Beta-propeller phytases in the aquatic environment

Phytate, which is one of the dominant organic phosphorus compounds in nature, is very stable in soils. Although a substantial amount of phytate is carried from terrestrial to aquatic systems, it is a minor component of organic phosphorus in coastal sediments. The ephemeral nature of phytate implies the rapid hydrolysis of phytate under aquatic conditions. Among the four classes of known phytases that have been identified in terrestrial organisms, only β-propeller phytase-like sequences have been identified in the aquatic environment. A novel β-propeller phytase gene (phyS), cloned from Shewanella oneidensis MR-1, was found to encode a protein with two beta-propeller phytase domains. The characterization of recombinant full-length PhyS and its domains demonstrated that Domain II was the catalytic domain responsible for phytate hydrolysis. The full-length PhyS displayed a K_m of 83 μM with a kcat of 175.9 min⁻¹ and the Domain II displayed a K_m of 474 μM with a kcat of 10.6 min⁻¹. These results confirm that the phyS gene encodes a functional β-propeller phytase, which is expressed in S. oneidensis under phosphorus deficienct condition. The presence of multiple sequences with a high similarity to phyS in aquatic environmental samples and the widespread occurrence of the Shewanella species in nature suggest that the β-propeller phytase family is the major class of phytases in the aquatic environment, and that it may play an important role in the recycling of phosphorus.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Cumulative effect of nitrogen and sulphur on <Emphasis Type="Italic">Brassica juncea</Emphasis> L. genotypes under NaCl stress

In the present study, N and S assimilation, antioxidant enzymes activity, and yield were studied in N and S-treated plants of Brassica juncea (L.) Czern. & Coss. (cvs. Chuutki and Radha) under salt stress. The treatments were given as follows: (1) NaCl₉₀ mM+N₀S₀ mg kg^-1 sand (control), (2) NaCl₉₀ mM+N₆₀S₀ mg kg^-1 sand, (3) NaCl₉₀ mM+N₆₀S₂₀ mg kg^-1 sand, (4) NaCl₉₀ mM+N₆₀S₄₀ mg kg^-1 sand, and (5) NaCl₉₀ mM+N₆₀S₆₀ mg kg^-1 sand. The combined application of N (60 mg kg⁻¹ sand) and S (40 mg kg⁻¹ sand) proved beneficial in alleviating the adverse effect of salt stress on growth attributes (shoot length plant⁻¹, fresh weight plant⁻¹, dry weight plant⁻¹, and area leaf⁻¹), physio-biochemical parameters (carbonic anhydrase activity, total chlorophyll, adenosine triphosphate-sulphurylase activity, leaf N, K and Na content, K/Na ratio, activity of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase, and content of glutathione and ascorbate), and yield attributes (pods plant⁻¹, seeds pod⁻¹, and seed yield plant⁻¹). Therefore, it is concluded that combined application of N and S induced the physiological and biochemical mechanisms of Brassica. The stimulation of antioxidant enzymes activity and its synergy with N and S assimilation may be one of the important mechanisms that help the plants to tolerate the salinity stress and resulted in an improved yield.     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Effect of phosphorus deficiency on acid phosphatase and phytase activities in common bean (Phaseolus vulgaris L.) under symbiotic nitrogen fixation

Changes in growth, symbiotic nitrogen fixation (SNF), acid phosphatase (ACP), and phytase activities to phosphorus availability (15 and 60 μmol KH₂PO₄ plant⁻¹ week⁻¹) were compared in two recombinant lines (115 and 147) of common bean. Plant growth, nodulation and SNF were genotype and P level-dependent. 147 was more affected by P shortage (15 μmol P) than 115. Four ACP types were revealed in the nodules of both lines, ACP1 exhibiting a higher specific activity under P shortage as compared to the 60 μmol P treatment, especially in 115. A single phytase was revealed for the nodules of both lines and was significantly enhanced by P deficiency. Three ACP types were found in roots and leaves, showing increasing activity under P deficiency, especially in 115. Regardless of P supply, leaf ACP specific activity was higher than that of nodules and roots in the both lines. Interestingly, phosphorus use efficiency for N₂ fixation significantly correlated to nodule ACP activity under P shortage in the both lines. The relatively better performance of 115 as compared to 147 under P deficiency could be partly ascribed to the ability of 115 to maintain higher ACP activity. This enzyme might be involved in the remobilization of the plant Pi and its utilization for SNF.     

