Uncovering the Ediacaran phosphorus cycle.

Phosphorus is a limiting nutrient that is thought to control oceanic oxygen levels to a large extent1-3. A possible increase in marine phosphorus concentrations during the Ediacaran Period (about 635-539 million years ago) has been proposed as a driver for increasing oxygen levels4-6. However, little is known about the nature and evolution of phosphorus cycling during this time4. Here we use carbonate-associated phosphate (CAP) from six globally distributed sections to reconstruct oceanic phosphorus concentrations during a large negative carbon-isotope excursion-the Shuram excursion (SE)-which co-occurred with global oceanic oxygenation7-9. Our data suggest pulsed increases in oceanic phosphorus concentrations during the falling and rising limbs of the SE. Using a quantitative biogeochemical model, we propose that this observation could be explained by carbon dioxide and phosphorus release from marine organic-matter oxidation primarily by sulfate, with further phosphorus release from carbon-dioxide-driven weathering on land. Collectively, this may have resulted in elevated organic-pyrite burial and ocean oxygenation. Our CAP data also seem to suggest equivalent oceanic phosphorus concentrations under maximum and minimum extents of ocean anoxia across the SE. This observation may reflect decoupled phosphorus and ocean anoxia cycles, as opposed to their coupled nature in the modern ocean. Our findings point to external stimuli such as sulfate weathering rather than internal oceanic phosphorus-oxygen cycling alone as a possible control on oceanic oxygenation in the Ediacaran. In turn, this may help explain the prolonged rise of atmospheric oxygen levels.

Phosphorus Nutrition in Songpu Mirror Carp (Cyprinus carpio Songpu) During Chronic Carbonate Alkalinity Stress: Effects on Growth, Intestinal Immunity, Physical Barrier Function, and Intestinal Microflora.

Carbonate alkalinity is a major environmental stress factor affecting aquatic feed configuration, which easily causes oxidative stress and hypoimmunity for fish. Hence, the purpose of the study is to assess the potential effect of phosphorus on growth, intestinal oxidation resistance, physical barrier function, and microflora for Songpu mirror carp (Cyprinus carpio Songpu) (initial average weight of 2.95 ± 0.21 g) reared at the high-concentration carbonate alkalinity environment. A two-factor, three-level (2 × 3) design was applied, in which diets with three different phosphorus levels (3.6, 7.0, and 10.5 g/kg dry matter) were randomly assigned to 0 and 15 mmol/L carbonate alkalinity groups with three replicate aquariums. After the 8-week trial, we found that weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and lipase and amylase activities in the intestine significantly (p < 0.05) declined with increasing carbonate alkalinity. Carbonate alkalinity of 15 mmol/L significantly reduced glutathione peroxidase (GSHPx) activities in the intestine (p < 0.05). The relative expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase 1a (GPX1a), Clautin3, Clautin11, and tumor necrosis factor ß (TNF-ß) in the intestine were markedly downregulated by increasing carbonate alkalinity levels (p < 0.05), whilst the relative expressions of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the intestine were markedly upregulated (p < 0.05). At the 15 mmol/L carbonate alkalinity treatment, Songpu mirror carp suffer from hypoimmunity status with failed digestion, antioxidant, inflammation, and immune response, thereby inducing impaired growth. Additionally, significant increments in the abundance of Proteobacteria and a significant decrease in the abundance of Fusobacteria and the Firmicutes/Bacteroidetes ratio were caused due to excessively high carbonate alkalinity (15 mmol/L) and excessively low dietary phosphorus supply (3.6 g/kg). Collectively, 7.0 g/kg dietary phosphorus supplementation was effective in promoting intestinal antioxidant enzyme activities and the corresponding gene expression via the Keap1-Nrf2 signaling pathway and in enhancing intestinal immunity by upregulating anti-inflammatory and downregulating pro-inflammatory genes. Appropriate dietary phosphorus supply could promote the formation of beneficial microflora in freshwater, and it has the potential ability to transfer the adverse effect of carbonate alkalinity stress to the structural composition of intestinal microflora. Hence, consideration should be given to suitable phosphorus supply for fish under the chronic carbonate alkalinity stress.

The biostimulating effects of viable microalgal cells applied to a calcareous soil: Increases in bacterial biomass, phosphorus scavenging, and precipitation of carbonates.

