Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacter xylinus in E . coli Nissle 1917 Abstract Based on cellulose biosynthesis pathway of Gluconacetobacterxylinus BPR2001 and E. coli Nissle 1917, bcsA and bcsB genes have been selected and bioinformatics studies done to the analyses of nucleotide and amino acid sequence alignment, stability of RNA, protein, and promotor power. We amplify and clone bcsA, bcsB, and bcsAB genes of G. xylinus BPR2001 in Escherichiacoli Nissle 1917 under the inducible tac promoter. Our results of bioinformatics predictions demonstrate similar active site and three-dimensional structure of BcsA and BcsB proteins in two different bacteria. In addition, our data reveal that BcsA and BcsB proteins of E. coli have weaker promotor power, RNA secondary structure, and protein stability than that of the same proteins in G. xylinus. Some of the reasons of BcsAB protein selection from G. xylinus and its heterologous expression in E. coli is the noted points. Production of the related proteins visualized using SDS-PAGE. We find out that Congo red absorbance at 490 nm has no significant difference in wild-type strain (E. coli Nissle 1917) compared to recombinants bcsA+ or bcsB+, but recombinant bcsAB+ could produce more cellulose than that of the wild-type strain. Furthermore, the measurement of cellulose dry weights of all samples confirms bacterial cellulose production enhancement in recombinant bcsAB+ (1.94 g l−1). The FTIR analysis reveals that the crystallinity indices do not change significantly after over expressing each of genes.

Biogas yields and composition from oil-extracted halophilic algae residues in conventional biogas plants operated at high salinities Abstract CO2-induced climate change drives the development of renewable processes for industrial products. Algae processes can actively fix and convert CO2 into value adding products, such as oils. Algae lipids hence counteract climate change and provide access to renewable commodities. In this context, valorization of algal residues remaining after oil extraction is a challenge for the emerging cyclic bioeconomy. This study focuses on the valorization of oil-extracted algae residues derived from the halophilic strain Scenedesmus obliquus via anaerobic digestion. We examined the effect of prior oil extraction on microbial digestibility and increasing salt content in the substrate with regard to biogas yield and composition. Our cumulative data demonstrate that the supercritical CO2 oil extraction acts as a physical pretreatment that facilitates enhanced hydrolysis of both polymeric call wall carbohydrates and cellular proteins, providing methane yields of 213.2 LN kg−1 VS day−1. Methane yields were 20% higher than literature values obtained with the same algae strain in the absence of prior oil extraction. We obtained these superior results albeit all lipids and nonpolar proteins had been extracted from the biogas substrate. Our data indicate that continuous anaerobic digestion without loss of fermentation efficiency is feasible up to a salt concentration of 2% w/v, if conventional, agricultural biogas plants are gradually adapted to the salt content of the substrate. Monofermentation of the investigated oil-extracted algae residue is technically feasible at loading rates of 1.5 kg VS m−3 day−1, but a supplementation with carbohydrate rich biomass would prove beneficial to alleviate ammonia inhibition.

Evaluation of carbon sources for the production of inulinase by Aspergillus niger A42 and its characterization Abstract Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q10 values of inulinase dropped below one. At 70 and 80 °C, t1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.

Novel fabrication of gelatin-encapsulated copper nanoparticles using Aspergillus versicolor and their application in controlling of rotting plant pathogens Abstract The fabrication of copper nanoparticles (CuNPs) with smallest size and more stability, with potential effects in plant disease management, may need a modified protocol for green synthesis. In this study, we could biosynthesize stable CuNPs extracellularly by an eco-friendly route using A. versicolor. The biosynthesis of nanoparticles was confirmed by UV–visible spectroscopy, Fourier transform infrared (FTIR), transmission electron microscope (TEM) and dynamic light scattering (DLS) techniques. CuNPs have a size range of 23–82 nm with round to polygonal shape. Antifungal study showed that CuNPs have potential antifungal activity against rotting plant pathogens, where 3.2 and 2.8 µg ml−1 of nanoparticle solution totally inhibited the growth of both Fusarium oxysporum and Phytophthora parasitica, respectively. Damaged hyphae with limited deformed spores were detected through scanning electron microscope (SEM) analysis after the treatment of both pathogens with CuNPs. Between all tested polymers, gelatin-encapsulated nanoparticles were characterized ‘by their smallest size, 7–33 nm, and regular spherical shape at all experimental conditions. After 6 months of storage, gelatin-CuNPs maintained full nanoscale and antifungal properties compared with uncoated particles which lost these properties after only 1 month. It is concluded that CuNPs can be biosynthesized by an eco-friendly cheap method using A. versicolor and can be preserved stably for a long time with the smallest size and full antifungal activity by their encapsulation with gelatin as a natural polymer. These nanoparticles can be used safely in the management of plant rotting fungi.

Rapid start-up of partial nitritation in aerobic granular sludge bioreactor and the analysis of bacterial community dynamics Abstract The rapid start-up of the partial nitritation process in a laboratory-scale aerobic granular sludge-sequencing batch reactor was successful by controlling low dissolved oxygen and gradually increasing the influent ammonia levels. The microbial community dynamics were analyzed by high-throughput sequencing and quantitative polymerase chain reaction. The microbial communities were significantly affected by the different influent NH4+-N concentrations (77.84, 119.42, 170.31, and 252.21 mg/L) in Phases I, II, III, and IV. The sludge Shannon index in Phases I, II, III, and IV was 3.9, 4.39, 3.47, and 2.13, respectively, which was higher than that of the inoculated sludge (1.62). The dominant class transformed from Alphaproteobacteria and Gammaproteobacteria in Phase I to Betaproteobacteria in Phase IV. Furthermore, Sphingobacteria and Clostridia were the dominant bacteria in Phases III and IV. The quantitative polymerase chain reaction (qPCR) results suggested that Nitrosomonadaceae_uncultured belonging to ammonia-oxidizing bacterium was the dominant species, but the relative abundance of nitrite-oxidizing bacteria (mainly Nitrospira and Nitrobacter) was extremely rare in Phase IV. Furthermore, Thauera, Denitratisoma, and Planctomycetacia were the dominant functional nitrogen removal microbes in Phases III and IV. Some nitrogen removal pathways such as partial nitritation, denitrification, and anaerobic ammonium oxidation co-existed in the partial nitritation process.

