Unexploited potential of some biotechnological techniques for biofertilizer production and formulation.

The massive application of chemical fertilizers to support crop production has resulted in soil, water, and air pollution at a global scale. In the same time, this situation escalated consumers' concerns regarding quality and safety of food production which, due to increase of fertilizer prices, have provoked corresponding price increase of food products. It is widely accepted that the only solution is to boost exploitation of plant-beneficial microorganisms which in conditions of undisturbed soils play a key role in increasing the availability of minerals that otherwise are inaccessible to plants. This review paper is focused on the employment of microbial inoculants and their production and formulation. Special attention is given to biotechniques that are not fully exploited as tools for biofertilizer manufacturing such as microbial co-cultivation and co-immobilization. Another emerging area includes biotechnological production and combined usage of microorganisms/active natural compounds (biostimulants) such as plant extracts and exudates, compost extracts, and products like strigolactones, which improve not only plant growth and development but also plant-microbial interactions. The most important potential and novel strategies in this field are presented as well as the tendencies that will be developed in the near future.

Stress-tolerant P-solubilizing microorganisms.

Drought, high/low temperature, and salinity are abiotic stress factors accepted as the main reason for crop yield losses in a world with growing population and food price increases. Additional problems create nutrient limitations and particularly low P soil status. The problem of phosphate fertilizers, P plant nutrition, and existing phosphate bearing resources can also be related to the scarcity of rock phosphate. The modern agricultural systems are highly dependent on the existing fertilizer industry based exclusively of this natural, finite, non-renewable resource. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using plant beneficial, including P-solubilizing, microorganisms. This short review paper summarizes the current and future trends in isolation, development, and application of P-solubilizing microorganisms in stress environmental conditions bearing also in mind the imbalanced cycling and unsustainable management of P. Special attention is devoted to the efforts on development of biotechnological strategies for formulation of P-solubilizing microorganisms in order to increase their protection against adverse abiotic factors.

The Biomarkers Consortium: practice and pitfalls of open-source precompetitive collaboration.

Precompetitive collaboration is a growing driver for innovation and increased productivity in biomedical science and drug development. The Biomarkers Consortium, a public-private platform for precompetitive collaboration specific to biomarkers, demonstrated that adiponectin has potential utility as a predictor of metabolic responses to peroxisome proliferator-activated receptor (PPAR) agonists in individuals with type 2 diabetes. Despite the challenges overcome by this project, the most important lesson learned is that cross-company precompetitive collaboration is a feasible robust approach to biomarker qualification.

Utility of adiponectin as a biomarker predictive of glycemic efficacy is demonstrated by collaborative pooling of data from clinical trials conducted by multiple sponsors.

This study, conducted under the Metabolic Disorders Steering Committee of the Biomarkers Consortium (a public-private partnership managed by the Foundation for the National Institutes of Health (FNIH)), analyzed blinded data on 2,688 type 2 diabetes (T2D) patients from randomized clinical trials conducted by four pharmaceutical companies. An increase in the levels of adiponectin was observed after peroxisome proliferator-activated receptor (PPAR)-agonist treatment (P < 0.0001), but not after treatment with non-PPAR drugs. This increase correlated with decreases in levels of glucose, hemoglobin A(1c) (Hb(A1c)), hematocrit, and triglycerides, and increases in levels of blood urea nitrogen, creatinine, and high-density lipoprotein cholesterol (HDL-C). Early (6-8 weeks) increases in levels of adiponectin after treatment with PPAR agonists showed a negative correlation (r = -0.21, P < 0.0001) with subsequent changes in levels of Hb(A1c). Changes in adiponectin level did not appear to be associated with baseline level of Hb(A1c). Logistic regression demonstrated that an increase in the level of adiponectin predicts a decrease in the level of Hb(A1c). These analyses confirm previously demonstrated relationships between adiponectin levels and metabolic parameters and support the robust predictive utility of adiponectin across the spectrum of glucose tolerance. Cross-company precompetitive collaboration is a feasible and powerful approach to biomarker qualification.

Experimental study of interaction of laser radiation with silver nanoparticles in SiO2 matrix.

