Anaerobic Fermentation of Silage from the Above-Ground Biomass of Jerusalem Artichoke (Helianthus tuberosus L.) and Maize (Zea mayse L.) as a New and Promising Input Raw Material for Biogas Production

Research of new input raw materials for biogas plants is a very actual topic. There are only a very few studies dealing with the possibility of using silage prepared from the above-ground parts of the Jerusalem artichoke (Helianthus tuberosus L.) and maize (Zea mayse L.) for methane production. This study deals with the determination of methane production and methane content in biogas during the fermentation of maize silage with dissimilar additions of the biomass of the Jerusalem artichoke (JA). Except for the effect of the JA's addition on the yield of methane, we also studied its potential influence on the inhibition of the process of anaerobic digestion and the bacterial and methanogenic archaeal composition of anaerobic digestate. There were five model silages prepared; two of them contained only maize or JAs, and the remaining three were mixtures of maize and JA silages (30%wt; 50%wt and 70%wt). The fermentation tests showed that the JA addition (from 30 to 70%wt) resulted in the production of biogas decreasing, on average, by 15%. Based on the performed metagenomic analysis, we cannot confirm an essential influence of JA biomass addition on the composition of the community of microorganisms during fermentation.O

Effect of biodegradable poly-3-hydroxybutyrate amendment on the soil biochemical properties and fertility under varying sand loads

Background: Poly-3-hydroxybutyrate (P3HB) is a bacterial intracellular carbon and energy storage polymer, used as a thermoplastic polyester in a wide array of industrial and agricultural applications. However, how the soil microbiome and fertility are altered by exogenously applied P3HB has been relatively unexplored. This study aimed to assess the effects of P3HB addition to nutrient restricted soil: its biological properties and lettuce (Lactuca sativa L. var. capitata L.) biomass production. The experiment was designed to evaluate impacts of spatial arrangement of the relatively organic-rich (soil organic matter, P3HB particles) versus poor fractions of the matrix with confounding factors such as variable microbial biomass, inherent nutrient/energy status, different water relations (due to variable hydrophysical properties of soil augmented by sand at different ratios). Results: The results revealed that P3HB in soils induced inconsistent to contradictory changes in the microbial abundance as well as in most enzymatic activities. The differences were conditioned by the sand content both under P3HB presence or absence. On the other hand, dehydrogenase, urease activities, basal and substrate-induced soil respirations were mostly enhanced by P3HB addition, directly with increasing sand content (several respiration types). Nevertheless, P3HB significantly inhibited lettuce biomass production. Conclusions: P3HB introduction to soil boosts the microbial activity owing to the preferential utilization of P3HB as C source, which depletes soil N and strongly inhibits the plant growth. Enhanced microbial activity in P3HB-amended soils with high sand content (60-80%) suggested that in nutrient-impoverished soil P3HB can temporarily replace SOM as a C source for microbial communities due to the shift of their structure to preferentially P3HB-degrading microbiome

Pre-activated biochar by fertilizers mitigates nutrient leaching and stimulates soil microbial activity

Background: Previous studies have suggested that the targeted application of biochars in agricultural soils may benefit soil health and crop production. Physico-chemical properties of soils after biochar addition have been explored, but less is known about how microbial parameters respond. Therefore, impact of biochar (NB), mineral fertilizer-activated biochar (AB), or mineral fertilizer (MF) application on selected chemical and microbial parameters of lettuce-planted soil was evaluated in a pot experiment. Results: In comparison to the control, soil enzymes activities, related to carbon (C), nitrogen (N), and phosphorus (P) cycling, and their content in plant biomass, were significantly increased by the addition of mineral fertilizer with or without biochar (MF, NB + MF). Conversely, microbial respiration (basal and substrate induced) was highly responsive to the activated biochar amendment (AB) as compared to other treatments. N, P, and potassium (K) concentrations in soil pore water were stabilized by the mineral fertilizer-activated biochar, indicating reduced leaching and the likelihood of increased longevity of these nutrients in soils. Enhanced carbon acquisition and mitigated nitrogen acquisition in soil of the most experimental treatments were coupled with higher crop (lettuce) biomass. Conclusions: Our study demonstrates that the application of biochar both with and without mineral fertilizer has the potential to enhance microbial activity and fertility in the tested agricultural soil, but that leaching of fertilizer-borne nutrients may be mitigated by the activation proces.O

Food and agricultural wastesderived biochars in combination with mineral fertilizer as sustainable soil amendments to enhance soil microbiological activity, nutrient cycling and crop production

