Appl Microbiol Biotechnol Research Articles

Purification Process for a Secreted Protein Produced in Insect Cells.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

Recovery of Bio-Based Fumaric Acid By Electrochemically Induced pH Shift Crystallization

The heterotrophic microbial production of platform chemicals is gaining increasing interest due to the growing need to replace fossil resources by renewable alternatives. Carboxylic acids such as fumaric acid are important building blocks for high-value bio-based chemicals [1]. Fumaric acid is used as an acidulant, as an antibacterial agent, and as a precursor for polymers and pharmaceuticals [2,3].A major challenge commercializing the microbial production route is the recovery and the purification of fumaric acid after fermentation. Bio-production of fumaric acid has not yet reached industrial scale due to high additive consumption during the acid- and base-induced pH shift in the downstream process. The inevitable co-production of saline waste deteriorates process economics further and raises ecological concerns.Therefore, we propose a new electrochemical downstream process, which reduces the use of pH adjustment agents [4]. This downstream process incorporates an electrolysis cell to adjust the pH value. During electrolysis, protons are produced at the anode and hydroxide ions are produced at the cathode by the electrochemical splitting of water. In conventional water electrolysis, protons or hydroxide ions migrate through a membrane to balance the charge in the electrolysis cell. Consequently, the pH value is constant in the electrolysis cell chambers. In pH shift electrolysis, a salt is added to the electrolyte. The salt provides additional ions to maintain electroneutrality in both electrolysis cell chambers. In this way, protons and hydroxide ions are retained in their respective chambers of the electrolysis cell and a pH difference is created between the anode and cathode. During acidification in the anode chamber, the fumaric acid salt from the fermentation is protonated. Subsequently, fumaric acid crystallizes in the electrolysis cell in high yields due to its low solubility. Afterwards, fumaric acid crystals can be easily separated from fermentation broth. Simultaneously, a base is produced at the cathode, which can be used to maintain a neutral pH in the fermentation. Consequently, saline waste production and its drawbacks are avoided.This work presents the pH shift with an electrolysis cell and the simultaneous crystallization of fumaric acid in the anode chamber. Next, the process is analyzed using on-line and off-line analytics such as Raman spectroscopy and high-performance liquid chromatography.[1] Bozell, J. J., & Petersen, G. R. (2010). Technology development for the production of biobased products from biorefinery carbohydrates - The US Department of Energy’s “Top 10” revisited. Green Chem, 12(4), 525-728. DOI: 10.1039/B922014C[2] Xu, Q., Li, S., Huang, H., & Wen, J. (2012). Key technologies for the industrial production of fumaric acid by fermentation. Biotechnol Adv, 30(6), 1685-1696. DOI: 10.1016/j.biotechadv.2012.08.007[3] Roa Engel, C. A., Straathof, A. J., Zijlmans, T. W., van Gulik, W. M., & van der Wielen, L. A. (2008). Fumaric acid production by fermentation. Appl Microbiol Biotechnol, 78, 379-389. DOI: 10.1007/s00253-007-1341-x[4] Kocks, C., Wall, D., & Jupke, A. (2022). Evaluation of a Prototype for Electrochemical pH-Shift Crystallization of Succinic Acid. Materials, 15(23), 8412. DOI: 10.3390/ma15238412

Screening of Spirulina Components for Anti-Parkinson's and Anti-Alzheimer's Activity byin Silico Methods and Docking Studies.

