Utilizing Cattle Manure Compost Increases Ammonia Monooxygenase A Gene Expression and Ammonia-oxidizing Activity of Both Bacteria and Archaea in Biofiltration Media for Ammonia Deodorization.

Malodorous emissions are a crucial and inevitable issue during the decomposition of biological waste and contain a high concentration of ammonia. Biofiltration technology is a feasible, low-cost, energy-saving method that reduces and eliminates malodors without environmental impact. In the present study, we evaluated the effectiveness of compost from cattle manure and food waste as deodorizing media based on their removal of ammonia and the expression of ammonia-oxidizing genes, and identified the bacterial and archaeal communities in these media. Ammonia was removed by cattle manure compost, but not by food waste compost. The next-generation sequencing of 16S ribosomal RNA obtained from cattle manure compost revealed the presence of ammonia-oxidizing bacteria (AOB), including Cytophagia, Alphaproteobacteria, and Gammaproteobacteria, and ammonia-oxidizing archaea (AOA), such as Thaumarchaeota. In cattle manure compost, the bacterial and archaeal ammonia monooxygenase A (amoA) genes were both up-regulated after exposure to ammonia (fold ratio of 14.2±11.8 after/before), and the bacterial and archaeal communities were more homologous after than before exposure to ammonia, which indicates the adaptation of these communities to ammonia. These results suggest the potential of cattle manure compost as an efficient biological deodorization medium due to the activation of ammonia-oxidizing microbes, such as AOB and AOA, and the up-regulation of their amoA genes.

Biosyntheses of geranic acid and citronellic acid from monoterpene alcohols by Saccharomyces cerevisiae.

Geraniol is one of the important aromatic ingredients in alcoholic beverages. Bioconversions of geraniol to other terpenoids and genes involved in the oxidation of geraniol were investigated. Geranic acid and citronellic acid were detected in yeast culture, where geraniol or nerol was added. Addition of citral, a mixture of geranial and neral, resulted in the production of geranic acid and citronellic acid, whereas the addition of citral or citronellal resulted in the production of citronellic acid, suggesting that citronellic acid might be produced through the conversion of citral to citronellal followed by the oxidation of citronellal. Consumption of geraniol and production of geranic acid, citronellic acid, and citronellol were affected in adh1Δ, adh3Δ, adh4Δ, and sfa1Δ yeast strains, which possess single deletion of a gene encoding alcohol dehydrogenase. This is the first report of the bioconversion of monoterpene alcohols, geraniol and nerol, to geranic acid and citronellic acid in yeast culture.

Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields.

Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6-8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.

Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus.

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.

Nitrogen fixation in Rhodopseudomonas palustris co-cultured with Bacillus subtilis in the presence of air.

Nitrogen fixation in purple non-sulfur bacteria (PNSB) does not take place even in N-free medium when they are cultured under aerobic conditions. It is assumed that PNSB might possess inadequate capability to protect their cellular components from exposure to air (20.95 vol.% oxygen). In this study, therefore, Bacillus subtilis was inoculated together with a purple non-sulfur bacterium Rhodopseudomonas palustris in N-free medium in order to examine whether nitrogen fixation in Rps. palustris takes place when the co-culture is exposed to 20.95 vol.% oxygen. Rps. palustris grew and formed biofilm only when it was inoculated together with B. subtilis. When the biofilm formed in the co-culture was inoculated in N-free medium, diazotrophic growth was observed in the sequential subcultures. Expression of nifH gene, derepression of nitrogenase activity, an increase of total nitrogen, and a decrease of C/N in the co-culture of Rps. palustris and B. subtilis demonstrated the occurrence of nitrogen fixation under aerobic conditions. The diazotrophic growth was suppressed at a lower medium-to-air ratio in a sealed culture vessel, and growth of B. subtilis preceded growth of Rps. palustris in the co-culture. These results suggest that growth of B. subtilis, which is usually accompanied with oxygen consumption, might cause a decrease of dissolve oxygen concentration in medium and contribute to the occurrence of nitrogenase activity in Rps. palustris.

Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

Effect of increased feeding of dietary α-linolenic acid by grazing on formation of the cis9,trans11-18:2 isoform of conjugated linoleic acid in bovine milk.

Feeding systems such as grazing affect the fatty acid profile of bovine milk fat. In addition, milk fat is formed as the product of fatty acid metabolism in cow bodies before being secreted into milk. However, how grazing influences milk fatty acid profile through the metabolism has not been completely characterized. When fatty acid concentrations in Holstein milk were compared between grazing and non-grazing periods, α-linolenic acid was significantly higher in the grazing period than in the non-grazing period. This could be explained with an increase in α-linolenic acid feeding with grazing. α-linolenic acid had a linear positive correlation with conjugated linoleic acid (9c,11t-18:2) (CLA) and vaccenic acid (VA) during the grazing period, whereas CLA had higher correlation with linoleic acid rather than with α-linolenic acid during the non-grazing period. These data indicate that the high content of dietary α-linolenic acid affects CLA and VA formation in milk of grazing periods via α-linolenic acid metabolism into VA.

Light-enhanced bioaccumulation of molybdenum by nitrogen-deprived recombinant anoxygenic photosynthetic bacterium Rhodopseudomonas palustris.

As molybdenum (Mo) is an indispensable metal for plant nitrogen metabolisms, accumulation of dissolved Mo into bacterial cells may connect to the development of bacterial fertilizers that promote plant growth. In order to enhance Mo bioaccumulation, nitrogen removal and light illumination were examined in anoxygenic photosynthetic bacteria (APB) because APB possess Mo nitrogenase whose synthesis is strictly regulated by ammonium ion concentration. In addition, an APB, Rhodopseudomonas palustris, transformed with a gene encoding Mo-responsive transcriptional regulator ModE was constructed. Mo content was most markedly enhanced by the removal of ammonium ion from medium and light illumination while their effects on other metal contents were limited. Increases in contents of trace metals including Mo by the genetic modification were observed. Thus, these results demonstrated an effective way to enrich Mo in the bacterial cells by the culture conditions and genetic modification.

Evaluation of bacterial communities by bacteriome analysis targeting 16S rRNA genes and quantitative analysis of ammonia monooxygenase gene in different types of compost.

Biofiltration technology based on microbial degradation and assimilation is used for the removal of malodorous compounds, such as ammonia. Microbes that degrade malodorous and/or organic substances are involved in composting and are retained after composting; therefore, mature composts can serve as an ideal candidate for a biofilter medium. In this study, we focused on different types of raw compost materials, as these are important factors determining the bacterial community profile and the chemical component of the compost. Therefore, bacterial community profiles, the abundance of the bacterial ammonia monooxygenase gene (amoA), and the quantities of chemical components were analyzed in composts produced from either food waste or cattle manure. The community profiles with the lowest beta diversity were obtained from single type of cattle manure compost. However, cattle manure composts showed greater alpha diversity, contained higher amounts of various rRNA gene fragments than those of food waste composts and contained the amoA gene by relative quantification, and Proteobacteria were abundantly found and nitrifying bacteria were detected in it. Nitrifying bacteria are responsible for ammonia oxidation and mainly belong to the Proteobacteria or Nitrospira phyla. The quantities of chemical components, such as salt, phosphorus, and nitrogen, differed between the cattle manure and food waste composts, indicating that the raw materials provided different fermentation environments that were crucial for the formation of different community profiles. The results also suggest that cattle manure might be a more suitable raw material for the production of composts to be used in the biofiltration of ammonia.

Mercury (II) sensor based on monitoring dissociation rate of the trans-acting factor MerR from cis-element by surface plasmon resonance.