Thin films of silica containing silver nanoparticles were deposited by magnetron co-sputtering followed by thermal annealing in air or Ar+2% H2. Laser fragmentation of the particles was carried out at two different wavelengths. The films were characterized by UV-VIS absorption spectroscopy and plasmon resonance numerical modeling based on the Mie theory, together with Rutherford backscattering elemental analysis, X-ray photoelectron spectroscopy chemical characterization, combined with statistical analysis of the transmission electron microscopy micrographs, and surface topography study by atomic force microscopy. It is demonstrated that the fragmentation is a result of a thermal process and its mechanism does not depend on the laser wavelength as long as the laser light is absorbed by the silver particles. Laser treatment with moderate fluences does not alter the precipitated metal content while fragmenting the particles. TEM study indicates that laser assisted silver particle modification can serve as a method for narrowing the particle size distribution.

Improvement of soil characteristics and growth of Dorycnium pentaphyllum by amendment with agrowastes and inoculation with AM fungi and/or the yeast Yarowia lipolytica.

The effectiveness of two microbiologically treated agrowastes [dry olive cake (DOC) and/or sugar beet (SB)] on plant growth, soil enzymatic activities and other soil characteristics was determined in a natural soil from a desertified area. Dorycnium pentaphyllum, a legume plant adapted to stress situations, was the test plant to evaluate the effect of inoculation of native arbuscular mycorrhizal (AM) fungi and/or Yarowia lipolytica (a dry soil adapted yeast) on amended and non-amended soils. Plant growth and nutrition, symbiotic developments and soil enzymatic activities were limited in non-amended soil where microbial inoculations did not improve plant development. The lack of nodules formation and AM colonization can explain the limited plant growth in this natural soil. The effectiveness and performance of inocula applied was only evident in amended soils. AM colonization and spores number in natural soil were increased by amendments and the inoculation with Y. lipolytica promoted this value. The effect of the inoculations on plant N-acquisition was only important in AM-inoculated plants growing in SB medium. Enzymatic activities as urease and protease activities were particularly increased in DOC amended soil meanwhile dehydrogenase activity was greatest in treatments inoculated with Y. lipolytica in SB added soil. The biological activities in rhizosphere of agrowaste amended soil, used as indices of changes in soil properties and fertility, were affected not only by the nature of amendments but also by the inoculant applied. All these results show that the lignocellulosic agrowastes treated with a selected microorganism and its further interaction with beneficial microbial groups (native AM fungi and/or Y. lipolytica) is a useful tool to modify soil physico-chemical, biological and fertility properties that enhance the plant performance probably by making nutrients more available to plants.

Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes.

Rock phosphate (RP) is an important natural material traditionally used for the production of phosphorus (P) fertilizers. Compared with chemical treatment, microbial solubilization of RP is an alternative environmentally mild approach. An overview of biotechnological techniques, mainly based on solubilization processes involving agro-industrial residues, is presented. Potential advantages of composting, solid-state fermentation, and liquid submerged fermentation employing free and immobilized microorganisms that produce organic acids and simultaneously solubilize RP are discussed. Subsequent introduction of the final fermented products into soil-plant systems promotes plant growth and P acquisition.

Application of free and Ca-alginate-entrapped Glomus deserticola and Yarowia lipolytica in a soil-plant system.

This study was performed to investigate the applicability of microbial inoculants entrapped in alginate gel. Glomus deserticola (AM) was inoculated into soil microcosms, enriched with rock phosphate, as either free form or entrapped in calcium alginate alone or in combination with a P-solubilizing yeast culture (Yarowia lipolytica). Plant dry weight, soluble P acquisition, and mycorrhizal index were equal in treatments inoculated with free and alginate-entrapped AM. Dual inoculation with entrapped G. deserticola and free cells of Y. lipolytica significantly increased all analyzed variables. Highest rates of the latter were obtained when both fungal microorganisms were applied co-entrapped in the carrier. The yeast culture behaved as a 'mycorrhiza helper microorganism' enhancing mycorrhization of tomato roots. These results indicate that dual inoculation with an AM fungus and a P-solubilizing microorganism co-entrapped in alginate can be an efficient technique for plant establishment and growth in nutrient deficient soils.