Spirulina platensis was first isolated from Lake Texcoco by Aztecs in the sixteenth century. In 2012, spirulina was considered to be safe dietary supplement by the Food and Drug Administration (FDA). Spirulina is a cyanophytic microalgae that is often considered as single cell protein. It contains many essential amino acids, proteins, fatty acids, antioxidant pigments, carotenoids, and cyanogenic pigments, that is, phycocyanobilins and phycocyanins (Eriksen, Appl Microbiol Biotechnol, 80(1):1-4, 2008). Components of spirulina are investigated for many health benefits and for pharmaceutical uses (Karkos et al., Spirulina in clinical practice: evidence-based human applications). Spirulina has been found to have a role in growth, immunity (Wu et al., Arch Toxicol, 90(8):1817-40, 2016), antioxidant (Wu et al., Arch Toxicol, 90(8):1817-40, 2016), antiviral (Ayehunie et al., J Acquir Immune Defic Syndr Hum Retrovirol, 18(1):7-12, 1998), antitoxicologic, anti-cancerogenic (Hirahashi et al., Int Immunopharmacol, 2(4):423-34, 2002), antidiabetic (Layam and Reddy, Diabetol Croat, 35(2):29-33, 2006), and neuroprotective properties. In this study, we focused on spirulina components in anti-Parkinson's and anti-Alzheimer's activity. Four potential targets, two for each activity, that is, structure of parkinE3 ligase (PDB ID:4I1H) and structure of BACE bound to 5-(3-(5-chloropyridin-3-yl)phenyl)-5-cyclopropyl-2-imino-3-methylimidazolidin-4one (PDBI D:4DJx) for anti-Parkinson's activity and structure of human MAO B in complex with selective inhibitor safinamide (PDB ID:2V5Z) and crystal structure of human BACE-1 in complex with CNP520(PDB ID:6EQM) for anti-Alzheimer's activity, have been selected. The in silico results and scoring of virtual screening, that is, molecular docking, were compared with commonly used marketed drugs such as levodopa for Parkinson's disease (PD) and rivastigmine (Rösler et al., BMJ, 318(7184):633-40, 1999) for Alzheimer's disease.

Single Crossover to Inactivate Target Gene in Cyanobacteria.

Anabaena sp. PCC 7120 (hereafter Anabaena 7120) is a model cyanobacterium for studying pathways such as photosynthesis and nitrogen fixation along with many other metabolic pathways common to plants. In addition, since Anabaena 7120 forms specialized N2-fixing cells, called heterocysts, to perform uniquely solar-powered, oxic nitrogen fixation under fixed-nitrogen depleted conditions, this cyanobacterium provides the unique opportunity to study cellular differentiation in bacteria. Since more than 155,810 sequenced prokaryotic genomes are currently available (Zhang et al., Microbiome 8(1):134, 2020), target gene inactivation, combined with analyses of the corresponding mutant's phenotype, has become a powerful tool to assess gene function through detecting a loss-of-function in the knockout mutant. In the method described here, a single crossover approach is used to knockout a target gene in Anabaena 7120. The method requires inserting an internal fragment of the target gene into the cyanobacterial integration vector pZR606 to create a knockout plasmid, and then is introduced to Anabaena 7120 via conjugative transformation. A single crossover, occurring via homologous recombination, disrupts the target gene, creating 3'- and 5'-deleted fragments (Fig. 1). The mutant containing the inactivated gene can then be studied to determine any loss of function, thereby defining the gene's function. This gene inactivation approach is based on an integrative vector pZR606 (Chen et al., Appl Microbiol Biotechnol 99:1779-1793, 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Effect of amphotericin B and voriconazole on the outgrowth of conidia of Aspergillus fumigatus followed by time-lapse microscopy