Transcriptional switches regulate gene expression in response to environmental changes surrounding cell. Many studies have focused on two fundamentally different models of transcriptional control by bacterial metalloregulatory protein. Distortion of the DNA fragment including cis-element, to which the trans-acting factor MerR binds, is accepted as the mechanism of gene expression regulation by Hg (II) while, in cases of the other trans-acting factors ArsR and CadC, events of association to and dissociation from cis-element are known to control transcription in response to As (III) and Cd (II), respectively. In this study, interactions between green-fluorescent-protein-tagged trans-acting factor and immobilized cis-element were analyzed on solid surface. Fluorescent measurements and surface plasmon resonance (SPR) responses revealed that although the equilibrium dissociation constant (KD) was much lower in MerR than in ArsR and CadC, the dissociation rate of MerR from DNA increased in response to Hg (II) at concentrations of 5-10(4) µg l(-1). These results firstly demonstrate an increase of KD between MerR and its recognition site in DNA by Hg (II), and possibility of rapid Hg (II) quantification with the low detection limit (5 µg l(-1)) and the high dynamic range (10(1)-10(4) µg l(-1)).

Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process.

We studied a process that enables simultaneous recovery of protein and phosphorus compounds from rice bran. Phosphorus substances in full-fat and defatted rice bran such as phytic acid and inorganic ions were solubilized under acidic conditions in the first step. After that, inorganic and/or organic phosphate salts were recovered in insoluble form under weak alkaline conditions. Furthermore, protein fractions obtained after phosphorus compounds had been removed were solubilized under alkaline conditions. After solubilization, protein fractions with high content were recovered by isoelectric precipitation (IP) followed by electrolyzed water treatment (EWT). The highest protein content (52.3 w/w%) was attained when machine defatted rice bran was treated through the process. Energy-dispersive X-ray spectroscopy (EDX) and inductively coupled plasma atomic emission spectrometry (ICP-AES) analyses demonstrated efficient desalting from the protein fractions by EWT and higher phosphorus contents (15.1-16.4 w/w% P) in the phosphorus fractions compared with commercial phosphate rock. In addition, no heavy metal ions in either protein or phosphorus fractions were detected. These results suggest that the newly developed process is suitable for practical recovery of highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants.

Population abundance of potentially pathogenic organisms in intestinal microbiome of jungle crow (Corvus macrorhynchos) shown with 16S rRNA gene-based microbial community analysis.

Jungle Crows (Corvus macrorhynchos) prefer human habitats because of their versatility in feeding accompanied with human food consumption. Therefore, it is important from a public health viewpoint to characterize their intestinal microbiota. However, no studies have been involved in molecular characterization of the microbiota based on huge and reliable number of data acquisition. In this study, 16S rRNA gene-based microbial community analysis coupled with the next-generation DNA sequencing techniques was applied to the taxonomic classification of intestinal microbiome for three jungle crows. Clustering of the reads into 130 operational taxonomic units showed that at least 70% of analyzed sequences for each crow were highly homologous to Eimeria sp., which belongs to the protozoan phylum Apicomplexa. The microbiotas of three crows also contained potentially pathogenic bacteria with significant percentages, such as the genera Campylobacter and Brachyspira. Thus, the profiling of a large number of 16S rRNA gene sequences in crow intestinal microbiomes revealed the high-frequency existence or vestige of potentially pathogenic microorganisms.

Acyclic carotenoid and cyclic apocarotenoid cleavage by an orthologue of lignostilbene-α,ß-dioxygenase in Rhodopseudomonas palustris.

Carotenoid cleavage oxygenases catalyse formation of apocarotenoids and the precursors of phytohormones, abscisic acid and strigolactones through oxidative cleavage at specific double bonds of carotenoids. A gene encoding a presumed bacterial oxygenase homologous to lignostilbene-α,ß-dioxygenases has been found in the genome of Rhodopseudomonas palustris. By analysing apocarotenoids in recombinant Escherichia coli strains, it was found that the presumed oxygenase catalyses the 15,15' double bond cleavage of lycopene and neurosporene. Cell lysate containing the recombinant protein cleaved all-trans-ß-apo-8'-carotenal at the 15,15' double bond into retinal and apo-8',15'-apocarotene-dial. These data demonstrate for the first time that the orthologue of lignostilbene-α,ß-dioxygenase found in the carotenogenic phototrophic bacterium has the 15,15' double bond cleavage activity towards both the acyclic carotenoids and cyclic apocarotenoid.