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition.

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.

Application of an encapsulated filamentous fungus in solubilization of inorganic phosphate.

Spores of Aspergillus niger were encapsulated in agar, calcium alginate and k-carrageenan and further applied in citric acid production during six repeated batch cultivations. Rock phosphate (RP) at concentrations of 3 g l-1 and 7 g l-1 was supplemented to the culture medium to test encapsulated-fungus solubilizing capability. The highest average citric acid productivity of 0.15 g l-1 h-1 was reached with alginate-bead-encapsulated A. niger on RP-free culture medium while agar seemed to be the most suitable carrier on RP-supplemented medium. Accordingly, the highest average soluble P concentration of 0.20 g l-1 batch-1 was obtained with agar-cell beads as compared with other encapsulated systems.

Interactions between histaminergic and cholinergic pathways of gastric motility regulation.

Smooth muscle preparations, isolated in a circular direction from guinea pig gastric fundus, were used to study the effects of H1 and H2 antagonists on acetylcholine (ACH)- and histamine (HA)-induced contractions as well as the effects of HA antagonists on spontaneous contractile activity. HA (1 x 10(-9) M to 1 x 10(-5) M) concentration-dependently enhanced the tone of the strips with ED50 = 3.5 x 10(-7) M. Applied 5 min before HA, the H1 antagonists (mepyramine, diphenhydramine, dimethpyrindene) and the H2 blockers (ranitidine, cimetidine, roxatidine) reduced HA-induced contractions. HA in concentrations of 1 x 10(-8) M to 1 x 10(-7) potentiated, and in higher concentrations (1 x 10(-6) M to 1 x 10(-5) M) inhibited, smooth muscle contractions evoked by low frequency electrical field stimulation (EFS). The H1 blockers (1 x 10(-6) M to 1 x 10(-4) M) concentration-dependently enhanced smooth muscle tone, the maximum contractions being about 50% smaller than the contractile responses to 1 x 10(-5) M ACH and 5 x 10(-5) M HA. Tetrodotoxin, atropine and indomethacin shifted to the right the concentration-response curve for mepyramine, reducing its maximum by 25, 58 and 62%, respectively. The H2 blocker ranitidine also suppressed (by 42%) mepyramine-evoked increase in the fundic strips tone. The H1 antagonists reduced ACH-induced contractions of the smooth muscle strips and did not affect the contractions in response to EFS. The H2 blockers had no effect on tone and ACH-evoked contractions of the smooth muscle strips but concentration-dependently enhanced both the contractions and [3H]-ACH release in response to EFS. The results demonstrate the presence of both H1 and H2 postsynaptic receptors which are involved in the direct myogenic action of HA on guinea pig gastric fundus smooth muscles. It also appears that HA might concentration-dependently modulate the cholinergic neurotransmission in gastric fundus. It could be suggested that H1 blockers have a direct myogenic effect on guinea pig gastric fundus smooth muscle and might also interact postsynaptically with muscarinic receptors in this tissue.

Different effects of H1 and H2 blockers on the tone and the contractile activity of guinea pig stomach fundus.

The action of H1 and H2 blockers on the spontaneous and evoked contractile activity of gastric fundus smooth muscles as well as the effects of H2 antagonists on the release of acetylcholine (ACh) from gastric myenteric neurons were studied. The experiments were performed on smooth muscle strips (25 x 3 mm) cut out in circular direction from guinea pig fundus region. In concentrations of 1 x 10(-7) M to 1 x 10(-4) M, the H1 blockers diphenhydramine (DPH), mepyramine (MEP) and dimethpyrindene (DMPD), but not the H2 blockers ranitidine (RAN), cimetidine (CIM) and roxatidine (ROX), increased in a concentration-dependent manner the smooth muscle tone, the maximum contractions being about 50% of the contractile effects of 1 x 10(-5) M ACH and 5 x 10(-5) M histamine (HA). The concentration-dependent contractions of the stomach fundus strips in response to electrical field stimulation (EFS) were enhanced by RAN, CIM and ROX (but not by MEP and DPH), all in concentrations of 1 x 10(-7) M to 1 x 10(-4) M. EFS increased the resting [3H]-ACh release by 67.8%, the S2/S1 ratio being 0.85 +/- 0.04. ROX in a concentration of 1 x 10(-5) M significantly increased (by 16.1%) the EFS-induced release with a S2/S1 ratio of 1.22 +/- 0.04. The ROX effect on the [3H]-ACh release was reduced or even abolished by 1 x 10(-6) M tetrodotoxin (TTX) and 1 x 10(-6) M scopolamine or in Ca(2+)-free medium, while 1 x 10(-6) M hexamethonium did not change it. It might be concluded that H2 blockers have no direct myogenic effect and do not interfere with muscarinic receptors in guinea pig stomach fundus. The H2 antagonists enhance the EFS-evoked contractions of the gastric smooth muscle most probably by increasing the release of ACH.

