Methanotrophy-driven accumulation of organic carbon in four paddy soils of Bangladesh

Biological methane oxidation is a crucial process in the global carbon cycle that reduces methane emissions from paddy fields and natural wetlands into the atmosphere.However,soil organic carbon accumulation associated with microbial methane oxidation is poorly understood.Therefore,to investigate methane-derived carbon incorporation into soil organic matter,paddy soils originated from different parent materials(Inceptisol,Entisol,and Alfisol) were collected after rice harvesting from four major rice-producing regions in Bangladesh.Following microcosm incubation with 5%(volume/volume)¹³ CH₄,soil¹³ C-atom abundances significantly increased from background level of 1.08% to 1.88%–2.78%,leading to a net methane-derived accumulation of soil organic carbon ranging from 120 to 307 mg kg^-1.Approximately 23.6%–60.0% of the methane consumed was converted to soil organic carbon during microbial methane oxidation.The phylogeny of¹³ C-labeled pmoA(enconding the alpha subunit of the particulate methane monooxygenase) and 16 S rRNA genes further revealed that canonical α(type II) and γ(type I) Proteobacteria were active methane oxidizers.Members within the Methylobacter-and Methylosarcina-affiliated type Ia lineages dominated active methane-oxidizing communities that were responsible for the majority of methane-derived carbon accumulation in all three paddy soils,while Methylocystis-affiliated type IIa lineage was the key contributor in one paddy soil of Inceptisol origin.These results suggest that methanotroph-mediated synthesis of biomass plays an important role in soil organic matter accumulation.This study thus supports the concept that methanotrophs not only consume the greenhouse gas methane but also serve as a key biotic factor in maintaining soil fertility.     

Fatty acid composition of ruminal bacteria and protozoa,and effect of defaunation on fatty acid profile in the rumen with special reference to conjugated linoleic acid in cattle

Objectives of this study were to compare fatty acid (FA) composition of ruminal bacterial (B) and protozoal (P) cells, and to investigate effect of protozoa on FA profile in the rumen of cattle. Three cows were used to prepare ruminal B and P cells. Four faunated and three defaunated cattle (half‐siblings) were used to study effect of protozoa on ruminal FA profile. Proportions of C16:0 and C18:0 in total fatty acids in B cells were 20.7% and 37.4%, whereas those in P cells were 33.4% and 11.6%, respectively. Proportions of trans‐vaccenic acid (VA) and cis‐9, trans‐11 conjugated linoleic acid (CLA) in B cells were 3.9% and 1.0%, and those in P cells were 5.5% and 1.6%, respectively, being higher in P cells. Proportions of C18:1, C18:2 and C18:3 in P cells were two to three times higher than in B cells. Proportions of unsaturated fatty acids, VA and CLA in B cells of faunated cattle were higher than those of defaunated. VA and CLA in the ruminal fluid of faunated were also 1.6 to 2.5 times higher than those of defaunated. This tendency was similar for cell‐free fraction of ruminal fluid. These results indicate that protozoa contribute greatly in VA and CLA production in the rumen.     

A quantitative study on arginine catabolism by mixed ruminal bacteria,protozoa and their mixture in vitro

The catabolism of arginine (Arg) by mixed rumen bacteria (B), mixed rumen protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system in order to confirm the metabolic pathway of Arg and provide basic information for enzymatic and molecular studies as well as an understanding of the quantitative distribution of metabolites. Rumen microbial suspensions (B, P, and BP) collected from fistulated goats were anaerobically incubated with or without 1 mmol/L Arg at 39°C for 12 h. Arg and other related compounds such as citrulline (Cit), ornithine (Orn), proline (Pro) and 5‐aminovaleric acid (5AV) in both supernatant and hydrolyzates of B, P, and BP suspensions were analyzed by HPLC. The metabolic pathways of Arg in mixed rumen bacteria and mixed rumen protozoa were considered to be as follows: rumen bacteria, Arg → Cit → Orn → Pro → 5AV → VFAs + NH₃; rumen protozoa, Arg → Cit → Orn → Pro → 5AV. The disappearance of Arg (1 mmol/L) was approximately 52.9 and 88.2% in B, 33.9 and 55.6% in P, and 52.8 and 85.2% in BP during 6 and 12 h incubations, respectively. When expressed in units of ‘per gram (g) of microbial nitrogen (MN)’, the net degradation rate of Arg in BP (50.3 µmol/g MN/h) was approximately 46% lower than that of B during a 12 h incubation period. The presence of protozoa tended to inhibit the production of Orn from Cit and the production of 5AV from Pro which were thought to be rate‐limiting steps of Arg metabolism in rumen microorganisms. As a result, protozoa appeared to have a saving effect on Arg metabolism, that is, protozoa protected Arg from wasteful exhaustion in the rumen.     

Quantitative studies of the in vitro production of pipecolic acid by rumen protozoa and its degradation by rumen bacteria

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species‐enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L‐lysine (1.0 mmol/L L‐Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L‐Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L‐Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D‐Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D‐Lys, though at a lower rate (1/3) compared with L‐Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L‐ or D‐Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D‐Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen‐produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.     