Combinatorial parallel display of polypeptides using bacteriophage T7 for development of fluorescent nano-bioprobes.

Application of nano-particles to diagnostic fields has attracted much attention. Biotechnology can contribute to produce useful nano-materials by engineering bacteriophage nano-particles, which are easily prepared by infecting phages to bacterial host cells. In this study, establishment of nano-bioprobes was demonstrated, based on the T7 phage display system, by constructing phage particles displaying a ligand polypeptide S-tag and a green fluorescent protein (GFP) at the same time on the surface of phage head. To achieve this purpose, two types of phage particles were tested: One displayed S-tag and GFP as a single polypeptide (tandem display), and another displayed these molecules as two different polypeptides (parallel display). Only the parallelly displayed phage could be detected with ligand blotting using S-protein and with immunoblotting using an anti-GFP antibody. S-protein-coated magnetic beads and nano-particles were successively labeled with fluorescence using the parallelly displayed phage but could not be labeled with the tandemly displayed phage. Thus, the parallel display of a ligand molecule and fluorescent protein on the head surface of bacteriophage T7 could provide a new scheme of producing fluorescent nano-bioprobes for diagnostic applications.

Thermoresponsive magnetic nano-biosensors for rapid measurements of inorganic arsenic and cadmium.

Green fluorescent protein-tagged sensor proteins, ArsR-GFP and CadC-GFP, have been produced as biosensors for simple and low-cost quantification of As(III) or Cd(II). In this study, the sensor protein-promoter DNA complexes were reconstructed on the surfaces of magnetic particles of different sizes. After the surface modification all the particles could be attracted by magnets, and released different amounts of GFP-tagged protein, according to the metal concentrations within 5 min, which caused significant increases in fluorescence. A detection limit of 1 µg/L for As(III) and Cd(II) in purified water was obtained only with the nanoparticles exhibiting enough magnetization after heat treatment for 1 min. Therefore, thermoresponsive magnetic nano-biosensors offer great advantages of rapidity and sensitivity for the measurement of the toxic metals in drinking water.

Fluorescent bioassays for toxic metals in milk and yoghurt.

BACKGROUND: From a human health viewpoint, contaminated milk and its products could be a source of long-term exposure to toxic metals. Simple, inexpensive, and on-site assays would enable constant monitoring of their contents. Bioassays that can measure toxic metals in milk or yoghurt might reduce the risk. For this purpose, the green fluorescent protein (GFP)-tagged trans factors, ArsR-GFP and CadC-GFP, together with their cis elements were used to develop such bioassays. RESULTS: ArsR-GFP or CadC-GFP, which binds either toxic metal or DNA fragment including cis element, was directly mixed with cow's milk or yoghurt within a neutral pH range. The fluorescence of GFP, which is reflected by the association/dissociation ratio between cis element and trans factor, significantly changed with increasing externally added As (III) or Cd (II) whereas smaller responses to externally added Pb (II) and Zn (II) were found. Preparation and dilution of whey fraction at low pH were essential to intrinsic zinc quantification using CadC-GFP. Using the extraction procedure and bioassay, intrinsic Zn (II) concentrations ranging from 1.4 to 4.8 mg/l for milk brands and from 1.2 to 2.9 mg/kg for yoghurt brands were determined, which correlated to those determined using inductively coupled plasma atomic emission spectroscopy. CONCLUSIONS: GFP-tagged bacterial trans factors and cis elements can work in the neutralized whole composition and diluted whey fraction of milk and yoghurt. The feature of regulatory elements is advantageous for establishment of simple and rapid assays of toxic metals in dairy products.

Genetic modification in Bacillus subtilis for production of C30 carotenoids.