Effect of neurotensin on the canine gallbladder motility: in vivo and in vitro experiments.

Neurotensin (NT) (10(-8)-10(-6)) exerted a dose-dependent increase in the tone and release of [3H]ACh in the guinea-pig gallbladder muscle strips but was inefficient in the canine gallbladder muscle strips. However, in conscious dogs NT (2.5-20 ng/kg intravenously (i.v.)) dose-dependently increased the gallbladder pressure. Similar was the effect of CCK8 (1-10 ng/kg i.v.) and carbachol (0.5-2 micrograms/kg i.v.). The NT- or CCK8-induced gallbladder pressure was inhibited by atropine (10-50 micrograms/kg i.v.) or hexamethonium (0.5-3 mg/kg i.v.). Somatostatin (1-2 micrograms/kg i.v.) or VIP (0.5-1 microgram/kg i.v.) also reduced or even abolished the NT- or CCK8-induced gallbladder pressure. The NT-induced increase of the tone of guinea-pig gallbladder preparations was accompanied by an increase of [3H]ACh release, suggesting the involvement of cholinergic innervation.

Production of gluconic acid by Aspergillus niger immobilized on polyurethane foam.

Production of gluconic acid by cells of Aspergillus niger immobilized on polyurethane foam was studied in repeated-batch shake-flask and bubble-column fermentations. For passive immobilization, various amounts of polyurethane foam and spore suspension were tested in order to obtain a suitable combination for optimal concentration of immobilized biomass. Immobilized cells were successfully reused with higher levels of product formation being maintained for longer period (65-70 h) than free cells. The highest gluconic acid concentration of about 143 g l-1 was reached on hydrol-based production medium with 0.3-cm3 foam cubes in the bubble column, where the effect of more suitable aeration and particle volume: medium volume ratio scheme was also investigated.

Effects of somatostatin and motilin on the motor and myoelectrical activity of the canine stomach and small intestine in vivo.

The experiments were performed on six starved dogs with chronic fistula in the stomach and small intestine and with chronically implanted electrodes on the smooth-muscle wall of these organs. Motilin (15-25 ng/kg i.v.) induced fastedtype peristaltic contractions accompanied by migrating myoelectrical spike activity in the stomach and small intestine. Somatostatin (0.5-2 micrograms/kg i.v.) inhibited the spontaneous contractile activity and the spike potentials in both organs. The antagonistic effect of somatostatin on the spontaneous and motilin-induced contractions resembled the effect of atropine and probably resulted from the decreased acetylcholine release in the cholinergic neurons.

Streptozotocin-induced diabetes in rat. III. Antioxidant protection of vascular complications by flunarizine and aligeron.

In rats with streptozotocin-induced diabetes antioxidant protection of diabetic angiopathy was performed by flunarizine (10 mg/kg/day) and aligeron (10 mg/kg/day), applied intraperitoneally during 2.5 months of diabetes. Diabetic vascular complications were assessed by morphologic determination of PAS-positive mucopolysaccharides and measurement of vascular wall thickness in addition to quantitative estimation of lipid hydroperoxides, thromboxane A2/prostacyclin disbalance and plasma beta-thromboglobulin changes. Both drugs prevented development of diabetic angiopathy in rats by inhibition of lipid peroxidation, prostanoid synthesis and platelet activity, but the effect of flunarizine was more pronounced, which could be explained by its additional blocking effect of abnormal calcium flux into vascular cells. The free radical scavenging action of flunarizine and aligeron was investigated.

