Production Of Acid Phosphatase Research Articles

Eggshell waste bioprocessing for sustainable acid phosphatase production and minimizing environmental hazards

BackgroundThe Environmental Protection Agency has listed eggshell waste as the 15th most significant food industry pollution hazard. Using eggshell waste as a renewable energy source has been a hot topic recently. Therefore, finding a sustainable solution for the recycling and valorization of eggshell waste by investigating its potential to produce acid phosphatase (ACP) and organic acids by the newly-discovered B. sonorensis was the target of the current investigation.ResultsDrawing on both molecular and morphological characterizations, the most potent ACP-producing B. sonorensis strain ACP2, was identified as a local bacterial strain obtained from the effluent of the paper and pulp industries. The use of consecutive statistical experimental approaches of Plackett–Burman Design (PBD) and Orthogonal Central Composite Design (OCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor, revealed an innovative medium formulation that substantially improved ACP production, reaching 216 U L−1 with an ACP yield coefficient Yp/x of 18.2 and a specific growth rate (µ) of 0.1 h−1. The metals Ag+, Sn+, and Cr+ were the most efficiently released from eggshells during the solubilization process by B. sonorensis. The uncontrolled pH culture condition is the most suitable and favoured setting for improving ACP and organic acids production. Quantitative and qualitative analyses of the produced organic acids were carried out using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Lactic acid, citric acid, and hydroxybenzoic acid isomer were the most common organic acids produced throughout the cultivation process. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of eggshell particles.ConclusionsThis study emphasized robust microbial engineering approaches for the large-scale production of a newly discovered acid phosphatase, accompanied by organic acids production from B. sonorensis. The biovalorization of the eggshell waste and the production of cost-effective ACP and organic acids were integrated into the current study, and this was done through the implementation of a unique and innovative medium formulation design for eggshell waste management, as well as scaling up ACP production on a bench-top scale.

Dynamics of acid phosphatase production by cell suspension system and its further characterization

The current study compares the influence of media engineered media on production of acid phophatase (APase) with its characterization in suspension cell cultures of Atropa acuminata, authenticated by rbcL and ITS. Various parameters like pH, inoculum size, temperature, nutrient sources in the medium were optimized for APase hyperproduction in in vitro conditions. APase production was accomplished to be evidently amplified at 5% inoculum size (94.90 U/gFW), pH 5.0(99.75 U/gFW) and incubation temperature of 30°C (91.5U/gFW). Sucrose as carbon and ammonium nitrate as nitrogen sources were eminently suited for maximum enzyme harvest (91.20 U/gFW and 94.56 U/gFW). The APase activity was observed to be distinctly increased in phosphate (Pi) starved suspension cells (197.84 U/gFW). Overall, 6.5 fold increase in APase activity was achieved as compared to the activity before optimization studies. Hence the present Apase production and its activity from Atropa acuminata suspension culture may have apparent use for future industrial, agricultural and biotechnological application. Keywords: Acuminata; Acid phosphatase; p-nitrophenylphosphate; Suspension cells; Pi-starvation

Long-term soil warming decreases microbial phosphorus utilization by increasing abiotic phosphorus sorption and phosphorus losses

Phosphorus (P) is an essential and often limiting element that could play a crucial role in terrestrial ecosystem responses to climate warming. However, it has yet remained unclear how different P cycling processes are affected by warming. Here we investigate the response of soil P pools and P cycling processes in a mountain forest after 14 years of soil warming (+4 °C). Long-term warming decreased soil total P pools, likely due to higher outputs of P from soils by increasing net plant P uptake and downward transportation of colloidal and particulate P. Warming increased the sorption strength to more recalcitrant soil P fractions (absorbed to iron oxyhydroxides and clays), thereby further reducing bioavailable P in soil solution. As a response, soil microbes enhanced the production of acid phosphatase, though this was not sufficient to avoid decreases of soil bioavailable P and microbial biomass P (and biotic phosphate immobilization). This study therefore highlights how long-term soil warming triggers changes in biotic and abiotic soil P pools and processes, which can potentially aggravate the P constraints of the trees and soil microbes and thereby negatively affect the C sequestration potential of these forests.

