Isolation and Characterization of Novel Yeasts from Rumen Fluids for Potential Use as Additives for Ruminant Feeding

We hypothesized that rumen fluid with yeast producing cellulase enzyme can occur and also produces a high biomass compared to S. cerevisiae. Therefore, the aim of this study was to screen and isolate yeast from rumen fluids with an experimental design method. We optimized a fermentation medium containing sugarcane molasses as a carbon source and urea as a nitrogen source to measure the efficiency of biomass production and cellulase activity. Two fistulated-crossbred Holstein Friesian steers, averaging 350 ± 20 kg body weight, were used to screen and isolate ruminal yeast. The two experiments were designed. A 12 × 3 × 3 factorial was used in a completely randomized design to determine biomass and carboxymethyl cellulase activity. Factor A was isolated yeasts and S. cerevisiae. Factor B was sugarcane molasses (M) concentration. Factor C was urea (U) concentration. Potential yeast was selected for identified and analyzed as a 4 × 3 factorial use in a completely randomized design including. Factor A was incubation times. Factor B was isolated yeast strains including code H-KKU20 (as P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (as Galactomyces sp.-KKU20). Isolation was under aerobic conditions, resulting in a total of 11 different colonies. We noted two appearances of colonies including, asymmetric colonies of isolated yeast (indicated as A, B, C, E, and J) and ovoid colonies (coded as D, F, G, H, I, and K). The highest biomass was observed in three yeasts including codes H, I, and C-KKU20 when inoculated in 25% molasses with 1% urea (M25+U1) (p <0.01). The highest CMCase activity was observed in yeast code H-KKU20 when inoculated in all media solutions (p <0.01). Ruminal yeasts strains H-KKU20, I-KKU20, and C-KKU20 were selected for their ability to produce biomass and their CMCase enzyme synthesis. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20, while C-KKU20 was identified as Galactomyces sp.-KKU20. Two strains provided maximum cell growth: P. kudriavzevii-KKU20 (9.78 and 10.02 Log cell/ml) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/ml) at 60 and 72 h of incubation time, respectively. The highest ethanol production was observed in S. cerevisiae: 76.4, 77.8, 78.5, and 78.6 g/L at 36, 48, 60, and 72 h of incubation time, respectively (p <0.01). The P. kudriavzevii-KKU20 yielded the least reducing sugar about 30.6 and 29.8 g/L at 60 and 72 h of incubation time, respectively. It could be concluded that screening and isolating yeast from rumen fluids resulted in 11 different characteristics of yeasts. The first novel yeasts discovered in the rumen fluid of cattle were Pichia kudriavzevii-KKU20, Candida tropicalis-KKU20, and Galactomyces sp.- KKU20. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of biomass production, cellulase enzyme activity, and cell number.

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Isolation and Characterization of Yeasts from Rumen Fluids for Potential Use as Additives in Ruminant Feeding

Veterinary Sciences ◽

10.3390/vetsci8030052 ◽

2021 ◽

Vol 8 (3) ◽

pp. 52

Author(s):

Chanon Suntara ◽

Anusorn Cherdthong ◽

Metha Wanapat ◽

Suthipong Uriyapongson ◽

Vichai Leelavatcharamas ◽

...

Keyword(s):