Potential of barnyard grass to remediate arsenic‐contaminated soil

The present study investigated the arsenic (As) remediation potential of barnyard grass (Echinochloa crus‐galli L. Beauv. var. formosensis Ohwi), with a special focus on the behavior of As in the soil in comparison with rice (Oryza sativa L. cv. Nipponbare). For both plants, very little growth inhibition was observed in the As‐contaminated soil. The amount of As in the soil was reduced by the plant's uptake and the level of As in the soil water from the rice‐growing pots was remarkably lower than that in the plant‐free soil water. In the soil with the barnyard grass, the amount of As in the soil water was higher than that in the plant‐free soil water, but the amount of As in the soil and the amount of As that was adsorbed on the soil solid were reduced by the plant's uptake. At the highest As level in the soil (100 mg kg^?1), 249.60 and 101.26 µg As pot^?1 were taken up by the rice shoot and barnyard grass shoot, respectively, and total amounts of 1468.65 and 1060.57 µg As pot^?1 were taken up by the barnyard grass and rice seedlings, respectively. At the same As level in the soil, the As concentrations were 14.99 and 37.76 µg g^?1 in the shoot of barnyard grass and rice, respectively, and 486.61 and 339.32 µg g^?1 in the root of barnyard grass and rice, respectively. Barnyard grass took up more As than rice, but the As concentration in the shoot of barnyard grass was lower than that in the shoot of rice. A considerable amount of As was taken up by both barnyard grass and rice, suggesting that the plant species have the potential to remediate As‐contaminated soil.     

Synthesis of citrulline from ornithine by the small intestinal mucosa of cattle

Three segments of cattle small intestine (duodenum, upper jejuno‐ileum and lower jejuno‐ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno‐ileum and lower jejuno‐ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at ?20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.     

Pipecolic acid in rumen fluid and plasma in ruminant animals

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen‐produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.     

Effects of Azolla species on weed emergence in a rice paddy ecosystem

The effects of Azolla pinnata R. Br. on weed emergence were evaluated in terms of plot area coverage by an A. pinnata mat, its biomass production and the amount of weed emergence, using fresh and dry weights, in a rice paddy field experiment. The experiment was conducted following a randomized block design with five combinations of fertilizer and A. pinnata treatments (control, A. pinnata  + superphosphate, A. pinnata  + urea, A. pinnata  + compound fertilizer, A. pinnata  + cow manure). The results revealed that after 18 days of inoculation, all superphosphate (T1) and cow manure (T4)-treated plots were fully covered by the A. pinnata mat. However, coverage of the urea (T2) and compound fertilizer (T3)-treated plots were only 80% and 70%, respectively. The full plot area coverage by the A. pinnata mat and the highest biomass production with superphosphate and cow manure-treated plots were able to completely inhibit two weed species ( Scirpus juncoides Roxb. var. hotarui Ohwi and Monochoria vaginalis Burm. f. Presl var. plantaginea (Roxb.) Solms-Laub) and significantly suppressed four other weeds ( Cyperus serotinus Rottb, Echinochla oryzicola Vasing, and Eclipta prostrata L.). In all the treatments, the fresh weight of weeds significantly reduced to 13, 29, 34, and 9%, respectively, for treatments T1, T2, T3, and T4. The dry weights also were significantly reduced to 10.00, 16.00, 22.00, and 7.26%, respectively, for treatments T1, T2, T3, and T4 over the control. The results revealed that there was a significant correlation among plot area coverage by the A. pinnata mat, its biomass production and weed emergence in a rice paddy field over the control. Azolla pinnata did not have any detrimental effect on the growth of rice plants.     

Effect of brewer's grain on rumen fermentation, milk production and milk composition in lactating dairy cows

Six lactating Holstein cows were divided into two groups (n = 3) and used in a double reversal trial with three periods of 14 days each to evaluate the rumen fermentation, milk production and milk composition of cows fed brewer's grain (BG). The control diets contained 14% chopped Sudangrass hay, 24% corn silage, 18% alfalfa hay cube, 34% concentrate mixture‐1 and 10% concentrate mixture‐2 (wheat bran, soybean meal and cottonseed). In the experimental diet, wet BG replaced the concentrate mixture‐2. The protozoal population, concentration of ammonia‐N and volatile fatty acids in the ruminal fluid did not differ between the control and BG diets. The molar percentage of acetic acid was significantly higher (P < 0.05) with the BG diet at 5 h after feeding. The milk yield, the percentage of protein, lactose, solids not‐fat and somatic cell counts of milk did not differ between the two diets. The percentage of milk fat tended to increase with the BG diet. The BG diet significantly increased the proportions of C18:0 and C18:1 in milk fat (P < 0.01, P < 0.05, respectively) and tended to increase that of conjugated linoleic acid.     

A quantitative study on arginine synthesis from argininosuccinic acid and citrulline by crude enzymes of cattle kidney

In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice‐cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl₂ to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.