Studies of morphological measurements from the outgrowth of cells to a network of hyphae have been extended from Candida albicans (Nagy et al. in Appl Microbiol Biotechnol 98(11):5185–5194. https://doi.org/10.1007/s00253-014-5696-5, 2014) to invasive conidiospores of Aspergillus fumigatus upon treatment with antifungal agents. The understanding of mycelial processes is important to optimize industrial processes such as fermentation and contributes to the fight against pathogenic fungi. This brief study combines TLS with digital image analysis. The TLS system was adapted to get information related to the adherence and growth dynamics of filamentous fungi. This approach was used earlier to distinguish among subphases of bacterial and fungal infections of mammal cells by detecting Mycoplasma infection in cell cultures causing serious damages in cell cultures. We describe changes in adherence, germination of spores, and hyphal growth of A. fumigatus, taking place in the absence and presence of amphotericin B (AMB) and voriconazole (VRC). These growth parameters were measured by TLS in CO2 incubator under physiological Photomicrography by TLS and extended for a longer period of time up to several weeks combined with image analysis represents a comfortable and reliable means to characterize the growth dynamism of A. fumigatus. The most important observation of medical importance related to the pathomechanism of VRC was that it did not adhere to conidiospores, i.e. that it did not contribute to the attachment of spores to the growth surface, and did not prevent germination but delayed hypha protrusion and elongation. In contrast AMB adhered to conidia, inhibited germination, hypha elongation and branching. It was concluded that AMB was efficient against the therapy of growth but not against the prevention of fungal infection.

Soluble Lactobacillus delbrueckii subsp. bulgaricus 92059 PrtB proteinase derivatives for production of bioactive peptide hydrolysates from casein.

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that β-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method.

A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477-7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629-8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5'end without changing the amino acid sequence. The syn-pds-int gene (1872bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.

Alternative fermentation pathway of cinnamic acid production via phenyllactic acid.

Cinnamic acid (CA) is the chemical basis for bulk production of flavoring reagents and chemical intermediates, and it can be fermented from biomass. Phenylalanine ammonia lyase (PAL) has been used exclusively in the bacterial fermentation of sugar biomass in which the fermentation intermediate phenylalanine is deaminated to CA. Here, we designed an alternative metabolic pathway for fermenting glucose to CA. An Escherichia coli strain that generates phenylalanine in this pathway also produces Wickerhamia fluorescens phenylpyruvate reductase and ferments glucose to D-phenyllactate (D-PhLA) (Fujita et al. Appl Microbiol Biotechnol 97: 8887-8894, 2013). Thereafter, phenyllactate dehydratase encoded by fldABCI genes in Clostridium sporogenes converts the resulting D-PhLA into CA. The phenyllactate dehydratase expressed by fldABCI in the D-PhLA-producing bacterium fermented glucose to CA, but D-PhLA fermentation and phenyllactate dehydration were aerobic and anaerobic processes, respectively, which disrupted high-yield CA fermentation in single batch cultures. We overcame this disruption by sequentially culturing the two strains under aerobic and anaerobic conditions. We optimized the incubation periods of the respective aeration steps to produce 1.7g/L CA from glucose, which exceeded the yield from PAL-dependent glucose fermentation to CA 11-fold. This process is a novel, efficient alternative to conventional PAL-dependent CA production.

Biochemical and genetic characterization of β-1,3 glucanase from a deep subseafloor Laceyella putida.

A β-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80°C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Strain and process development for poly(3HB-co-3HP) fermentation by engineered Shimwellia blattae from glycerol.

Poly(3-hydroxybytyrate-co-3-hydroxypropionate), poly(3HB-co-3HP), is a possible alternative to synthetic polymers such as polypropylene, polystyrene and polyethylene due to its low crystallinity and fragility. We already reported that recombinant strains of Shimwellia blattae expressing 1,3-propanediol dehydrogenase DhaT as well as aldehyde dehydrogenase AldD of Pseudomonas putida KT2442, propionate-CoA transferase Pct of Clostridium propionicum X2 and PHA synthase PhaC1 of Ralstonia eutropha H16 are able to accumulate up to 14.5% (wtPHA/wtCDW) of poly(3-hydroxypropionate), poly(3HP), homopolymer from glycerol as a sole carbon source (Appl Microbiol Biotechnol 98:7409-7422, 2014a). However, the cell density was rather low. In this study, we optimized the medium aiming at a more efficient PHA synthesis, and we engineered a S. blattae strain accumulating poly(3HB-co-3HP) with varying contents of the constituent 3-hydroxypropionate (3HP) depending on the cultivation conditions. Consequently, 7.12, 0.77 and 0.32 gPHA/L of poly(3HB-co-3HP) containing 2.1, 8.3 and 18.1 mol% 3HP under anaerobic/aerobic (the first 24 hours under anaerobic condition, thereafter, aerobic condition), low aeration/agitation (the minimum stirring rate required in medium mixing and small amount of aeration) and anaerobic conditions (the minimum stirring rate required in medium mixing without aeration), respectively, were synthesized from glycerol by the genetically modified S. blattae ATCC33430 strains in optimized culture medium.