Microalgae used in wastewater treatment may be applied to soil as a biofertilizer - this is a novel strategy for recycling of nutrients in the circular economy. There is little information about how the application of large concentrations of unicellular algae to soil will affect soil biochemistry, particularly when they are living algal cells with the potential to form a soil biofilm, whereas soil biofilms are expected to influence plant-microbe interactions. Chlorophyte unicellular algae of the Chlorella genera are widely employed in algae-based water treatment systems, and Chlorella sorokiniana has proven to be highly adaptable for this purpose. We applied three filtrates of a Chlorella sorokiniana culture to soil microcosms, separating the microalgae from other microorganisms, as well as a sterile control filtrate without biological activity. Bacterial biomass in soils receiving the non-filtered (NF) slurry with viable algal cells was increased by 25% in the soil surface (0-8mm), and heterotrophic activity in those treatments increased as measured by CO2-C evolution. Total soil carbon concentrations were increased in the treatment with living algal cells (NF) by 0.4%, but no differences in organic carbon were measured; instead, it was found that inorganic carbon (CaCO3) concentrations increased by 0.6% in the NF treatment only. Soil phosphorus availability was also reduced in the surface of the NF treatment, indicating an increased biological demand. The results show that, when applied to soil, microalgae and associated biofilms will have relevant direct and indirect effects on soil quality and nutrients of agricultural importance.

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions.

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Soil carbonate drives local adaptation in Arabidopsis thaliana.

High soil carbonate limits crop performance especially in semiarid or arid climates. To understand how plants adapt to such soils, we explored natural variation in tolerance to soil carbonate in small local populations (demes) of Arabidopsis thaliana growing on soils differing in carbonate content. Reciprocal field-based transplants on soils with elevated carbonate (+C) and without carbonate (-C) over several years revealed that demes native to (+C) soils showed higher fitness than those native to (-C) soils when both were grown together on carbonate-rich soil. This supports the role of soil carbonate as a driving factor for local adaptation. Analyses of contrasting demes revealed key mechanisms associated with these fitness differences. Under controlled conditions, plants from the tolerant deme A1(+C) native to (+C) soil were more resistant to both elevated carbonate and iron deficiency than plants from the sensitive T6(-C) deme native to (-C) soil. Resistance of A1(+C) to elevated carbonate was associated with higher root extrusion of both protons and coumarin-type phenolics. Tolerant A1(+C) also had better Ca-exclusion than sensitive T6(-C) . We conclude that Arabidopsis demes are locally adapted in their native habitat to soils with moderately elevated carbonate. This adaptation is associated with both enhanced iron acquisition and calcium exclusion.

The impact of operational strategies on the performance of a photo-EBPR system.

A novel Phototrophic - Enhanced Biological Phosphorus Removal (Photo-EBPR) system, consisting of a consortium of photosynthetic organisms and polyphosphate accumulating organisms (PAOs), was studied in this work. A sequencing batch reactor was fed with a mixture of acetate and propionate (75%-25%) and subjected to dark/light cycles in order to select a photo-EBPR system containing PAOs and photosynthetic organisms, the latter likely providers of oxygen to the system. The results from the selection period (stage 1) showed that the photo-EBPR culture was capable of performing P release in the dark and P uptake in the presence of light, under limited oxygen concentrations. During the optimization period, the aeration period, which was initially provided at the end of the light phase, was gradually reduced until a non-aerated system was achieved, while the light intensity was increased. After optimization of the operational conditions, the selected consortium of photosynthetic organisms/PAOs showed high capacity of P removal in the light phase in the absence of air or other electron acceptor. A net P removal of 34 ± 3 mg-P/L was achieved, with a volumetric P removal rate of 15 ± 2 mg-P/L.h, and 79 ± 8% of P removal from the system. Also, in limiting oxygen conditions, the P uptake rate was independent of the PHA consumption, which demonstrates that the organisms obtained energy for P removal from light. These results indicated that a photo-EBPR system can be a potential solution for P removal with low COD/P ratios and in the absence of air, prospecting the use of natural sunlight as illumination, which would reduce the costs of EBPR operation regarding aeration.

Enhancement of total lipid yield by nitrogen, carbon, and iron supplementation in isolated microalgae.

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG-11 containing sodium carbonate concentration at 0.03 g · L-1 , and in normal BG-11 containing iron concentration (IC) at 0.009 or 0.012 g · L-1 . Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L-1 under the IC of 0.012 g · L-1 . Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties.

The effect of cation source and dietary cation-anion difference on rumen ion concentrations in lactating dairy cows.

