Microbiological interactions with cold plasma

There is a diverse range of microbiological challenges facing the food, healthcare and clinical sectors. The increasing and pervasive resistance to broad‐spectrum antibiotics and health‐related concerns with many biocidal agents drives research for novel and complementary antimicrobial approaches. Biofilms display increased mechanical and antimicrobial stability and are the subject of extensive research. Cold plasmas (CP) have rapidly evolved as a technology for microbial decontamination, wound healing and cancer treatment, owing to the chemical and bio‐active radicals generated known collectively as reactive oxygen and nitrogen species. This review outlines the basics of CP technology and discusses the interactions with a range of microbiological targets. Advances in mechanistic insights are presented and applications to food and clinical issues are discussed. The possibility of tailoring CP to control specific microbiological challenges is apparent. This review focuses on microbiological issues in relation to food‐ and healthcare‐associated human infections, the role of CP in their elimination and the current status of plasma mechanisms of action.     

Pectinase production by <Emphasis Type="Italic">Aspergillus giganteus</Emphasis> in solid-state fermentation: optimization,scale-up,biochemical characterization and its application in olive-oil extraction

The application of pectinases in industrial olive-oil processes is restricted by its production cost. Consequently, new fungal strains able to produce higher pectinase titers are required. The aim of this work was to study the capability of Aspergillus giganteus NRRL10 to produce pectinolytic enzymes by SSF and evaluate the application of these in olive-oil extraction. A. giganteus was selected among 12 strains on the basis of high pectinolytic activity and stability. A mixture composed by wheat bran, orange, and lemon peels was selected as the best substrate for enzyme production. Statistical analyses of the experimental design indicated that pH, temperature, and CaCl₂ are the main factors that affect the production. Subsequently, different aeration flows were tested in a tray reactor; the highest activity was achieved at 20 L min^?1 per kilogram of dry substrate (kgds). Finally, the pectinolytic enzymes from A. giganteus improved the oil yield and rheological characteristics without affecting oil chemical properties.     

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.     

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered with inulinase expression through cell surface display

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.     

Different responses in the expression of alginases,alginate polymerase and acetylation genes during alginate production by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> under oxygen-controlled conditions

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate \((q_{{{\text{O}}_{ 2} }} )\) from 10.9 to 45.3 mmol g^?1 h^?1 by changes in the dilution rate (D) from 0.06 to 0.10 h^?1, whereas under 1% DOT the \(q_{{{\text{O}}_{ 2} }}\) was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.     

Frequency of a natural truncated allele of <Emphasis Type="Italic">MdMLO19</Emphasis> in the germplasm of <Emphasis Type="Italic">Malus domestica</Emphasis>

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.     

Development of genome-wide SSR markers in horsegram and their use for genetic diversity and cross-transferability analysis

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] commonly known as kulthi or Madras gram is an important drought tolerant legume crop used as food and fodder in India and across the globe. Horsegram is tolerant to many biotic and abiotic stresses and considered a potential future food legume. Despite being a multiutility crop, insufficient genomic information is available in this species, which is otherwise required for genetic improvement. Hence, in the present work we used next-generation sequencing (NGS) technology for genome-wide development and characterization of novel simple sequence repeat (SSR) markers in horsegram. In all, 2458 SSR primer pairs were designed from NGS data and 117 SSRs were characterized in 48 diverse lines of horsegram. Cross-transferability of these markers was also checked in nine related legume species. The polymorphic SSRs revealed high diversity measures such as mean values of expected heterozygosity (He; 0.54), observed heterozygosity (Ho; 0.64), and polymorphism information content (PIC; 0.46). Analysis of molecular variance (AMOVA) revealed high degree of genetic variance within the populations. Dendrogram based on Jaccard’s similarity coefficient and principal component analysis (PCA) revealed two groups in the analyzed accessions. This observation was further confirmed by Bayesian genetic STRUCTURE analysis. The SSR markers developed herein can be used in diverse genetic analysis including association mapping in this crop and also in related legume crops with limited marker resources. Hence, this new SSR dataset can be useful for molecular breeding research in this underutilized pulse crop. In addition, genetic diversity estimates of analyzed germplasm can be important for devising future breeding programmes in horsegram.     

QTL mapping for tuberous stem formation of kohlrabi (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">gongylodes</Emphasis> L.)

The tuberous stem of kohlrabi is an important quantitative trait, which affects its yield and quality. Genetic control of this trait has not yet been unveiled. To identify the QTLs controlling stem swelling of kohlrabi, a BC₁ population of 92 plants was developed from a cross of broccoli DH line GCP04 and kohlrabi var. Seine. A wide range of variation in tuberous stem diameter was observed among the mapping populations. We constructed a genetic map of nine linkage groups (LGs) with different types of markers, spanning a total length of 913.5 cM with an average marker distance of 7.55 cM. Four significant QTLs for radial enlargement of kohlrabi stem, namely, REnBo1, REnBo2, REnBo3, and REnBo4 were detected on C02, C03, C05, and C09, respectively, and accounted for the phenotypic variation of 59% for the stem diameter and 55% for the qualitative grading of tuberous stem in classes. Then, we confirmed the stability of identified QTLs using BC₁S₁ populations derived from the BC₁ plants having heterozygous alleles at the target QTL and homozygous kohlrabi alleles at the remaining QTLs. REnBo1and REnBo2 using 128 plants of BC₁68S₁ and 94 plants of BC₁43S₁, respectively, and REnBo3 and REnBo4 using 152 plants of BC₁57S₁ were detected at the same positions as the respective QTLs of the BC₁ population. Confirmation of QTLs in two successive generations indicates that the QTLs are persistent. The QTLs obtained in this study could be useful in marker-assisted selection of kohlrabi breeding, and to understand the genetic mechanisms of stem swelling and storage organ development in kohlrabi and other Brassica species.