A sustainable and effective bioprocessing approach for improvement of acid phosphatase production and rock phosphate solubilization by Bacillus haynesii strain ACP1

There is indeed a tremendous increase in biotechnological production on a global scale, more and more innovative bioprocesses, therefore, require to perform ideally not only in a small lab- but also on large production scales. Efficient microbial process optimization is a significant challenge when accomplishing a variety of sustainable development and bioengineering application objectives. In Egypt's mines, several distinct types of rock phosphate (RP) are utilized as a source of phosphate fertilizers in agriculture. It is more ecologically beneficial to utilize RP bio-solubilization than acidulation. Therefore, this work aimed to strategically scale up the acid phosphatase (ACP) production and RP bio-solubilization by the newly-discovered Bacillus haynesii. The use of consecutive statistical experimental approaches of Plackett–Burman Design (PBD), and Rotatable Central Composite Design (RCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor revealed an innovative medium formulation. These approaches substantially improved ACP production, reaching 207.6 U L−1 with an ACP yield coefficient Yp/x of 25.2 and a specific growth rate (µ) of 0.07 h−1. The metals Na, Li, and Mn were the most efficiently released from RP during the solubilization process by B. haynesii. The uncontrolled pH culture condition is the most suitable setting for simultaneously improving the ACP and organic acids production. The most abundant organic acid produced through the cultivation process was lactic acid, followed by glutamic acid and hydroxybenzoic acid isomer. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of RP particles.

EXPLORING POTENTIAL OF METAL RESISTANT AND PGP ATTRIBUTES OF PHOSPHATE SOLUBILIZING RHIZOBACTERIA ISOLATED FROM SOUTHERN PUNJAB, PAKISTAN

Phosphorus is highly required nutrient for proper plant functioning and growth but due to its highly reactive nature it changed its form after short period of time when we applied as fertilizer and become unavailable for plant uptake while Phosphate solubilizing rhizobacteria are powerful candidates that solubilize the P that is already present in soil. So, the present research based on the hypothesis to isolating, screening and optimizing the phosphate solubilizing rhizobacteria with potential of metalresistant and multiple PGPR attributes. Total 55 PSR strains were isolated out of which 20 strains (WUSB1,WUSB2, WUSB3, WUSB4, WUSB5, WUSB6, WUSB7, WUSB8, WUSB9, WUSB10, WUSB11, WUSB12, WUSB13, WUSB14, WUSB15, WUSB16, WUSB17, WUSB18, WUSB19, WUSB20) were selected with 4-8 (PSI) and 30-122?g/ml phosphate solubilization in NBRIP medium for one weekincubation at 370C. These strains were further analyzed for their physiochemical attributes, metal resistant, extracellular enzyme production and PGP attributes. Final results showed that one candidate designated the name as WUSB16 produce significant results i.e. having capacity to solubilize P 5.46 (PSI) on agar plate assay and 105?g/ml in broth assay, Acid and alkaline phosphatase production 20?mol PNPb ml-1 hr-1 or 15?mol PNP ml-1 hr-1 respectively. It showed metal resistant against ZnSO4, MnSO4 and CuSO4. Evaluation of PGP attributes indicated that the capacity of WUSB16 strain for auxin production was 100?g/ml, for siderophore and nitrogen fixation was stronger (+++), for Hydrogen Cyanide and ammonia production was medium (++) and for ACC deaminase was negative (-). From above results we can conclude that WUSB16 PSR strain with multifaceted PGP traits is the best candidate that have potential to utilized as Phosphate solubilizing bioinoculant.

Acid Phosphatase Immobilization and Production Study by Trichoderma spp. in Soybean Molasses

AbstractAcid phosphatase production was carried out by Trichoderma spp. using treated soybean molasses at different concentrations. Results showed higher acid phosphatase activity when the soybean molasses concentration increased for the microorganisms Trichoderma harzianum and Trichoderma reesei. The acid phosphatase activity performance of Trichoderma asperellum was also evaluated. In general, the polyethyleneimine glyoxyl agarose support showed much better performance and final recovery of activity than DEAE Toyopearl 650s.