Incubation Time ◽

Cellulase Activity ◽

The Other ◽

Pichia Kudriavzevii ◽

Factor B ◽

Yeast Strains ◽

Randomized Design ◽

Holstein Friesian ◽

The Media ◽

Completely Randomized Design

Saccharomyces cerevisiae is a yeast strain often used to improve the feed quality of ruminants. However, S. cerevisiae has limited capacity to provide biomass when inoculated with carbon sources and a low ability to produce cellulase enzymes. Here, we hypothesized that yeast in the rumen produces a large amount of biomass and could release cellulase enzymes to break down fiber content. Therefore, the aim of this study was to screen, isolate and identify yeast from the rumen fluids of Holstein Friesian steers and measure the efficiency of biomass production and cellulase activity. A fermentation medium containing sugarcane molasses as a carbon source and urea as a nitrogen source was optimized. Two fistulated–crossbred Holstein Friesian steers averaging 350 ± 20 kg body weight were used to screen and isolate the ruminal yeast. Two experiments were designed: First, a 12 × 3 × 3 factorial was used in a completely randomized design to determine biomass and carboxymethyl cellulase activity. Factor A was the isolated yeast and S. cerevisiae. Factor B was sugarcane molasses (M) concentration. Factor C was urea (U) concentration. In the second experiment, potential yeasts were selected, identified, and analyzed for 7 × 4 factorial use in a completely randomized design. Factor A was the incubation times. Factor B was the isolated yeast strains, including codes H-Khon Kaen University (KKU) 20 (as P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (as Galactomyces sp.-KKU20). Isolation was imposed under aerobic conditions, resulting in a total of 11 different colonies. Two appearances of colonies including asymmetric colonies of isolated yeast (indicated as A, B, C, E, and J) and ovoid colonies (coded as D, F, G, H, I, and K) were noted. Isolated yeast from the rumen capable of providing a high amount of biomass when inoculant consisted of the molasses 15% + urea 3% (M15 + U3), molasses 25% + urea 1% (M25 + U1), molasses 25% + urea 3% (M25 + U3), and molasses 25% + urea 5% (M25 + U5) when compared to the other media solution (p < 0.01). In addition, 11 isolated biomass-producing yeasts were found in the media solution of M25 + U1. There were 4 isolates cellulase producing yeasts discovered in the media solution of M25 + U1 and M25 + U5 whereas molasses 5% + urea 1% (M5 + U1), molasses 5% + urea 3% (M5 + U3), molasses 5% + urea 5% (M5 + U5), molasses 15% + urea 1% (M15 + U1), molasses 15% + urea 3% (M5 + U3), and M25 + U3 were found with 2, 3, 1, 2, 1, and 2 isolates, respectively. Ruminal yeast strains H-KKU20, I-KKU20, and C-KKU20 were selected for their ability to produce biomass. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20 while C-KKU20 was identified as Galactomyces sp.-KKU20. Two strains provided maximum cell growth: P. kudriavzevii-KKU20 (9.78 and 10.02 Log cell/mL) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/mL) at 60 and 72 h of incubation time, respectively. The highest ethanol production was observed in S. cerevisiae at 76.4, 77.8, 78.5, and 78.6 g/L at 36, 48, 60, and 72 h of incubation time, respectively (p < 0.01). The P. kudriavzevii-KKU20 yielded the least reducing sugar at about 30.6 and 29.8 g/L at 60 and 72 h of incubation time, respectively. The screening and isolation of yeasts from rumen fluids resulted in 11 different yeasts being obtained. The potential yeasts discovered in the rumen fluid of cattle were Pichia kudriavzevii-KKU20, Candida tropicalis-KKU20, and Galactomyces sp.-KKU20. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of biomass production, cellulase enzyme activity, and cell number.

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Comparison on biomass production of three fodder germplasms

Bangladesh Journal of Animal Science ◽

10.3329/bjas.v42i1.15772 ◽

2013 ◽

Vol 42 (1) ◽

pp. 35-39 ◽

Cited By ~ 3

Author(s):

AR Kanak ◽

MJ Khan ◽

MR Debi ◽

ZH Khandakar ◽

MK Pikar

Keyword(s):

Organic Matter ◽

Plant Height ◽

Biomass Production ◽

Crude Protein ◽

Biomass Yield ◽

Fresh Biomass ◽

Randomized Design ◽

Dry Biomass ◽

Completely Randomized Design ◽

Brachiaria Mutica

The experiment was conducted to study the comparison on biomass production of fodder germplasm. Para (Brachiaria mutica Stapf.), German (Echinochloa crusgalli L.) and Dhal (Hymenachne pseudointerrupta C. Muell) grasses were cultivated in a completely randomized design (CRD). The whole area was divided into nine plots. The area of each unit plot was 6 m x 6 m. Number of cuttings were 16,000 /hectare where Plant to Plant distance was 16 cm and row and raw distance was 16 cm. Equal amount of organic and chemical fertilizer were applied in all cutting. The fodders were first harvested after 60 days of planting, second and third after successive 60 days of re-growth. The findings of the study showed that fresh biomass (p<0.01) and dry biomass yield (p<0.05) of three fodder germplasm differed significantly. Crude protein and organic matter yield were significant (p<0.01) only in the second cutting. German grass was showed significantly higher in CP and OM yield (p<0.01) at second cutting than other grasses. However, no significant effect on plant height was observed among three grasses. From the above findings it may be concluded that among the three fodder germplasm, German fodder showed best result in respect of biomass production. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15772 Bang. J. Anim. Sci. 2013. 42 (1): 35-39