Many studies have focused on the influence of dietary cation-anion difference (DCAD) on animal performance but few have examined the effect of DCAD on the rumen ionic environment. The objective of this study was to examine the effects of DCAD, cation source (Na vs. K), and anion source (Cl vs. bicarbonate or carbonate) on rumen environment and fermentation. The study used 5 rumen-fistulated dairy cows and 5 dietary treatments that were applied using a 5×5 Latin square design with 2-wk experimental periods. Treatments consisted of (1) the basal total mixed ration (TMR); (2) the basal TMR plus 340mEq/kg of Na (dry matter basis) using NaCl; (3) the basal TMR plus 340mEq/kg of K using KCl; (4) the basal TMR plus 340mEq/kg of Na using NaHCO3; and (5) the basal TMR plus 340mEq/kg of K using K2CO3. On the last day of each experimental period, rumen samples were collected and pooled from 5 different locations at 0, 1.5, 3, 4.5, 6, 9, and 12h postfeeding for measurement of rumen pH and concentrations of strong ions and volatile fatty acids (VFA). Dietary supplementation of individual strong ions increased the corresponding rumen ion concentration. Rumen Na was decreased by 24mEq/L when K was substituted for Na in the diet, but added dietary Na had no effect on rumen K. Rumen Cl was increased by 10mEq/L in diets supplemented with Cl. Cation source had no effect on rumen pH or total VFA concentration. Increased DCAD increased rumen pH by 0.10 pH units and increased rumen acetate by 4mEq/L but did not increase total VFA. This study demonstrated that rumen ion concentrations can be manipulated by dietary ion concentrations. If production and feed efficiency responses to DCAD and ionophores in the diet are affected by rumen Na and K concentrations, then manipulating dietary Na and K could be used either to enhance or diminish those responses.

The effect of dietary cation-anion difference concentration and cation source on milk production and feed efficiency in lactating dairy cows.

Feed costs currently account for 55% or more of the total cost of milk production in US dairy herds, and dairy producers are looking for strategies to improve feed efficiency [FE; 3.5% fat-corrected milk (FCM) per dry matter (DM) intake]. Increasing dietary cation-anion difference [DCAD; Na+K-Cl (mEq/kg of DM)] has been shown to increase milk production, FCM, and FE. However, the optimal DCAD concentration for maximal FE has yet to be determined. The objectives of this research were to test the effects of DCAD concentration and cation source on dairy FE. Sixty Holstein dairy cows (20 cows per experiment) were used in three 4×4 Latin square design experiments with 3-wk experimental periods. In experiments 1 and 2, we tested the effect of DCAD concentration: cows were fed a basal diet containing ~250 mEq/kg of DM DCAD that was supplemented with potassium carbonate at 0, 50, 100, and 150 mEq/kg of DM or 0, 125, 250, and 375 mEq/kg of DM in experiments 1 and 2, respectively. In experiment 3, we tested the effect of cation source: sodium sesquicarbonate replaced 0, 33, 67, and 100% of the supplemental potassium carbonate (150 mEq/kg of DM DCAD). The DCAD concentration had no effect on milk production, milk protein concentration, or milk protein yield in experiments 1 and 2. Dry matter intake was not affected by DCAD concentration in experiment 1 or by cation source in experiment 3. However, DMI increased linearly with increasing DCAD in experiment 2. We detected a linear increase in milk fat concentration and yield with increasing DCAD in experiments 1 and 2 and by substituting sodium sesquicarbonate for potassium carbonate in experiment 3. Increased milk fat concentration with increasing DCAD led to increases in 3.5% FCM in experiments 1 and 2. Maximal dairy FE was achieved at a DCAD concentration of 426 mEq/kg of DM in experiments 1 and 2 and by substituting Na for K in experiment 3. The results of these experiments suggest that both DCAD concentration and the cation source used to alter DCAD concentration have effects on milk fat content and yield and dairy FE.

Growth of Desulfovibrio vulgaris when respiring U(VI) and characterization of biogenic uraninite.

The capacity of Desulfovibrio vulgaris to reduce U(VI) was studied previously with nongrowth conditions involving a high biomass concentration; thus, bacterial growth through respiration of U(VI) was not proven. In this study, we conducted a series of batch tests on U(VI) reduction by D. vulgaris at a low initial biomass (10 to 20 mg/L of protein) that could reveal biomass growth. D. vulgaris grew with U(VI) respiration alone, as well as with simultaneous sulfate reduction. Patterns of growth kinetics and solids production were affected by sulfate and Fe(2+). Biogenic sulfide nonenzymatically reduced 76% of the U(VI) and greatly enhanced the overall reduction rate in the absence of Fe(2+) but was rapidly scavenged by Fe(2+) to form FeS in the presence of Fe(2+). Biogenic U solids were uraninite (UO2) nanocrystallites associated with 20 mg/g biomass as protein. The crystallite thickness of UO2 was 4 to 5 nm without Fe(2+) but was <1.4 nm in the presence of Fe(2+), indicating poor crystallization inhibited by adsorbed Fe(2+) and other amorphous Fe solids, such as FeS or FeCO3. This work fills critical gaps in understanding the metabolic utilization of U by microorganisms and formation of UO2 solids in bioremediation sites.