High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains

Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533-4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.

RETRACTED ARTICLE: Cell-specific delivery of messenger RNA and microRNA by recombinant MS2 virus-like particles carrying cell-penetrating peptide.

This article “Cell-specific delivery of messenger RNA and microRNA by recombinant MS2 virus-like particles carrying cell penetrating peptide” published in Appl Microbiol Biotechnol DOI 10.1007/s00253-014-6274-6 has been retracted at the request of the Authors. There was a conflict of intellectual property ownership of the published material. Professor Li Jinming who also contributed to the research made for this article did not give his approval for publication.

Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300.

Few studies concerning the nutritional requirements of Deinococcus geothermalis DSM 11300 have been conducted to date. Three defined media compositions have been published for the growth of this strain but they were found to be inadequate to achieve growth without limitation. Furthermore, growth curves, biomass concentration and growth rates were generally not available. Analysis in Principal Components was used in this work to compare and consequently to highlight the main compounds which differ between published chemically defined media. When available, biomass concentration, and/or growth rate were superimposed to the PCA analysis. The formulations of the media were collected from existing literature; media compositions designed for the growth of several strains of Deinococcaceae or Micrococcaceae were included. The results showed that a defined medium adapted from Holland et al. (Appl Microbiol Biotechnol 72:1074-1082, 2006) was the best basal medium and was chosen for further studies. A growth rate of 0.03 h(-1) and a final OD600nm of 0.55 were obtained, but the growth was linear. Then, the effects of several medium components on oxygen uptake and biomass production by Deinococcus geothermalis DSM 11300 were studied using a respirometry-based method, to search for the nutritional limitation. The results revealed that the whole yeast extract in the medium with glucose is necessary to obtain a non-limiting growth of Deinococcus geothermalis DSM 11300 at a maximum growth rate of 0.64 h(-1) at 45 °C.

Electrochemical Study of the Chalcopyrite Biooxidation in Presence and Absence of Sulfuro- and Ironoxidizing Microorganisms