Effects of Three Widely Used Antibiotics and Their Mixture on the Haemocytes of the Clam Ruditapes philippinarum

Although the presence of pharmaceutical and personal care products in aquatic ecosystems is well documented, little information is available about their sublethal effects, on aquatic invertebrates. From an ecotoxicological point of view, the use of in vitro approaches has been recommended as a tool to assess adverse effects and to understand the mechanisms of action of chemicals at the cellular level. In the present in vitro study, the effects of Amoxicillin (AMX), Trimethoprim (TMP) and Ciprofloxacin (CIP) (1 µg/L, each) were tested alone and—for the first time—as a mixture (MIX) on haemocytes of the clam Ruditapes philippinarum. After the exposure, a battery of cellular parameters was evaluated, such as haemocyte viability, lysosomal membrane stability, superoxide anion production, acid phosphatase activity, the frequency of micronuclei and chromosomal aberrations. The results demonstrated that AMX, TMP, CIP and MIX affected lysosomal membrane stability, as well as superoxide anion and acid phosphatase production, and promoted chromosomal aberrations. This study highlighted that Manila clam haemocytes are a sensitive cell model to assess the effects of exposure to pharmaceutical products on non-target species. Our study demonstrated that the effects of pharmaceutical mixtures on marine species should be experimentally evaluated because they are not predictable from single exposures as the compounds can interact in different ways on the various biological endpoints considered.

Nutrient Alteration Drives the Impacts of Seawater Acidification on the Bloom-Forming Dinoflagellate Karenia mikimotoi.

Seawater acidification and nutrient alteration are two dominant environmental factors in coastal environments that influence the dynamics and succession of marine microalgae. However, the impacts of their combination have seldom been recorded. A simulated experimental system was set up to mimic the effects of elevated acidification on a bloom-forming dinoflagellate, Karenia mikimotoi, exposed to different nutrient conditions, and the possible mechanism was discussed. The results showed that acidification at different pH levels of 7.6 or 7.4 significantly influenced microalgal growth (p<0.05) compared with the control at pH 8.0. Mitochondria, the key sites of aerobic respiration and energy production, were impaired in a pH-dependent manner, and a simultaneous alteration of reactive oxygen species (ROS) production occurred. Cytochrome c oxidase (COX) and citrate synthase (CS), two mitochondrial metabolism-related enzymes, were actively induced with acidification exposure, suggesting the involvement of the mitochondrial pathway in coping with acidification. Moreover, different nutrient statuses indicated by various N:P ratios of 7:1 (N limitation) and 52:1 (P limitation) dramatically altered the impacts of acidification compared with those exposed to an N:P ratio of 17:1 (control), microalgal growth at pH 7.4 was obviously accelerated with the elevation of the nutrient ratio compared to that at pH 8.1 (p<0.05), and nutrient limitations seemed beneficial for growth in acidifying conditions. The production of alkaline phosphatase (AP) and acid phosphatase (AcP), an effective index indicating the microalgal growth status, significantly increased at the same time (p<0.05), which further supported this speculation. However, nitrate reductase (NR) was slightly inhibited. Hemolytic toxin production showed an obvious increase as the N:P ratio increased when exposed to acidification. Taken together, mitochondrial metabolism was suspected to be involved in the process of coping with acidification, and nutrient alterations, especially P limitation, could effectively alleviate the negative impacts induced by acidification. The obtained results might be a possible explanation for the competitive fitness of K. mikimotoi during bloom development.

Fine roots stimulate nutrient release during early stages of leaf litter decomposition in a Central Amazon rainforest

PurposeLarge parts of the Amazon rainforest grow on weathered soils depleted in phosphorus and rock-derived cations. We tested the hypothesis that in this ecosystem, fine roots stimulate decomposition and nutrient release from leaf litter biochemically by releasing enzymes, and by exuding labile carbon stimulating microbial decomposers.MethodsWe monitored leaf litter decomposition in a Central Amazon tropical rainforest, where fine roots were either present or excluded, over 188 days and added labile carbon substrates (glucose and citric acid) in a fully factorial design. We tracked litter mass loss, remaining carbon, nitrogen, phosphorus and cation concentrations, extracellular enzyme activity and microbial carbon and nutrient concentrations.ResultsFine root presence did not affect litter mass loss but significantly increased the loss of phosphorus and cations from leaf litter. In the presence of fine roots, acid phosphatase activity was 43.2% higher, while neither microbial stoichiometry, nor extracellular enzyme activities targeting carbon- and nitrogen-containing compounds changed. Glucose additions increased phosphorus loss from litter when fine roots were present, and enhanced phosphatase activity in root exclusions. Citric acid additions reduced litter mass loss, microbial biomass nitrogen and phosphorus, regardless of fine root presence or exclusion.ConclusionsWe conclude that plant roots release significant amounts of acid phosphatases into the litter layer and mobilize phosphorus without affecting litter mass loss. Our results further indicate that added labile carbon inputs (i.e. glucose) can stimulate acid phosphatase production by microbial decomposers, highlighting the potential importance of plant-microbial feedbacks in tropical forest ecosystems.