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil in solvent-free system.

The enzymatic coproduction of biodiesel and glycerol carbonate by transesterification of soybean oil and dimethyl carbonate (DMC) has been studied in a solvent-free system. The effects on biodiesel and glycerol carbonate conversion of reaction conditions including the kind of enzyme, the amount of enzyme, the molar ratio of DMC to soybean oil, the reaction temperature, and water addition were investigated. The optimal conditions for biodiesel and glycerol carbonate were 20% Novozym 435, 10:1 molar ratio of DMC to soybean oil, and 0.7% water addition. Under these conditions, the conversions of 96.4% biodiesel and 92.1% glycerol carbonate have been achieved after 48h.

Raman spectroscopy demonstrates Amifostine induced preservation of bone mineralization patterns in the irradiated murine mandible.

PURPOSE: Adjuvant radiotherapy in the management of head and neck cancer remains severely debilitating. Fortunately, newly developed agents aimed at decreasing radiation-induced damage have shown great promise. Amifostine (AMF) is a compound, which confers radio-protection to the exposed normal tissues, such as bone. Our intent is to utilize Raman spectroscopy to demonstrate how AMF preserves the mineral composition of the murine mandible following human equivalent radiation. METHODS: Sprague Dawley rats were randomized into 3 experimental groups: control (n=5), XRT (n=5), and AMF-XRT (n=5). Both XRT and AMF groups underwent bioequivalent radiation of 70Gy in 5 fractions to the left hemimandible. AMF-XRT received Amifostine prior to radiation. Fifty-six days post-radiation, the hemimandibles were harvested, and Raman spectra were taken in the region of interest spanning 2mm behind the last molar. Bone mineral and matrix-specific Raman bands were analyzed using one-way ANOVA, with statistical significance at p<0.05. RESULTS: The full-width at half-maximum of the primary phosphate band (FWHM) and the ratio of carbonate/phosphate intensities demonstrated significant differences between AMF-XRT versus XRT (p<0.01) and XRT versus control (p<0.01). There was no difference between AMF-XRT and control (p>0.05) in both Raman metrics. Computer-aided spectral subtraction further confirmed these results where AMF-XRT was spectrally similar to the control. Interestingly, the collagen cross-link ratio did not differ between XRT and AMF-XRT (p<0.01) but was significantly different from the control (p<0.01). CONCLUSION: Our novel findings demonstrate that AMF prophylaxis maintains and protects bone mineral quality in the setting of radiation. Raman spectroscopy is an emerging and exceptionally attractive clinical translational technology to investigate and monitor both the destructive effects of radiation and the therapeutic remediation of AMF on the structural, physical and chemical qualities of bone.

Effect of carbonate on anaerobic acidogenesis and fermentative hydrogen production from glucose using leachate as supplementary culture under alkaline conditions.

Carbonate was added into a co-culture of glucose and fresh leachate under alkaline condition to enhance batched acidogenesis and fermentative hydrogen production simultaneously. Results indicated carbonate has positive effect on both H(2) production and acetic acid generation. The highest hydrogen yield (about 1.40 mol/mol glucose) was obtained at [CO(3)(2-)] = 280 mg/L with pH 8.0 and [CO(3)(2-)] = 560 mg/L with pH 9. The dominant liquid metabolites were ethanol, acetic and butyric acid. The highest total volatile fatty acid yield (0.38 g/g glucose) was achieved at [CO(3)(2-)] = 560 mg/L with pH 9. In this case, the acetic acid yield reached 0.13 g/g glucose. Verification tests using simulated wastewater as substrate were also carried out at pH 9. Results demonstrated calcium ions inhibit hydrogenogens activity while carbonate addition can alleviate the suppression effect caused by Ca(2+).

Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate.

Biodiesel [fatty acid methyl esters (FAMEs)] and glycerol carbonate were synthesized from corn oil and dimethyl carbonate (DMC) via transesterification using lipase (Novozyme 435) in solvent-free reaction in which excess DMC was used as the substrate and reaction medium. Glycerol carbonate was also simultaneously formed from DMC and glycerol. Conversions of FAMEs and glycerol carbonate were examined in batch reactions. The FAMEs and glycerol carbonate reached 94 and 62.5% from oil and DMC (molar ratio of 1:10) with 0.2% (v/v) water and 10% (w/w) Novozyme 435 (based on oil weight) at 60 °C. When Novozyme 435 was washed with acetone after each reaction, more than 80% activity still remained after seven recycling.