The chalcopyrite (CuFeS2) is one of the main mineral sulfides (MS) used for the leaching of Cu; during its oxidation, generates insoluble sulfur species or secondary phases of sulfur (by example S°, Sn 2- and intermediate products ) that may form a passivation layer on the mineral surface, which decreases the dissolution kinetics.[ 1-7 ] Iron and sulfur oxidizing microorganisms (IOM and SOM, respectively) may prevent such effect throughout the biooxidation of reduced sulfur species [8, 9]. It has been found that the electrochemical formation of S° and Sn 2- phases on mineral surface facilitates initial attachment of microorganisms; in addition, it has been reported that the electrochemical activity degree of sulfur species, directly influences on the physicochemical and biological characteristics of the biofilm developed during the biooxidation[10,11]. This research assessed the biooxidation process of CuFeS2, correlating the initial presence of S° and/or Sn 2- with changes in the hydrophobic domains (HD) of extracellular polymeric substances (EPS) of the formed biofilm. For this, the behavior of two different microbial cultures were compared, one SOM culture (containing only Acidithiobacillus thiooxidans) and a mixed SOM-IOM culture (Acidithiobacillus thiooxidans + Leptospirillum sp.). Massive Electrodes of chalcopyrite were electrochemically oxidized to form the sulfur secondary species (S° and Sn 2-) previous to the mineral-bacteria contact. The evolution of secondary [RL1] species during biooxidation process in each culture was followed by Raman Spectroscopy. The spacetime changes of the biofilm and interfacial electrochemical study were evaluate by Electrochemical Impedance Spectroscopy (EIS and Scanning Electron Microscopy (SEM). The previous results showed that there are important differences in the biooxidation process, depending on the sulfur species produced initially, the type of culture used, and the incubation time; in addition, it was found that both types of cultures efficiently remove S° and Sn 2- by dissimilar mechanisms that seem to be associated with the IOM role in the bioleaching process. Key Words chalcopyrite, passivation layer, IOM, SOM, biooxidation.1.- Habashi F. (1978). Chalcopyrite. Its chemistry and metallurgy. McGraw-Hill.2.- Dutrizac JE (1989) Elemental sulphur formation during the ferric sulphate leaching ofchalcopyrite. Can Metall Q 28:337–44.3.-Dutrizac JE (1992)The leaching of sulphide minerals in chloride media. Hydrometallurgy 29:1–45.3.- Antonijevic MM, Jankovic Z, Dimitrijevic M (1994) Investigation of the kinetics ofchalcopyriteoxidation by potassium dichromate. Hydrometallurgy 35:187–201.4.- Harmer SL (2002) Surface layer control for improved copper recovery for chalcopyriteleaching. University of South Australia.5. Xian YJ, Wen SM, Deng JS, Liu J, Nie Q (2012) Leaching chalcopyrite with sodium chlorate inhydrochloric acid solution. Can Metall Q 51:133–40.6.- Parker AJ, Paul RL, Power GP (1981) Electrochemistry of the oxidative leaching of copperfrom chalcopyrite. J Electroanal Chem Interfacial Electrochem 118:305–16.7.- Hackl RP., Dreisinger DB, Peter SE, King JA (1995). Passivation of chalcopyrite duringoxidative leaching in sulfate media. Hydrometallurgy 39: 25–48.8.- Pan HD, Yang HY, Tong LL, Zhong CB, Zhao YS (2012) Control method of chalcopyritepassivation in bioleaching. Trans. Nonferrous Met Soc China 22:2255−2260.9.- Anjum F, Shahid M, Akcil, A (2012) Biohydrometallurgy techniques of low grade ores: Areview on black shale. Hydrometallurgy (1-12):117-118 doi:10.1016/j.hydromet.2012.01.007.10.-González D, Lara R, Valdez-Pérez D, Alvarado K, Navarro-Contreras H, Cruz R, García-MezaJV (2012) Evolution of biofilms during the colonization process of pyrite by Acidithiobacillus thiooxidans. Appl Microbiol Biotechnol 93:763–775.11.- Lara R, García-Meza V, González I, Cruz R (2012) Influence of the surface speciation onbiofilm attachment to chalcopyrite by Acidithibacillus thiooxidans. Appl Microbial Biotechnol.doi 10.1007/s00253-0112-4099-8.

Effect of Humic Acid on the Electrochemical Performance of Microbial Fuel Cells (MFCs)

Microbial fuel cell (MFC) are bioelectrochemical system using exoelectrogenic microorganisms to produce electricity, electrons are transferred through a biofilm growth on the anode surface (Sund et al., 2007), the humic acid had optimal biofilm formation, reducing the lag-time and improve the performance of the MFCs. The aim of this work was to evaluate the effect of humic acid addition on the performance of MFC. The biofilm growth with and without humic acid was evaluated by cyclic voltammetry (CV), chronopotentiometry at open circuit potential and scanning electron microscopy (SEM), the performance of MFC was evaluated by polarization curves and values of voltage, current density and power density achieved with data acquisition system. Triplicate measurements made in a MFC with two chambers of 375 mL, carbon cloth electrodes and proton exchange membrane. From the 5th day, the MFC without humic acid showed a voltage of 0.1 V in response to biofilm wrowth and a steady voltage of 0.34 V after 40 days, cyclic voltammetry showed reduction signs in -0.284 to 0.259 V Vs SHE and oxidation signs in -0.643 to 0.060 V Vs SHE, showing electroactivity biofilms. Optimal biofilm formed reduce the internal resistance from 50 to 4.5 kµÙ, also the anode potential changed from -0.251 to -0.512 V Vs SHE for better performance of MFC. The MFC showed a maximum power density of 1.78E-03 W/m2, current density of 3.6 mA/m2 and power density of 1.4 W/m2. The optimal biofilm formation is important for the performance of MFCs (Martin et al., 2013) and humic acid had effect on the electrode surface supported the growth of biofilm due to functional groups with redox properties (Wandruszka Palmer, 2010). References Sund et al., 2007. Appl Microbiol Biotechnol. 76, 561-568.Palmer y Wandruszka, 2010. Environmental Science & Pollution Research. 17, 1362-1370.Martin et al., 2013. J. Appl. Electrochem. DOI 10.1007/510800-013-0537-2.