Phosphorus Deficiency Promoted Hydrolysis of Organophosphate Esters in Plants: Mechanisms and Transformation Pathways.

The biotransformation of organophosphate esters (OPEs) in white lupin (Lupinus albus) and wheat (Triticum aestivum L.) was investigated in hydroponic experiments with different phosphorus (P)-containing conditions. The hydrolysis rates of OPEs followed the order of triphenyl phosphate (TPHP) > tri-n-butyl phosphate (TnBP) > tris(1,3-dichloro-2-propyl) phosphate (TDCPP). Hydrolysis of OPEs was accelerated at P-deficient conditions, and faster hydrolysis took place in white lupin than in wheat. Coincidingly, the production of acid phosphatase (ACP) in both plants was promoted, and much higher intracellular and extracellular ACPs were observed in white lupin under P-deficient conditions. In vitro experiments revealed that ACP was a key enzyme to hydrolyze OPEs. The hydrolysis rates of OPEs were significantly correlated with the Hirshfeld charges, calculated by density functional theory, of the oxygen atom in the single P-O bond. Using ultra-high-performance liquid chromatography coupled with Orbitrap Fusion mass spectrometer, 30 metabolites were successfully identified. Some of these metabolites, such as sulfate-conjugated products, hydration of cysteine-conjugated products of TPHP, and reductively dechlorinated metabolites of TDCPP, were observed for the first time in plants. It is noteworthy that OPEs may transform into many hydroxylated metabolites, and special attention should be paid to their potential environmental effects.

Low Intensity Vibration Treatment Reduces Myeloma Cell-Induced Osteoclast Formation By Inhibiting IL-6 and GFI-1 Production By MM Cells

Multiple myeloma (MM) bone disease is characterized by increased osteoclast-mediated resorption of bone adjacent to tumor growth. Fusion and activation of osteoclasts (OC), indicated by elevated tartrate-resistant acid phosphatase (TRAP) production, are induced by a myriad of inflammatory factors; e.g. TGF-β, growth factor independent 1 transcription factor (GFI-1), RANKL, and IL-6 that are produced by myeloma cells and cells in the marrow microenvironment. Current therapies reduce tumor burden and block osteoclast formation and activity, thereby decreasing bone destruction. However, MM bones rarely heal. While the anabolic effects of exercise on healthy bone are well-documented, patients with compromised skeletal tissue, such as in MM, may not be able to participate in regimented exercise for fear of developing a fracture. Low intensity vibrations (LIV) that deliver subtle mechanical signals, on the order of those induced by fast-twitch muscle fibers on bone, can enhance mesenchymal stem cell differentiation towards osteoblasts. LIV also significantly preserves trabecular bone while reducing tumor burden in femora of mice harboring U266 MM cells. We hypothesize that LIV is both anti-resorptive and anabolic, through differential effects on MM cells and bone cells. To test this hypothesis, we used a vertically oscillating, vibratory platform that delivered mechanical signals (<1 x Earth's gravitational acceleration; where 1g=9.8m/s2) to culture plates.5TGM1 murine MM cells (5x105 cells/well) were cultured in RPMI with 10% FCS and subjected to LIV or Sham-LIV. Each 6-well plate received either LIV (frequency=90Hertz, acceleration=0.3g) or Sham-LIV (SH; unpowered, static platform) twice per day for 20min per treatment for 48hrs. MM cell lysates were then collected using RIPA buffer. IL-6, GFI-1, and RANKL levels were determined via Western Blot using specific monoclonal antibodies. GAPDH was used as the loading control. Conditioned media (CM) were isolated from SH- and LIV-treated MM cultures. Purified primary murine OC precursors (non-adherent marrow mononuclear cells) were expanded with 10ng/mL of MCSF for 3 days and then these cells were isolated by trypsinization and replated in 12-well plates at a density of 5x105 cells/well in α-MEM, 10% FCS, and 30% v/v CM. All cultures were then incubated for 36hrs. OC number and morphology were confirmed via TRAP staining (counterstained with hematoxylin) of the cultures. TRAP+ OC's containing at least three nuclei were quantified in the cultures by microscopy.Expression levels of IL-6 and GFI-1 were 53% (p<0.01) and 50% (p<0.02) lower, respectively, in lysates from LIV-exposed 5TGM1 cells as compared to those that were SH-treated. Expression of RANKL was not significantly different between these groups. TRAP+ OC's were 61% (p<0.005) lower in LIV-treated cultures compared with SH cultures. The morphology of OC's exposed to LIV-conditioned media from MM cultures was distinct from control cultures, with fewer filopodia than OC's in cultures treated with SH-conditioned media. Based on these data we then examined the direct effect of LIV-treatment on committed OC-precursors treated with RANKL (50ng/mL) to induce formation of mature OC's. After 48hrs of LIV-treatment, committed OC-precursors had 36% (p<0.0001) fewer nuclei per OC than in SH-treated cultures. The cultures of committed OC-precursors directly treated with LIV had fewer filopodia in comparison to SH-treated cultures upon examination of OC morphology.These findings suggest that mechanical signals in the form of LIV may influence the secretion of IL-6 and GFI-1 by myeloma cells, which, in turn, can decrease the capacity of MM cells to induce OC formation. The morphology of OC's and expression of TRAP by OC's exposed to LIV further suggest a distinct mechanosensitive effect on osteoclast formation and fusion that may impact their bone resorbing capacity. Thus, LIV has a significant effect on the capacity of MM cells to secrete inflammatory cytokines as well as directly affect OC formation. These results further suggest that low intensity vibrations may be a useful, non-invasive approach for treating myeloma bone disease. DisclosuresRoodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Biodiversity, current developments and potential biotechnological applications of phosphorus-solubilizing and -mobilizing microbes: A review