Microbiota adaptation after an alkaline pH perturbation in a full-scale UASB anaerobic reactor treating dairy wastewater Abstract The aim of this study was to understand how the microbial community adapted to changes, including a pH perturbation, occurring during the start-up and operation processes in a full-scale methanogenic UASB reactor designed to treat dairy wastewater. The reactor performance, prokaryotic community, and lipid degradation capacity were monitored over a 9-month period. The methanogenic community was studied by mcrA/mrtA gene copy-number quantification and methanogenic activity tests. A diverse prokaryotic community characterized the seeding sludge as assessed by sequencing the V4 region of the 16S rRNA gene. As the feeding began, the bacterial community was dominated by Firmicutes, Synergistetes, and Proteobacteria phyla. After an accidental pH increase that affected the microbial community structure, a sharp increase in the relative abundance of Clostridia and a decrease in the mcrA/mrtA gene copy number and methanogenic activity were observed. After a recovery period, the microbial population regained diversity and methanogenic activity. Alkaline shocks are likely to happen in dairy wastewater treatment because of the caustic soda usage. In this work, the plasticity of the prokaryotic community was key to surviving changes to the external environment and supporting biogas production in the reactor.

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14 Abstract Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K2 biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K2 production significantly increased compared to control. The maximum vitamin K2 concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K2 production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

Cockroach wings-promoted safe and greener synthesis of silver nanoparticles and their insecticidal activity Abstract Simpler and biocompatible greener approaches for the assembly of nanoparticles (NPs) have been the focus lately which have minimum environmental damage and often entails the use of natural biomolecules to synthesize NPs. Such greener synthesis of nanoparticles has capitalized on the use of microbes, fungi, and plants using biological resources. In this study, Periplaneta americana (American cockroach) wings' extract (chitin-rich) is studied as a novel biomaterial for the first time to synthesize silver NPs (less than 50 nm); chitin is the second most abundant polymer after cellulose on earth. The physicochemical properties of these NPs were analyzed using UV–visible spectroscopy, X-ray diffraction, and transmission electron microscopy (TEM). The insecticidal effect of ensuing NPs was examined on the mortality of Aphis gossypii under laboratory conditions; 48 h after treatments of A. gossypii with silver NPs (100 μg/ml), the mortality rate in treated aphids was about 40% (an average), while an average percentage of losses in the control sample was about 10%. These results indicate the lethal effect of green-synthesized silver NPs on A. gossypii, in vitro. Graphic abstract Greener synthesis of silver nanoparticles using American cockroach wings and their insecticidal activities.

Application of an efficient indole oxygenase system from Cupriavidus sp. SHE for indigo production Abstract Indigo, one of the most widely used dyes, is mainly produced by chemical processes, which generate amounts of pollutants and need high energy consumption. Microbial production of indigo from indole has attracted much attention; however, the indole oxygenase has never been explored and applied for indigo production. In the present study, the indole oxygenase indAB genes were successfully cloned from Cupriavidus sp. SHE and heterologously expressed in Escherichia coli BL21(DE3) (designated as IND_AB). Strain IND_AB produced primarily indigo in tryptophan medium by high-performance liquid chromatography–mass spectroscopy (HPLC–MS) analysis. The preferable conditions for indigo production were pH 6.5 (normal pH), 30 °C, 150 rpm, strain inoculation concentration OD600 0.08, and induction with 1 mM IPTG at the time of inoculation. The optimal culture medium compositions were further determined as tryptophan 1.0 g/L, NaCl 3.55 g/L, and yeast extract 5.12 g/L based on single-factor experiment and response surface methodology. The highest indigo yield was 307 mg/L, which was 4.39-fold higher than the original value. This is the first study investigating indigo production using the indole oxygenase system and the results highlighted its potential in bio-indigo industrial application.

N 2 O production from hydroxylamine oxidation and corresponding hydroxylamine oxidoreductase involved in a heterotrophic nitrifier A . faecalis strain NR Abstract N2O production from NH2OH oxidation involved in a heterotrophic nitrifier Alcaligenes faecalis strain NR was studied. 15N-labeling experiments showed that biological NH2OH consumption by strain NR played a dominant role in N2O production, although chemical reaction between NH2OH and O2 indeed existed. Hydroxylamine oxidoreductase (HAO) from strain NR was partially purified by (NH4)2SO4 fractionation and DEAE Cartridge chromatography. The maximum activity of HAO was 9.60 mU with a specific activity of 92.04 mU/(mg protein) when K3Fe(CN)6 was used as an electron acceptor. The addition of Ca2+ promoted the HAO activity, while the presence of Mn2+ inhibited the enzyme activity. The optimal temperature and pH for HAO activity were 30 °C and 8. Analysis of enzyme-catalyzed products demonstrated that NH2OH oxidation catalyzed by HAO from strain NR played significant role in the production of N2O.