Oligocarbonate molecular transporters: oligomerization-based syntheses and cell-penetrating studies.

A new family of guanidinium-rich molecular transporters featuring a novel oligocarbonate backbone with 1,7-side chain spacing is described. Conjugates can be rapidly assembled irrespective of length in a one-step oligomerization strategy that can proceed with concomitant introduction of probes (or by analogy drugs). The new transporters exhibit excellent cellular entry as determined by flow cytometry and fluorescence microscopy, and the functionality of their drug delivery capabilities was confirmed by the delivery of the bioluminescent small molecule probe luciferin and turnover by its intracellular target enzyme.

Bone and shell contribution to lactic acid buffering of submerged turtles Chrysemys picta bellii at 3 degrees C.

To evaluate shell and bone buffering of lactic acid during acidosis at 3 degrees C, turtles were submerged in anoxic or aerated water and tested at intervals for blood acid-base status and plasma ions and for bone and shell percent water, percent ash, and concentrations of lactate, Ca(2+), Mg(2+), P(i), Na(+), and K(+). After 125 days, plasma lactate concentration rose from 1.6 +/- 0.2 mM (mean +/- SE) to 155.2 +/- 10.8 mM in the anoxic group but only to 25.2 +/- 6.4 mM in the aerated group. The acid-base state of the normoxic animals was stable after 25 days of submergence. Plasma calcium concentration (¿Ca(2+)) rose during anoxia from 3.2 +/- 0.2 to 46.0 +/- 0.6 mM and ¿Mg(2+) from 2.7 +/- 0.2 to 12.2 +/- 0.6 mM. Both shell and bone accumulated lactate to concentrations of 135.6 +/- 35.2 and 163.6 +/- 5.1 mmol/kg wet wt, respectively, after 125 days anoxia. Shell and bone ¿Na(+) both fell during anoxia but the fate of this Na(+) is uncertain because plasma ¿Na(+) also fell. No other shell ions changed significantly in concentration, although the concentrations of both bone calcium and bone potassium changed significantly. Control shell water (27.8 +/- 0.6%) was less than bone water (33.6 +/- 1.1%), but neither changed during submergence. Shell ash (44.7 +/- 0.8%) remained unchanged, but bone ash (41.0 +/- 1.0%) fell significantly. We conclude that bone, as well as shell, accumulate lactate when plasma lactate is elevated, and that both export sodium carbonate, as well as calcium and magnesium carbonates, to supplement ECF buffering.

Detoxication of phenol in annual plant seedlings.

Transformation of phenol (14C6H5OH) penetrating through the roots of mung bean (Phaseolus aureus) and wheat (Triticum vulgare) sterile seedlings has been studied. Phenol was coupled to low-molecular-weight peptides, producing phenol-peptide conjugates. Hydrolytic cleavage of the conjugates liberated initial labeled phenol and some unlabeled amino acids. Phenol- glutathione and phenol-homoglutathione were not found among the peptide conjugates. It is suggested that the conjugation is carried out via the hydroxyl group of phenol and functional groups of peptides. Conjugation with low-molecular-weight peptides is considered to be the main pathway for phenol detoxication, since about 60% of phenol absorbed by plants conjugates with peptides. In the plants treated with phenol, the amount of low-molecular-weight peptides is increased. The increase in peptide synthesis in plants seems to be induced by the penetration of toxic phenol molecules into the cell. The small amount of phenol molecules assimilated through roots is transformed via aromatic ring cleavage and bibasic carbonic acid formation.

Effect of casein-based semi-synthetic food on renal acid excretion and acid-base state of blood in dogs.

Urinary acid excretion and blood acid-base state were determined in dogs fed a casein-based semi-synthetic food (SSF), to which different amounts of salts had been added, in comparison with feeding normal dog food. Net acid excretion (NAE) and inorganic acid excretion (IAE) increased during SSF feeding. IAE was higher than the acid load calculated from the sulphur and phosphorus content of the casein. This higher IAE appeared to be due to the presence of calcium and magnesium phosphate in the diet, because calcium and magnesium may be in part precipitated as carbonate, leaving phosphate to be absorbed as phosphoric acid. Acid excretion decreased by addition of CaO. When no neutral Na+ and K+ salts were added, the increase in NAE was accompanied by a metabolic acidosis. K+ was more effective in attenuating the acidosis than Na+. On the basis of these findings a diet can be made which imposes a known acid load, and provides stable baseline values. Hence, any additions that influence the acid-base balance can be properly studied. The data obtained in these and future studies utilising this diet may be of help in optimising the composition of nutrient solutions to be used in the care of critically ill patients.