As one of the most important and essential macronutrients next to nitrogen, phosphorus (P) is important for plant development, but it is the least mobile nutrient element in plant and soil. Globally, P is mined from geological sediments and added to agricultural soils so as to meet the critical requirements of crop plants for agronomic productivity. Phosphorus exists in soil in both organic and inorganic forms. The various inorganic forms of the element in soil are salts with calcium, iron, and aluminum, whereas the organic forms come from decaying vegetation and microbial residue. There is a huge diversity of plant microbiomes (epiphytic, endophytic, and rhizospheric) and soil microbiomes that have the capability to solubilize the insoluble P and make it available to plant. The main mechanism for the solubilization of inorganic P is by the production of organic acids, which lowers soil pH, or by the production of acid and alkaline phosphatases, which causes the mineralization of organic P. The P-solubilizing and -mobilizing microorganisms belong to all three domains, comprising archaea, bacteria, and eukarya. The strains belonging to the genera Arthrobacter, Bacillus, Burkholderia, Natrinema, Pseudomonas, Rhizobium, and Serratia have been reported as efficient and potential P solubilizers. The use of P solubilizers, alone or in combination with other plant growth-promoting microbes as an eco-friendly microbial consortium, could increase the P uptake of crops, increasing their yields for agricultural and environmental sustainability.

Efficacy of phosphate solubilizing Actinobacteria to improve rock phosphate agronomic effectiveness and plant growth promotion