Utilization of phytate by some yeasts

Summary Of 21 yeast strains screened for ability to hydrolyse phytic acid salts, nine strains grew on sodium phytate as sole source of inorganic phosphate. Of the five most interesting strains for their growth parameters tested and for their phytase activity in batch-culture,Schwanniomyces castellii CBS 2863 had the highest phytase activity in presence of 5 g phytate I^–1.     

Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.     

Effect of salinity on germination,phytase activity and phytate content in lettuce seedling

Seeds of four lettuce (Lactuca sativa L.) varieties (Romaine, Augusta, Vista and Verte) differing in their salt sensitivity were sown at 0 (Control), 50, 100 and 150 mM NaCl. The final germination percentage decreased with the increasing salinity and was annulated at the highest salt concentration in Vista and Verte, the most sensitive varieties. However, in the less sensitive ones, Romaine and Augusta, it was slightly modified at 50 and 100 mM NaCl and then decreased by 50% compared with the control, at 150 mM. The effects of NaCl 100 mM on seedling growth, phytase activities, phytate and inorganic phosphorus contents were studied in Romaine and Vista showing different behaviours towards salinity. Radicle and hypocotyl length and fresh and dry weights were reduced by salt treatment in both varieties. In addition, radicle phytase activity exhibited an increase in Romaine (less sensitive) and a decrease in Vista (more sensitive). In hypocotyl, this activity showed no difference with the control in the two varieties. However, in cotyledons, and during early hours after germination, salinity decreased phytase activity in both varieties whereas in the later hours (72–96 h) this activity reached the value of the control in Romaine. The enhancement of phytase activity was concomitant with an increase in orthophosphate content and a decrease in phytate reserve. These results suggest that salt presence in the medium delays Pi remobilization from phytate stock, but stimulates assimilation of phosphorus more than its accumulation in the organs of the two lettuce varieties.     

Acid phosphatases and growth of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars under diverse phosphorus nutrition

The morphological and physiological responses of barley to moderate Pi deficiency and the ability of barley to grow on phytate were investigated. Barley cultivars (Hordeum vulgare L., Promyk, Skald and Stratus) were grown for 1–3 weeks on different nutrient media with contrasting phosphorus source: KH₂PO₄ (control), phytic acid (PA) and without phosphate (−P). The growth on −P medium strongly decreased Pi concentration in the tissues; culture on PA medium generally had no effect on Pi level. Decreased content of Pi reduced shoot and root mass but root elongation was not affected; Pi deficit had slightly greater impact on growth of barley cv. Promyk than other varieties. Barley varieties cultured on PA medium showed similar growth to control. Extracellular acid phosphatase activities (APases) in −P roots were similar to control, but in PA plants were lower. Histochemical visualization indicated for high APases activity mainly in the vascular tissues of roots and in rhizodermis. Pi deficiency increased internal APase activities mainly in shoot of barley cv. Stratus and roots of cv Promyk; growth on PA medium had no effect or decreased APase activity. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoforms may be affected by Pi deficiency or growth on PA medium; two of four isoforms in roots were strongly induced by conditions of Pi deficit, especially in barley cv. Promyk. In conclusion, barley cultivars grew equally well both on medium with Pi and where the Pi was replaced with phytate and only slightly differed in terms of acclimation to moderate deficiency of phosphate; they generally used similar pools of acid phosphatases to acquire Pi from external or internal sources.     

