bacterial communities was Firmicutes-58.61% and 36.49% in bioreactors 1 and 2, respectively. Phylum Actinobacteria were found in significant quantities (22.91%) in the microbial community in Bioreactor 1, whereas in Bioreactor 2, they were 2.1%. Bacteroidetes are present in both bioreactors. Phylum Euryarchaeota made up 0.4% of the contents in the first bioreactor and 11.4% in the second. As the dominant genera among methanogenic archaea are Methanothrix (8.03%) and Methanosarcina (3.39%), the main fungal representatives were Saccharomyces cerevisiae. New knowledge of anaerobic digestion mediated by novel microbial consortia could be widely used to convert different wastes to green energy.

Biogas Production Potential of Thermophilic Anaerobic Biodegradation of Organic Waste by a Microbial Consortium Identified with Metagenomics.

Anaerobic digestion (AD) is a widespread biological process treating organic waste for green energy production. In this study, wheat straw and corn stalks without any harsh preliminary treatment were collected as a renewable source to be employed in a laboratory-scale digester to produce biogas/biomethane. Processes parameters of temperature, pH, total solids, volatile solid, concentration of volatile fatty acids (VFA), and cellulose concentration, were followed. The volume of biogas produced was measured. The impact of organic loading was stated, showing that the process at 55 °C tolerated a higher substrate load, up to 45 g/L. Further substrate increase did not lead to biogas accumulation increase, probably due to inhibition or mass transfer limitations. After a 12-day anaerobic digestion process, cumulative volumes of biogas yields were 4.78 L for 1 L of the bioreactor working volume with substrate loading 30 g/L of wheat straw, 7.39 L for 40 g/L and 8.22 L for 45 g/L. The degree of biodegradation was calculated to be 68.9%, 74% and 72%, respectively. A fast, effective process for biogas production was developed from native wheat straw, with the highest quantity of daily biogas production occurring between day 2 and day 5. Biomethane concentration in the biogas was 60%. An analysis of bacterial diversity by metagenomics revealed that more than one third of bacteria belonged to class Clostridia (32.9%), followed by Bacteroidia (21.5%), Betaproteobacteria (11.2%), Gammaproteobacteria (6.1%), and Alphaproteobacteria (5%). The most prominent genera among them were Proteiniphilum, Proteiniborus, and Pseudomonas. Archaeal share was 1.37% of the microflora in the thermophilic bioreactor, as the genera Methanocorpusculum, Methanobacterium, Methanomassiliicoccus, Methanoculleus, and Methanosarcina were the most abundant. A knowledge of the microbiome residing in the anaerobic digester can be further used for the development of more effective processes in conjunction with theidentified consortium.

Bacterial Natural Disaccharide (Trehalose Tetraester): Molecular Modeling and in Vitro Study of Anticancer Activity on Breast Cancer Cells.

Isolation and characterization of new biologically active substances affecting cancer cells is an important issue of fundamental research in biomedicine. Trehalose lipid was isolated from Rhodococcus wratislaviensis strain and purified by liquid chromatography. The effect of trehalose lipid on cell viability and migration, together with colony forming assays, were performed on two breast cancer (MCF7-low metastatic; MDA-MB231-high metastatic) and one "normal" (MCF10A) cell lines. Molecular modeling that details the structure of the neutral and anionic form (more stable at physiological pH) of the tetraester was carried out. The tentative sizes of the hydrophilic (7.5 Å) and hydrophobic (12.5 Å) portions of the molecule were also determined. Thus, the used trehalose lipid is supposed to interact as a single molecule. The changes in morphology, adhesion, viability, migration, and the possibility of forming colonies in cancer cell lines induced after treatment with trehalose lipid were found to be dose and time dependent. Based on the theoretical calculations, a possible mechanism of action and membrane asymmetry between outer and inner monolayers of the bilayer resulting in endosome formation were suggested. Initial data suggest a mechanism of antitumor activity of the purified trehalose lipid and its potential for biomedical application.

Characterization and potential antitumor effect of a heteropolysaccharide produced by the red alga Porphyridium sordidum.

Taking into account the rising trend of the incidence of cancers of various organs, effective therapies are urgently needed to control human malignancies. However, almost all chemotherapy drugs currently on the market cause serious side effects. Fortunately, several studies have shown that some non-toxic biological macromolecules, including algal polysaccharides, possess anti-cancer activities or can increase the efficacy of conventional chemotherapy drugs. Polysaccharides are characteristic secondary metabolites of many algae. The efficacy of polysaccharides on the normal and cancer cells is not well investigated, but our investigations proved a cell specific effect of a newly isolated extracellular polysaccharide from the red microalga Porphyridium sordidum. The investigated substance was composed of xylose:glucose and galactose:manose:rhamnose in a molar ratio of 1:0.52:0.44:0.31. Reversible electroporation has been exploited to increase the transport through the plasma membrane into the tested breast cancer tumor cells MCF-7 and MDA-MB231. Application of 75 µg/mL polysaccharide in combination with 200 V/cm electroporation induced 40% decrease in viability of MDA-MB231 cells and changes in cell morphology while control cells (MCF10A) remained with normal morphology and kept vitality.

Examination of Humicola lutea immobilized in sol-gel matrices: effective source of alpha-galactosidase.

Alpha-Galactosidase production by the fungus Humicola lutea 120-5 immobilized in a hybrid sol-gel matrix, consisting of tetraethylorthosilicate (TEOS) as a precursor and a mixture of polyethyleneglycol (PEG) and polyvinylalcohol (PVA), was investigated under semicontinuous shake flask cultivation and compared to the enzyme secretion by free cells. The influence of the carrier weight on the alpha-galactosidase biosynthesis in repeated batch experiments was followed. Best results were obtained with 2 g of the sol-gel particles per culture flask using 144-h runs. The growth behaviour of the immobilized mycelium during both the growth and productive phases was observed by scanning electron microscopy. The presence of abundant mycelial growth of intact hyphae correlated with a 2-fold higher enzyme activity compared to free cells. The obtained biocatalyst retained a high level of enzyme titer exceeding the activity of free cells during four cycles of operation (24 days). This result is confirmed by the micrographs showing the retained viability of the growing vegetative cells due to the protective role of the carrier.