In the rhizosphere, plant growth is facilitated by plant-growth-promoting rhizobacteria (PGPR). PGPRs like Actinobacteria can increase level of phosphate available to plants through P solubilization or mineralization. In this study we evaluated the ability of nine Phosphate Solubilizing Actinobacteria (PSA) to release P from rock phosphate (RP) and to promote maize growth and yield under in vitro and greenhouse conditions. Plate bioassay and pots culture experiment were conducted to investigate the effects of these PSA on maize plants development. High reactive rock phosphate (BG4, from Gantour phosphate mine, Morocco) and triple superphosphate (TSP) from Morocco were used as positive controls. BG4, is a reactive RP directly used to increase the P status of soil according to the Office Chérifien des Phosphates (OCP). For the other treatments, plants were grown in soil amended with low reactive RP obtained from the Khouribga phosphate mine (Morocco) and inoculated with selected PSA strains. Our findings have indicated that, PSA inoculation resulted in increasing of the assessed growth parameters and nutrient content in maize seedlings. Four of the tested strains (BC3, BC10, BC11 and P18) showed high performance efficiency in RP-solubilization and improved plant P nutrition and growth under low-P conditions i.e. amended with low reactive RP. Furthermore, inoculation with these latter strains also showed an important production of acid and alkaline phosphatase in the rhizosphere of the maize. The relative agronomic effectiveness (RAE) index showed that BC3(Streptomyces griseorubens) and BC11 (Norcardiopsis alba) increased the yield by 135.33 and 126.91% over the positive controls BG4. These strains can be considered as the most efficient to use rock phosphate as a crude phosphate fertilizer. Overall, our results demonstrate that PSA could constitute a promising solution to valorize low reactive RP in agriculture. The 16 S rRNA gene sequencing and phylogenetic analysis identified these isolates belong to Streptomyces and Nocardiopsis genera.

Mobilization of recalcitrant phosphorous and enhancement of pepper P uptake and yield by a new biocontrol and bioremediation bacterium Burkholderia cepacia CQ18.

Phosphorus (P) is a finite resource and inoculation of phosphorus-mobilizing bacteria (PMB) is a promising approach for the enhancement of soil P availability and plant P uptake. This drives scientists to search for the microbes effective in mobilizing legacy P in soils. The current incubation and greenhouse pot experiments were conducted to investigate P mobilization and pepper P uptake as affected by a new biocontrol and bioremediation bacterium Burkholderia cepacia CQ18. This bacterium converted Ca3 (PO4 )2 , FePO4 , AlPO4 , and lecithin into soluble inorganic P in the culture solutions and increased available P (including water-soluble P and Olsen P) in the soil. There were positive correlations between the soluble inorganic phosphorus and the exudates (protons, organic acids (oxalate and gluconate), siderophores and phosphatases) in culture solutions. Pepper plant biomass, fruit yield and P uptake changed in the sequence: chemical fertilizers plus bacterial inoculant >only chemical fertilizers >only bacterial inoculant >blank control. Taking into account the wide spectrums of P mobilization and simultaneous production of acid, neutral and alkaline phosphatases at a given pH, B.cepacia CQ18 may be a potential PMB used in soils with wide pH ranges. The mechanisms employed by this bacterium in the solubilization of recalcitrant inorganic P could be the efflux of protons, organic acids (oxalate and gluconate) and siderophores. Phosphatases could be of utmost importance in the mineralization of the organic P. The production of siderophores and phosphatases by of B.cepacia CQ18 could thus be crucial for not only the antagonism against plant pathogens but also the mobilization of soil sparingly available P. Burkholderia cepacia CQ18 could be potentially developed into a biofertilizer.

Dietary nucleotides in the diets of on-growing grass carp (Ctenopharyngodon idella) suppress Aeromonas hydrophila induced intestinal inflammation and enhance intestinal disease-resistance via NF-κB and TOR signaling

The beneficial influences of nucleotides on fish intestinal immune responses and the mechanisms underlying the regulation of on-growing grass carp (Ctenopharyngodon idella) were evaluated using A. hydrophila induced intestinal inflammation model in vivo. Sixty days growth trial was carried out and fish were fed with mixed nucleotides diets: 0, 200.0, 400.0, 600.0, 800.0 and 1000.0 mg/kg diet. At the end of growth trial, grass carp were faced with infection trials with the challenge of A. hydrophila over a period of 14 days. Studies demonstrated that nucleotides supplementation had a powerful effect on inhibition of enteritis morbidity and inflammation caused by A. hydrophila in fish intestine. Dietary nucleotides elevated the production of intestinal acid phosphatase, lysozyme (LZ) activities, complements, immunoglobulin M and increased intestinal antimicrobial peptides (hepcidin, β-defensin-1, liver expressed antimicrobial peptide 2A and 2B) and mucin2 [only in distal intestine (DI)] gene expression. Nucleotides inhibited intestinal pro-inflammatory cytokines mRNA levels [interferon γ2 (IFN-γ2), tumor necrosis factor α (TNF-α), IL-6, IL-15, IL-17D, IL-12p35 and IL-12p40], which were correlated with nuclear factor kappa B (NF-κB) signaling [IκB kinase (IKK-β and IKK-γ)/inhibitor of (κBα) (IκBα)/NF-kB (p65 and c-Rel)]. Nucleotides increased intestinal anti-inflammatory cytokines [IL-11, transforming growth factor β1 (TGF-β1), IL-4/13A and IL-4/13B] gene expression partly associated with target of rapamycin (TOR) signaling [TOR/ribosomal protein S6 kinases 1 (S6K1) and eIF4E-binding proteins (4E-BP1)]. In summary, dietary nucleotides could enhance intestinal immune responses of fish via the inhibition of NF-κB and the promotion of TOR signaling. Furthermore, based on the parameters of enteritis morbidity and LZ activity in proximal intestine (PI), middle intestine (MI) and DI, optimal nucleotides supplementation levels for on-growing grass carp were estimated to be 580.35, 622.53, 613.21 and 617.98 mg/kg diet, respectively.