Reversibility of phosphorus sorption by ferruginous nodules

Ferruginous nodules sorb significant amounts of available soil and fertiliser phosphate. The effect of this sorption on phosphorus availability of an agricultural soil was tested by sequential extraction and by exhaustive cropping with millet (Pennisetum typhoides) in a greenhouse trial following fertilisation of the original soil containing 70% nodules and of prepared samples containing various mixes of separated soil fines and nodules. Phosphorus sorption maxima by the soil fines and nodules were 190 mg kg⁻¹ and 380 mg kg⁻¹ respectively. Samples of fines and nodules which had sorbed 110 and 194 mg kg⁻¹ were submitted to 8 successive extractions with 0.01 M KCl, after which P desorption amounted to 117 mg kg⁻¹ and 103 mg kg⁻¹ respectively. Hysteresis between sorption and desorption was negligible for the soil fines and increased with increasing nodule content of the samples. In the greenhouse experiment, P uptake at the first cropping was highest in the soil fines at all levels of phosphorus applied. Subsequent croppings, however, showed higher P uptake in the concretionary soils. These results indicate a higher initial P release from the soil fines with cropping followed by an earlier exhaustion of phosphorus. At the end of the greenhouse experiment, yields were low in spite of the large quantities of P still remaining in the soils. Phosphorus fractionation showed that, of the P left in the soil after cropping 20% was in labile, 29% in Fe or Al-associated, and 51% in low-availability forms.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg⁻¹) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg⁻¹) were less than that of the wild-type phytase (3,524 U mg⁻¹), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.     

Effect of exogenous factors on metabolism of phosphorous compounds in the chicken tissues

The activity of plant and microbic phytases depending on the medium pH was studied. The factors have been investigated as follows: decomposition efficiency of seed ingredients and the releasing of phytate phosphorus; the efficiency of the adsorption of phosphorus under the in situation of microbic phytase; the influence of microbic phytase feeding in the ratios with low content of accessible phosphorus for assimilation of phytate phosphorus and poultry production indices. It has been stated that the microbic phytase has a wider optimum of action depending on pH value. The microbic phytase positive action on the metabolism of phosphorus in the chicken organism has been determined through the experimental investigations.     

Purification and Characterization of a Bacterial Phytase Whose Properties Make it Exceptionally Useful as a Feed Supplement

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K _M = 0.15 mmol/l and k _cat = 1164 s⁻¹ at pH 4.5 and 37°C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.     

Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l⁻¹ h⁻¹; 0.8 to 2.3 g l⁻¹ h⁻¹). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol d-lactate (90 g) per 100 g sugar (glucose or sucrose). Revisions requested 17 January 2006; Revisions received 7 February 2006     

Somatic organogenesis and plant regeneration in <Emphasis Type="Italic">Ricinus communis</Emphasis>

An in vitro propagation system was developed for castor-bean (Ricinus communis L. cv. TMV 6) through cotyledon derived callus cultures. The impact of different concentrations of auxins, cytokinins, additives, amino acids and sugars were evaluated for callus induction and shoot proliferation. Green compact nodular organogenic callus was obtained on the medium fortified with Murashige and Skoog (MS) salts, B5 vitamins, 2.0 mg dm⁻³ 6-benzyladenine and 0.8 mg dm⁻³ α-naphthalene acetic acid (NAA). Multiple shoot proliferation from the callus cultures was achieved on the medium with MS salts, B5 vitamins, 2.5 mg dm⁻³ thidiazuron (TDZ), 0.4 mg dm⁻³ NAA and 15 mg dm⁻³ glutamine. During multiple shoot induction the phenolic secretion was controlled by the addition of 15 mg dm⁻³ polyvinylpyrolidone. The proliferated shoots were elongated on the medium comprising MS salts, B5 vitamins, 1.5 mg dm⁻³ TDZ and 0.3 mg dm⁻³ gibberellic acid. The elongated shoots were rooted on the medium containing MS salts, B5 vitamins, 0.3 mg dm⁻³ indole-3-butyric acid and 0.6 mg dm⁻³ silver nitrate. After root induction, the plants were hardened in earthen pots containing sand, soil and vermiculite.     