Streptozotocin-induced diabetes in rat. II. Lipid and lipid peroxide changes of lipoprotein fractions in diabetes complicated by hypertension and myocardial infarction.

Lipid peroxide levels and plasma lipids were studied in plasma lipoprotein fractions of streptozotocin diabetic rats, spontaneous hypertensive rats (SHR) + diabetes, and in myocardial infarction rats (MIR) + diabetes. The duration of diabetes in all experimental groups was 2.5 months. We found a tendency of elevation of cholesterol in VLDL and fall in HDL2 but the differences were not significant. Total plasma triglycerides were increased in the three diabetic groups, and the increase was due to LDL fraction but again the differences were not significant. The lipid peroxide (LP) level in total plasma showed a significant increase in the three diabetic groups: in Wistar diabetic rats LP increased 3 times, in MIR + diabetes 3.5 times, and in SHR + diabetes 5 times. The increase of LP in the three diabetic groups was due to LDL with good correlation (r = 0.60) between LP and triglycerides in LDL of the three diabetic groups. The results are in agreement with the concept of the importance of lipoprotein fraction changes: increased cholesterol, triglycerides and lipid peroxides in atherogenic (VLDL and LDL) fractions, and decreased levels in antiatherogenic (HDL, HDL2) fractions in diabetes mellitus.

Streptozotocin-induced diabetes in rat. I. Influence of hypertension and myocardial infarction on the development of vascular complications.

Streptozotocin diabetes in rats was complicated by spontaneous hypertension (SHR) and myocardial infarction (MIC), considered as "risk groups". Renal function was assessed on the basis of blood urea nitrogen (BUN) and albuminuria. BUN increased by 36% in Wistar diabetic group, by 100% in SHR + diabetes, and by 51% in MIR + diabetes. Morphologic changes were assessed by estimation of PAS-positive glycosaminoglycans and measurement of vascular wall thickness of glomerular arterioles. The risk groups showed exaggerated tendency for development of diabetic angiopathy. A significant imbalance between TXA2 and prostacyclin was found, which was reflected by TXB2/6-keto-PGF1 alpha (the stable metabolites of TXA2 and prostacyclin, respectively) ratio, which increased by 38% in Wistar diabetic rats, by 61% in SHR + diabetes, and by 133% in MIR + diabetes. These changes correlated very well with increased platelet aggregability (r = 0.70; p less than 0.05) and with increased lipid peroxide level (r = 0.60; p less than 0.05), but neither with total plasma cholesterol (r = 0.20), nor with plasma triglycerides (r = 0.34). Lipid peroxides increased 5-fold in Wistar diabetic rats, 6-fold in SHR + diabetes, and 5.5-fold in MIR + diabetes. A causative relationship between TXA2/PGI2 imbalance and lipid peroxide changes on one hand, and diabetic angiopathy, on the other, was suggested.

Pathogenesis of cardiovascular disorders in streptozotocin-induced diabetes in rat. I. Cardiovascular, renal and morphologic changes in different stages of diabetes.

Some cardiovascular (heart rate and mean arterial pressure), and renal (glomerular filtration rate-GFR; renal plasma flow-RPF; filtration fraction-FF; blood urea nitrogen-BUN and albuminuria) parameters, coupled with morphologic examination, was undertaken in early (2 months) and late (6 months) stage of streptozotocin-induced diabetes mellitus in rats. The results showed a temporally (early) bradycardia and gradually increase of blood pressure with morphologic changes typical for diabetic cardiopathy. The increased GFR (by 92%), associated with significantly decreased RPF (by 37%), increased FF (by 133%), increased kidney weight/body weight ratio (by 88%), increased BUN (by 52%) and distinct albuminuria (13.53 +/- 2.08 mg/24 h/100 g b. w.), together with typical morphologic changes, suggested the development of diabetic nephropathy which was progressive with the duration of the disease.