Salt stress and hydroxyectoine enhance phosphate solubilisation and plant colonisation capacity of Kosakonia radicincitans

Gram-negative bacterial endophytes have attracted research interest caused by their advantageous over epiphytic bacteria in plant nutrition and protection. However, research on these typically Gram-negative endophytes has deficiencies concerning the role of cultivation and pre-formulation strategies on further plant colonisation capabilities. Besides, the influence of cultivation conditions and osmotic stress within bacterial endophytes on their phosphate solubilising ability has not yet been addressed. By pre-conditioning cells with an osmoadaptation and a hydroxyectoine accumulation approach, this research aimed at enhancing the capability of the plant growth promoting bacterium Kosakonia radicincitans strain DSM 16656T to both solubilise phosphate and colonise plant seedlings. The results showed that halotolerant bacterial phenotypes increased the root-colonising capability by approximately 3-fold and presented growth-promoting effects in radish plants. Interestingly, findings also demonstrated that salt stress in the culture media along with the accumulation of hydroxyectoine led to an increase in the in vitro phosphate-solubilising ability by affecting the production of acid phosphatases, from 1.24 to 3.34 U mg-1 for non-salt stressed cells and hydroxyectoine-added cells respectively. Thus, this approach provides a useful knowledge upon which the salt stress and compatible solutes in bacteria endophytes can confer phenotypic adaptations to support the eco-physiological performance concerning phosphate-solubilising abilities and endosphere establishment.

Effects of nitrogen and phosphorus fertilization on the ratio of activities of carbon-acquiring to nitrogen-acquiring enzymes in a primary lowland tropical rainforest in Borneo, Malaysia

ABSTRACTPrevious meta-analyses revealed that the ratio of activities of carbon (C)-acquiring enzyme to nitrogen (N)-acquiring enzymes in tropical forest ecosystems was nearly identical to those in other ecosystems, despite of the N-rich condition in tropical forests. This could be explained by microbes in tropical forest soils, which require a large amount of N to produce N-rich acid phosphatase (AP) for catalyzation of the organic form of phosphorus (P) and compensation for poor P availability in soils. Based on this idea, we hypothesized that experimental P fertilization would reduce the allocation to N-acquiring enzymes compared with that of C-acquiring enzymes, i.e. that it would increase the ratios of activities of β-1,4-glucosidase (BG) to β-1,4-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP). We tested this hypothesis using an experimental fertilization site with factorial N (100 kg ha−1 yr−1) and P (50 kg ha−1 yr−1) addition in a primary tropical lowland forest in Bornean Malaysia, where our earlier work demonstrated that P fertilization reduced AP activity. Contrary to our hypothesis, the BG:NAG and BG:(NAG + LAP) ratios were not altered by either N or P fertilizations. This result indicated that AP production was not a reason for the maintenance of a relatively high investment in N-acquiring enzyme at our study site. Rather, NAG and LAP production was likely driven by C acquisition, rather than N acquisition, as the target substrates contained C as well as N. This idea was supported by the fact that neither the BG:NAG ratio nor the BG:(NAG + LAP) ratio was elevated by N addition. We propose that the ratios of activities of BG to NAG and LAP do not necessarily indicate the ratio of C:N acquisition, at least in our N-rich tropical forest ecosystem.