C30 carotenoids, which have shorter backbones than C40 carotenoids, are known to be produced in the pathogenic bacterium Staphylococcus aureus that causes opportunistic infection. The first committed enzyme in the C30 carotenoid synthetic pathway is dehydrosqualene synthase CrtM. CrtM converts farnesyl pyrophosphate to dehydrosqualene. Dehydrosqualene desaturase CrtN then converts dehydrosqualene to the yellow C30 carotenoid, 4,4'-diaponeurosporene. This chapter describes a method to synthesize C30 carotenoids in Bacillus subtilis, which is generally recognized as a safe (GRAS) organism. Introduction of S. aureus crtM and crtN genes into B. subtilis results in yellow pigmentation. The B. subtilis transformant accumulates two C30 carotenoids, 4,4'-diapolycopene and 4,4'-diaponeurosporene. Furthermore, together with crtMN, introduction of S. aureus crtP and crtQ genes, which encode mixed function oxidase and glycosyltransferase, respectively, donates the ability to produce glycosylated C30 carotenoic acid. Thus, carotenoid biosynthesis genes of S. aureus is applicable to genetically modify B. subtilis in order to construct a safe organism producing C30 carotenoids.

Solid phase biosensors for arsenic or cadmium composed of A trans factor and cis element complex.

The presence of toxic metals in drinking water has hazardous effects on human health. This study was conducted to develop GFP-based-metal-binding biosensors for on-site assay of toxic metal ions. GFP-tagged ArsR and CadC proteins bound to a cis element, and lost the capability of binding to it in their As- and Cd-binding conformational states, respectively. Water samples containing toxic metals were incubated on a complex of GFP-tagged ArsR or CadC and cis element which was immobilized on a solid surface. Metal concentrations were quantified with fluorescence intensity of the metal-binding states released from the cis element. Fluorescence intensity obtained with the assay significantly increased with increasing concentrations of toxic metals. Detection limits of 1 µg/L for Cd(II) and 5 µg/L for As(III) in purified water and 10 µg/L for Cd(II) and As(III) in tap water and bottled mineral water were achieved by measurement with a battery-powered portable fluorometer after 15-min and 30-min incubation, respectively. A complex of freeze dried GFP-tagged ArsR or CadC binding to cis element was stable at 4 °C and responded to 5 µg/L As(III) or Cd(II). The solid phase biosensors are sensitive, less time-consuming, portable, and could offer a protocol for on-site evaluation of the toxic metals in drinking water.

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array.

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.

Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions.

Environmental toxic metals cause serious global public health problems. On-site monitoring protects people from exposure to such harmful elements. In this study, the bacterial transcriptional switches were applied to monitoring of toxic metals. ArsR and CadC, trans factors of Escherichia coli and Staphylococcus aureus, were fused to GFP. The fusion proteins, ArsR-GFP and CadC-GFP, associated with cis elements, P(ars)-O(ars) and P(cad)-O(cad), respectively and dissociated from those upon recognition of As(III) or Pb/Cd. Cell lysates containing ArsR-GFP were pre-incubated with As(III) standard solutions for 15 min and loaded into P(ars)-O(ars)-immobilized microplate wells. Cell lysates containing CadC-GFP were pre-incubated with Pb or Cd solutions and loaded into P(cad)-O(cad)-immobilized wells. The cell lysates were incubated for 15 min and removed from the wells. Fluorescence intensity in the wells dose-dependently decreased in response to As(III) up to 200 µg/l or Pb/Cd up to 100 µg/l. Detection limits were 10 µg/l for As(III) 10 µg/l for Cd, and 20 µg/l for Pb with a microplate fluororeader, whereas 5.0 µg/l for As(III), 1.0 µg/l for Cd, and 10 µg/l for Pb with a handheld fluorometer. This method was available to detect Pb/Cd or As(III) in water containing soil extracts. This is the first demonstration of a simple and rapid fluorometry to detect analytes based on in vitro interaction between a cis element and a trans factor.