Engineered, Modular Microbial Communities for Simultaneous Wastewater Decontamination and Energy Generation

We propose a strategy to produce biocatalytic wastewater cleaning materials that incorporate microorganisms simultaneously removing organic and inorganic contaminants while creating electricity (or hydrocarbon fuel) for energy generation in support of a sustainable and portable system.Central to our work will be then encapsulation of dissimilatory metal reducing bacteria for energy generation on the anode of a microbial biofuel cell (MFC) . We have previously demonstrated both great stability and greater current densities produced Shewanella oneidensis when biofilms of this organism are immobilized on a carbon anode. 1While our published results indicate full operation for a week, one such device was active for a full 6 months in our laboratories, indicating the stability and functionality of such systems. While DMRBs are able to remove a plethora of organic compounds, including pesticides, inorganic compounds, such as ammonia, phosphate and heavy metals must also be removed to produce clean water. Immobilized algae have recently been shown to effectively remove phosphorous and nitrogen from wastewater. 2 An advantage of an encapsulated system is demonstrated by the immunity of this system to population fluctuation, the presence of other solids, or competing bacteria that frequently plague phosphate removal in particular.3 Such a system could be combined with, for example, Shewanella oneidensis in a second module and Geobacter sp. in a third, and used to produce a self-regererating carbon neutral electrical system. Alternatively, the Geobacter module could be replaced by a module containing an acetate/methanogen syntrophic community to generate methane.Other example modules include algae to remove heavy metals 4, Acinetobacter venetianus to remove oil spills 5, or pesticide remediating heterotrophs 6.Research areas for this program will include metabolic activity of constituent organisms upon encapsulation, optimization of the transport characteristics of the encapsulating silica and the integration of electrodes or other energy collection modalities.(1) Luckarift, H., Sizemore, S., Roy, J., Lau, C., Gupta, G., Atanassov, P., Johnson, G. Chem.Comm. 2010, 46, 6048. (2) Shi, J.; Padola, B.; Melkonian, M. Bioresource Tech 2014 154, 260.(3) Tu, Y.; Schuler, A. J. Environ Sci Technol 2013, 47, 3816.(4) Montiero, C. M.; Castro, P. M. L.; Makcata, F. X. Biotechnol Prog 2012, 28, 299.(5) Luckarift, H. R.; Sizemore, S. R.; Farrington, K. E.; Fulmer, P. A.; Biffinger, J. C.; Nadeau, L. J.; Johnson, G. R. Biotechnol Prog 2011, 27, 1580.(6) Nunez, O. C.; Lopes, A. R.; Manaia, C. M. Appl Microbiol Biotechnol 2013, 97, 10275.