Growth promotion and root colonisation in pepper plants by phosphate‐solubilising Chryseobacterium sp. strain ISE14 that suppresses Phytophthora blight

AbstractPreviously, we selected bacterial strain ISE14 through a sequential selection procedure that included radicle, seedling, and in planta assays and field tests. This strain not only suppressed a destructive soilborne disease, Phytophthora blight, caused by Phytophthora capsici but also increased fruit yields of pepper plants in the fields. This study was conducted to identify strain ISE14 by 16S rRNA gene sequence analysis and to characterise biocontrol and plant growth promotion activities of the strain in pepper plants. Strain ISE14, identified as Chryseobacterium sp., significantly reduced disease severity in plants inoculated with Ph. capsici and promoted plant growth (lengths and dry weights of shoots and roots) compared with those in plants treated with Escherichia coli DH5α (negative control) or MgSO4 solution (untreated control). This strain effectively colonised pepper plant roots as assessed by bacterial population analysis and confocal laser scanning microscopy; it enhanced soil microbial activity and biofilm formation, but not the production of indole acetic acid. Strain ISE14 also solubilised organic or inorganic phosphate by production of acid and alkaline phosphatases or reduction in pH, resulting in enhanced pepper plant growth. This strain exhibited similar or greater activity in disease control and plant growth promotion tests compared with positive control strains Paenibacillus polymyxa AC‐1 (biocontrol) and Bacillus vallismortis EXTN‐1 (plant growth). Therefore, Chryseobacterium sp. ISE14 may be a phosphate‐solubilising and plant growth‐promoting rhizobacterium (PGPR) strain that suppresses Phytophthora blight of pepper. To our knowledge, this is the first report of a phosphate‐solubilising PGPR strain of Chryseobacterium sp. that suppresses the pepper disease.

Validating novel oligonucleotide primers targeting three classes of bacterial non-specific acid phosphatase genes in grassland soils

Microbially driven mineralization of organic phosphorus forms is of particular importance in the soil environment, where it becomes available to plants as inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs; E.C. 3.1.3.2) which act on the most common forms of organic P in the soil. Our understanding of phosphorus turnover in soils would greatly benefit from an improvement in research tools targeting these genes. Thus, in this study we developed two novel oligonucleotide PCR primer sets, that will enable researchers to target the present and active communities of bacteria with the genetic potential of acid phosphatase production. A total of three primer sets were validated to target the three classes of NSAPs. Utilizing Illumina MiSeq, amplicons from grassland pasture soils were sequenced. The resulting target specificity was high for all three groups; CAAP (97.2%), CBAP (99.5%), and CCAP (94.8%). Quantification of target genes by qPCR indicated measurable differences between classes, ranging from 5 log to 7.5 log for CAAP, 6 log to 8 log for CBAP, and 4 log to 5 log for CCAP. The validated primer sets were specific to the target genes and identified potential quantitative differences between the NSAP classes.

Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.

Phosphorus is an essential element for all life forms. Phosphate solubilizing bacteria are capable of converting phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-37, was isolated from mangrove soil of the Mahanadi river delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Serratia sp. The maximum phosphate solubilizing activity of the strain was determined to be 44.84 μg/ml, accompanied by a decrease in pH of the growth medium from 7.0 to 3.15. During phosphate solubilization, various organic acids, such as malic acid (237 mg/l), lactic acid (599.5 mg/l) and acetic acid (5.0 mg/l) were also detected in the broth culture through HPLC analysis. Acid phosphatase activity was determined by performing p-nitrophenyl phosphate assay (pNPP) of the bacterial broth culture. Optimum acid phosphatase activity was observed at 48 h of incubation (76.808 U/ml), temperature of 45 °C (77.87 U/ml), an agitation rate of 100 rpm (80.40 U/ml), pH 5.0 (80.66 U/ml) and with glucose as a original carbon source (80.6 U/ml) and ammonium sulphate as a original nitrogen source (80.92 U/ml). Characterization of the partially purified acid phosphatase showed maximum activity at pH 5.0 (85.6 U/ml), temperature of 45 °C (97.87 U/ml) and substrate concentration of 2.5 mg/ml (92.7 U/ml). Hence the present phosphate solubilizing and acid phosphatase production activity of the bacterium may have probable use for future industrial, agricultural and biotechnological application.