The ever-increasing human population associated with high rate of waste generation may pose serious threats to soil ecosystem. Nevertheless, conversion of agricultural and food wastes to biochar has been shown as a beneficial approach in sustainable soil management. However, our understanding on how integration of biochar obtained from different wastes and mineral fertilizers impact soil microbiological indicators is limited. Therefore, in the present study the effects of agricultural (AB) and food waste derived (FWB) biochars with and without mineral fertilizer (MF) on crop growth and soil health indicators were compared in a pot experiment. In particular, the impacts of applied amendments on soil microbiological health indicators those related to microbial extracellular (C, N and P acquiring) enzymes, soil basal as well as different substrate induced respirations along with crop’s agronomic performance were explored. The results showed that compared to the control, the amendment with AB combined with MF enhanced the crop growth as revealed by higher above and below ground biomass accumulation. Moreover, both the biochars (FWB and AB) modified soil chemical properties (pH and electric conductivity) in the presence or absence of MF as compared to control. However, with the sole application of MF was most influential strategy to improve soil basal and arginin-induced respiration as well as most of the soil extracellular enzymes, those related to C, N and P cycling. Use of FWB resulted in enhanced urease activity. This suggested the role of MF and FWB in nutrient cycling and plant nutrition. Thus, integration of biochar and mineral fertilizers is recommended as an efficient and climate smart package for sustainable soil management and crop production

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σHin vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters

Biochar and Sulphur Enriched Digestate: Utilization of Agriculture Associated Waste Products for Improved Soil Carbon and Nitrogen Content, Microbial Activity, and Plant Growth

A number of agriculture residues may be used either directly or after suitable treatment as amendments to improve soil quality. Such materials include biochar made of agriculture residues, digestate or elemental sulphur obtained from biogas desulphurisation. The joint use of these materials via pre-incubation may be more advantageous than only mixing prior the application to soil. In this study, digestates were mixed with amendments and incubated for 6 weeks before application to soil in a short-term pot experiment with lettuce (Lactuca sativa). The following treatments were tested: control digestate, digestate + biochar, digestate + elemental sulphur, digestate + biochar + elemental sulphur. The biochar-enriched digestate significantly increased soil microbial biomass, soil C:N, fresh above ground biomass, fresh and dry root biomass. Elemental sulphur-enriched digestate caused highest arylsulfatase and phosphatase, increased urease, microbial biomass in soil and fresh root biomass. Amendment of digestate + biochar + sulphur led to the significantly highest total soil carbon, microbial biomass, fi-glucosidase, urease, and increased C:N ratio, arylsulfatase in soil and root biomass. It mitigated the adverse effect of either biochar or elemental sulphur on soil respiration. Properties of digestates were apparently affected by pre-incubation. This approach in digestate fertilizer production may contribute to sustainable farming

Modulace genové exprese mutagenezí v promotorech Corynebacterium glutamicum

Studies of structure and function of promoters in C. glutamicum were used in graded expression of genes involved in biosynthetic pathway of valine (ilvBNC, ilvD, ilvE) and in competing or branching-off pathways (ilvA, leuA). Combination of particular promoter mutants resulted in tuning the whole pathway to overall higher flux of the intermediates through the pathway and in high production of valin

Study of regulation of branched-chain amino acid biosynthesis in C.glutamicum and construction of valine producing strain

L-valine is an essential amino acid with a great medical and industoial importance and the need for efficient bacterial producersof this amino acid is increasing. Corynebacteriumglutamicum is a suitable organism for construction of such productionstrainsandthusbiosynthesisofvaline and expressionofgenes involved in its pathway are intensively studed. In this PhD. thesis, analysis of regulationand modulationof expressionwas carriedout with: (l) genes,which code for common enzymesof all branched-chainamino acids; (2) genes,whose products are involved in the competing biosynthesis pathways of L-isoleucine and L-leucine; and (3) the genethatdeterminesthe key enzyme in the catabolism of pyruvate. Results were used for construction of C. glutamicum production shains with deregulatedbiosynthesisof valine, restrictedbiosynthesisof isoleucine and leucine and optimum distributionof intermediatesof valine. The production strains were manipulated via modification of promoters in the chromosome. Promoters of the ilvA, leuA, ilvD, ilvE, and aceE genes were modified by site-directedmutagenesisof their -10 regions. All these alterationsincreased production of valine by the modified strains. Introduction of weak P-ilvA and P-leuA promotersinto the C. glutamicumchromosomeresultedin an intrinsic lowJevel biosynthesisof..