Metabolic Engineering of Saccharomyces cerevisiae for Isoprenoid Production

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of either therapeutic or industrial interest. However, heterologous protein productivity is often low due to limitations of the selected host strain (1). In the case of the α-amylase, overproduction by S. cerevisiae may lead to an intracellular accumulation of reactive oxygen species (ROS) as a consequence of the formation of disulfide bonds during protein folding in the endoplasmic reticulum, and therefore causing a significant decrease in the cell performance which ultimately affects the protein titer and productivity (2). In order to reduce the internal ROS levels, we describe a strategy based on the overexpression of the Hap1 transcription factor, known to be responsible for both the activation of a set of genes involved in ROS detoxification and the oxidative stress response, as well as genes related to the respiratory metabolism (3). This strategy resulted to have a positive effect in the protein production by increasing the final titer compared to the control strains, while keeping the ROS levels constant during batch cultivations. 1. Hou J, Osterlund T, Liu Z, Petranovic D, Nielsen J. 2013. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 97:3559-3568. 2. Liu Z, Osterlund T, Hou J, Petranovic D, Nielsen J. 2013. Anaerobic alpha- amylase production and secretion with fumarate as the final electron acceptor in Saccharomyces cerevisiae. Appl Environ Microbiol 79:2962- 2967. 3. Zhang L, Hach A. 1999. Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator. Cell Mol Life Sci 56:415-426.

Acetobacter pasteurianus DSM 3509 produces cobalamin

Seed coat is an important tissue for the regulation of imbibition and maintenance of the integrity of seed, and it is also the first seed barrier encountered by pests and pathogens. Seed cotyledons contain an array of proteins that may be involved in the protection of quiescent seeds against fungi. Now we know that seed coat from Theobroma cacao L. seeds contains an antifungal activity. In the present study inhibition tests of seed coat extracts against microorganisms isolated from cocoa bean fermentations were performed. Seed coat was extracted using surfacesterilization and filter-sterilization. To determine antifungal activity in seed coat extract agar diffusion and broth microdilution tests were used. Seed coat extract can inhibit growth of fungi (e.g. Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, Mortierella isabellina, Penicillium citrinum, Penicillium purpurogenum, Penicillium roquefortii). and yeasts (e.g. Candida krusei, Candida lipolytica, Candida guilliermondii, Cryptococcus laurentii, Rhodotorula mucilaginosa, Rhodotorula rubra, Saccharomyces cerevisiae, Schizosaccharomyces pombe). No inhibition effect could be detected against gram positive bacteria (e.g. Bacillus subtilis, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, Propionibacterium freudenreichii) and gram negative bacteria (e.g. Escherichia coli, Acetobacter pasteurianus, Acetobacter tropicalis, Acetobacter pomorum, Gluconobacter frateurii, Acetobacter orientalis). The minimum inhibitory concentration (MIC) of seed coat extract was determined. 25 mg/mL of seed coat extract can inhibit growth of fungi (e.g. Penicillium citrinum, Aspergillus niger, Penicillium purpurogenum), and 10 mg/mL seed coad extract can inhibit growth of yeasts (e.g. Saccharomyces cerevisiae, Rhodotorula rubra, Candida lipolytica). Nisin production in tofu by fermentation Nicole Illas, Raphael Cziskus, Caterina Hunniger, Agata Segieda, Xuan Zhu, Markus Fischer, Bernward Bisping University of Hamburg, Institute of Food Chemistry, Division of Food Microbiology and Biotechnology, Biocenter Klein Flottbek, Ohnhorststr. 18, D-22609 Hamburg, Germany Division of Food Chemistry, Bundesstr. 45, D-20146 Hamburg, Germany Tofu has a high nutritional content but on the other hand it can easily be spoiled. In Indonesia tofu production takes place mostly in small factories without an opportunity for pasteurization. Nisin production in tofu by fermentation with Lactococcus lactis ssp. lactis DSM 20729 may be a possibility to preserve tofu. The fermentation of tofu cubes (2*2 cm) submerged in water was performed for two days. Two methods of detection of nisin in fermented tofu were tested and compared by using inhibition test (modified method of Pongtharangkul and Demirci 2004), and a liquid chromatography electrospray ionization tandem mass spectrometry method (LC-ESI-MS/MS), based on ISO/TS 27106:2009. The detection limit was significantly lower by using the inhibition test. The nisin content was 0.19 mg/kg in tofu and 0.06 mg/L in liquid substrate. In comparison to this the detection limit for nisin using LC-ESI-MS/MS was 0.34 mg/kg in tofu. Matrix calibration of the liquid substrate could not be carried out by LC-ESI-MS/MS, because the background noise of the matrix was too high. Furthermore the production and distribution of nisin in tofu cubes was investigated. The nisin concentration on the surface of tofu was 2.64 mg/kg and so it was nine times higher compared to the interior of tofu. The optimum storage condition for fermented tofu was a combination of low temperature and low pH-value. An alternative to preserve tofu may be fermentation of tofu with L. latis ssp. lactis. Nisin concentration of complete tofu cubes could be successfully determined by using both methods. The inhibition test proved to be more sensitive for nisin detection in tofu. Pongtharangkul T, Demirci A (2004) Evaluation of agar diffusion bioassay for nisin quantification. Appl Microbiol Biotechnol 65: 268-272 International Organization for Standardization: ISO/TS 27106 (2009) Cheesedetermination of nisin A content by LC-MS and LC-MS/MS. Geneva, Switzerland