Effect of salinity and its composition on the accumulation of selenium by alfalfa

Alfalfa (Medicago sativa L.) was grown in greenhouse sand culture to examine the effect of salinity composition and concentration on Se accumulation by plants. In a 2×2×4 factorial experiment, salinity was added as either C1⁻ or SO ₄ ²⁻ salts to the irrigating solution to achieve an electrical conductivity of 0.5, 1.5–3.0, or 6.0 dS m⁻¹. Selenium was added to the nutrient solution at a concentration of 0.25 or 1.0 mg Se(VI)I⁻¹. Following the third cutting, the roots were washed and all plant material analyzed for dry weight and Se. Plant biomass production decreased with additions of either Se or salinity, regardless of composition. In the presence of Se, the yield reduction was greater with Cl⁻ salinity than with SO ₄ ²⁻ salinity. Plant Se accumulation was reduced from 948 mg Se kg⁻¹ to 6 mg Se kg⁻¹ in the presence of SO ₄ ²⁻ salts (0.5 mmol SO ₄ ²⁻ l⁻¹ vs. 40 mmol SO ₄ ²⁻ l⁻¹) due to an apparent Se(VI) −SO ₄ ²⁻ antagonism. This Se−SO ₄ ²⁻ antagonism prevented accumulation of Se and reduced Se-induced toxicity. A lesser antagonistic effect on Se accumulation was observed between Cl⁻, and Se. A synergistic interaction between SO ₄ ²⁻ and Se(VI) increased plant S concentrations in the presence of the relatively low basal SO ₄ ²⁻ concentrations but not at the higher solution SO ₄ ²⁻ concentrations. In many areas, soil and water containing high Se concentrations also contain large amounts of SO ₄ ²⁻ . The occurrence of SO ₄ ²⁻ with Se reduces plant accumulation of Se(VI) and may lower the risk of Se overexposure to animals feeding on forage material grown in high Se−SO ₄ ²⁻ regions.     

Characterization of phytase produced byAspergillus niger

The extracellular activity ofAspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor. The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55°C and high pH and temperature stability. TheK _m for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively. The enzyme is noncompetively inhibited by inorganic monophosphate (K _i=2.85 mmol/L) and by Cu²⁺, Zn²⁺, Hg²⁺, Sn²⁺, Cd²⁺ ions and strongly by F⁻ ones; it is activated by Ca²⁺, Mg²⁺ and Mn²⁺ ions. The substrate specificity of phytase is broad with the highest affinity to calcium phytate.     

Soil particle accumulation in termite (Macrotermes bellicosus) mounds and the implications for soil particle dynamics in a tropical savanna Ultisol

This study investigated the influence of mound-building termites on soil particle dynamics on the land surface and in soil-forming processes by examining the amount of soil particles in mound structures of Macrotermes bellicosus in a highly weathered Ultisol of tropical savanna. Soil particle turnover via the mounds was estimated using particle stock data and soil turnover data from previous studies. A 4-ha study plot with six mounds of relatively uniform shape and size was investigated. Soil mass constituting the mounds was 6,166 ± 1,581 kg mound⁻¹ within which the mound wall and nest body accounted for 5,002 ± 1,289 and 1,164 ± 293 kg, respectively. The mound wall contained a significantly larger amount of clay (252 ± 9.97 g kg⁻¹) balanced with a lower sand content (676 ± 26.5 g kg⁻¹) than in the adjacent surface (Ap1) horizon, (46.4 ± 12.8 g clay kg⁻¹; 866 ± 83.2 g sand kg⁻¹); the nest body had much higher clay content (559 ± 51.0 g kg⁻¹) but less sand (285 ± 79.2 g kg⁻¹) than the mound wall. As a result, the mounds of M. bellicosus accumulated clay of 2,874 ± 781 kg ha⁻¹ (corresponding to 2.52% of clay stock in the Ap1 horizon) along with an estimated clay turnover rate of 169 kg ha⁻¹ year⁻¹. These findings suggest a positive feedback effect from termite mound-building activity on soil particle dynamics in tropical savanna ecosystems: M. bellicosus preferentially use subsoil material for mound construction, resulting in relocation of illuvial clay in the subsoil to the land surface where clay eluviation from the surface soil and its illuviation in the subsoil are major soil-forming processes.