Study of regulation of branched-chain amino acid biosynthesis in C.glutamicum and construction of valine producing strain

L-valine is an essential amino acid with a great medical and industoial importance and the need for efficient bacterial producersof this amino acid is increasing. Corynebacteriumglutamicum is a suitable organism for construction of such productionstrainsandthusbiosynthesisofvaline and expressionofgenes involved in its pathway are intensively studed. In this PhD. thesis, analysis of regulationand modulationof expressionwas carriedout with: (l) genes,which code for common enzymesof all branched-chainamino acids; (2) genes,whose products are involved in the competing biosynthesis pathways of L-isoleucine and L-leucine; and (3) the genethatdeterminesthe key enzyme in the catabolism of pyruvate. Results were used for construction of C. glutamicum production shains with deregulatedbiosynthesisof valine, restrictedbiosynthesisof isoleucine and leucine and optimum distributionof intermediatesof valine. The production strains were manipulated via modification of promoters in the chromosome. Promoters of the ilvA, leuA, ilvD, ilvE, and aceE genes were modified by site-directedmutagenesisof their -10 regions. All these alterationsincreased production of valine by the modified strains. Introduction of weak P-ilvA and P-leuA promotersinto the C. glutamicumchromosomeresultedin an intrinsic lowJevel biosynthesisof...Souhrn L-valin je esenciáIníaminokyselin4 kteú má vellcý význam v medicíně a průmyslu. Proto stale roste potřeba účinnéhobakteriálního producenta této aminokyseliny. Druh Corynebacterium glutamiczzr je vhodným organismem pro konstrukci takovéhoprodukěního kmene a biosyntézavalinu i exprese genů zučastněnýchvjeho biosyntetické drrázejsou tudížintenzivně studovany. V této disertačnípráci byla provedena analýza regulace a modulace exprose genů"které: (1) kódují enzymy společnépro biosyntézuvšech tří větvených aminokyselin; (2) determinujíreakce v konkurenčníchbiosynteticlcých drahách L.isoleucinu a L- leucinu; a (3) genu, kteý kóduje klíčoý enzym katabolismupyruvátu.Výsledky byly využiý pro konstrukci produkčníchhnenů C. glutamicum s deregulovanou biosyntézou valinu, omezenou biosyntézou isoleucinu a leucinu a s optimáIní distribucí intermediátůvalinu. Produkčníkmeny byly manipulovany modifikací promotonl v chÍomosomu.Promotory gen.ůilvA, leuA, ilvD, ilvE a ace:E by|y modifikovfury místně-specifickoumutagenezíjejich oblastí -10. Všechny ýto změny zvýšily u mutantrích kmenůprodukci valinu. Zavedeni slabých promotorů P.ilvA a P.leuA do chromosomu C' glutamicum zpttsobilo sníženíbiosyntézy isoleucinu nebo leucinu a niíslednoubradytrofii na tyto aminokyseliny, která je ýhodnější neŽ auxotrofie navozsná...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

E1 Enzyme of the Pyruvate Dehydrogenase Complex in Corynebacterium glutamicum: Molecular Analysis of the Gene and Phylogenetic Aspects

The E1p enzyme is an essential part of the pyruvate dehydrogenase complex (PDHC) and catalyzes the oxidative decarboxylation of pyruvate with concomitant acetylation of the E2p enzyme within the complex. We analyzed the Corynebacterium glutamicum aceE gene, encoding the E1p enzyme, and constructed and characterized an E1p-deficient mutant. Sequence analysis of the C. glutamicum aceE gene and adjacent regions revealed that aceE is not flanked by genes encoding other enzymes of the PDHC. Transcriptional analysis revealed that aceE from C. glutamicum is monocistronic and that its transcription is initiated 121 nucleotides upstream of the translational start site. Inactivation of the chromosomal aceE gene led to the inability to grow on glucose and to the absence of PDHC and E1p activities, indicating that only a single E1p enzyme is present in C. glutamicum and that the PDHC is essential for the growth of this organism on carbohydrate substrates. Surprisingly, the E1p enzyme of C. glutamicum showed up to 51% identity to homodimeric E1p proteins from gram-negative bacteria but no similarity to E1 α- or β-subunits of heterotetrameric E1p enzymes which are generally assumed to be typical for gram-positives. To investigate the distribution of E1p enzymes in bacteria, we compiled and analyzed the phylogeny of 46 homodimeric E1p proteins and of 58 α-subunits of heterotetrameric E1p proteins deposited in public databases. The results revealed that the distribution of homodimeric and heterotetrameric E1p subunits in bacteria is not in accordance with the rRNA-based phylogeny of bacteria and is more heterogeneous than previously assumed