A genetically modified broad-spectrum strain of Bacillus thuringiensis toxic against Holotrichia parallela, Anomala corpulenta and Holotrichia oblita

Cry8Ea1 from Bacillus thuringiensis strain Bt185 has insecticidal activity against Holotrichia parallela. Cry8Ca2 from strain HBF-1 is effective against Anomala corpulenta. Cry8Ga1 from strain HBF-18 is toxic to H. oblita. Given the need to broaden the spectrum of B. thuringiensis, a broad-spectrum coleopteran active strain of B. thuringiensis, BIOT185, engineered to express multiple cry genes, including cry8Ea1, cry8Fa1 and cry8Ca2, was created by homologous recombination introducing the cry8Ca2 into the B. thuringiensis strain Bt185 by Liu et al. (Appl Microbiol Biotechnol 87:243-249, 2010). To further broaden the spectrum, an engineered B. thuringiensis strain BIOT1858G was constructed by introducing the recombinant plasmid pSTK-8G containing cry8Ga1 into the engineered B. thuringiensis strain BIOT185. PCR and Southern blotting demonstrated that the cry8Ga1 gene was transferred to the novel strain BIOT1858G. SDS-PAGE and RT-PCR confirmed the normal expression and transcription of the cry8Ga1 gene in addition to the cry8Ea1, cry8Fa1 and cry8Ca2 genes. Bioassays of BIOT1858G indicated that the recombinant strain broadened the spectrum against not only H. parallela susceptible to the Cry8E protein and A. corpulenta susceptible to the Cry8C protein but also H. oblita susceptible to the Cry8G protein. The pesticide could also decrease the cost of production and field labor.

Progress in bioleaching: part B: applications of microbial processes by the minerals industries

This review presents developments and applications in bioleaching and mineral biooxidation since publication of a previous mini review in 2003 (Olson et al. Appl Microbiol Biotechnol 63:249-257, 2003). There have been discoveries of newly identified acidophilic microorganisms that have unique characteristics for effective bioleaching of sulfidic ores and concentrates. Progress has been made in understanding and developing bioleaching of copper from primary copper sulfide minerals, chalcopyrite, covellite, and enargite. These developments point to low oxidation-reduction potential in concert with thermophilic bacteria and archaea as a potential key to the leaching of these minerals. On the commercial front, heap bioleaching of nickel has been commissioned, and the mineral biooxidation pretreatment of sulfidic-refractory gold concentrates is increasingly used on a global scale to enhance precious metal recovery. New and larger stirred-tank reactors have been constructed since the 2003 review article. One biooxidation-heap process for pretreatment of sulfidic-refractory gold ores was also commercialized. A novel reductive approach to bioleaching nickel laterite minerals has been proposed.