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Integrated management of tough lovegrass (Eragrostis plana Nees): associating chemical control tools and plant physiology

Ciência Rural ◽

10.1590/0103-8478cr20200271 ◽

2021 ◽

Vol 51 (2) ◽

Author(s):

Eduardo Avelino Faleiro ◽

Diego Martins Chiapinotto ◽

Fabiane Pinto Lamego ◽

Carlos Eduardo Schaedler ◽

Eduardo Bohrer de Azevedo

Keyword(s):

Solar Radiation ◽

Integrated Management ◽

Dry Mass ◽

Radiation Factor ◽

Factor B ◽

Randomized Design ◽

Completely Randomized Design ◽

Factor C ◽

Resource Conditions ◽

And Control

ABSTRACT: The objective of the study was to evaluate the efficiency of herbicides use in the control of tough lovegrass according to the availability of solar radiation and the presence or absence of flooded. Two experiments were conducted in a completely randomized design, in a 2x2x4 and 2x2x5 factorial scheme, where factor A was equivalent to the environment (natural or reduced radiation); factor B to water condition (with a 2 cm flooded or without); and, factor C the herbicides: cyhalofop butyl (315 g ha-1), glyphosate (1080 g ha-1), cyhalofop butyl + glyphosate (315 g ha-1 + 1080 g ha-1) and control (without application) in the first experiment. In the second experiment, the herbicides used were cyhalofop butyl (315 g ha-1), glyphosate (1080 g ha-1), sethoxydim (184g ha-1), imazethapyr (106 g ha-1) and control (without application). Glyphosate efficiently controls plants of tough lovegrass with four tillers (>90%), being superior to cyhalofop, imazethapyr and sethoxydim; independently, of resource conditions. Reduction in the availability of solar radiation generates less shoot dry mass production from the weed, and improves the control only by imazethapyr and cyhalofop. In general, a flooded condition does not affect tough lovegrass control by herbicides.

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PSVII-14 Feeding carbohydrases to grazing beef cattle under different nutritional strategies on dry season: Ruminal fermentation

Journal of Animal Science ◽

10.1093/jas/skab235.801 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 449-450

Author(s):

Anderson Acosta ◽

Guilherme S Vasconcellos ◽

Alexandre Perdigão ◽

Victor Valério de Carvalho ◽

Tiago S Acedo ◽

...

Keyword(s):

Rumen Fluid ◽

Latin Square ◽

Experimental Period ◽

Dry Season ◽

Ruminal Fermentation ◽

Beta Glucanase ◽

Randomized Design ◽

Nutritional Strategy ◽

Completely Randomized Design ◽

Nutritional Strategies

Abstract The aim with this trial was to evaluate two nutritional plans with the addition of carbohydrases enzyme blend (CEB) on ruminal fermentation of grazing Nellore bulls in dry season. Five rumen cannulated Nellore steers (BW = 350 kg) were used. Animals were randomly assigned to a treatment sequence in a 5×5 Latin square experiment with 15 d-periods (10 d to diet adaptation and 5 d for data collection), following a completely randomized design, being the treatments: mineral-proteic supplementation (Fosbovi® Proteico 35; PS) offered at 0,1% BW (0 or 4.75 g/animal/day of CEB) or mineral-protein-energetic supplementation (Fosbovi® Proteico-Energético 25; PES) offered at 0.3% BW (0, 4.75 or 9.50 g/animal/day of CEB). The CEB was mainly composed by beta-glucanase and xylanase enzymes (Ronozyme® VP and Ronozyme® WX). Both enzymes and supplements were provided by DSM Nutritional Products Brasil S.A. Rumen fluid samples were collected on the last day of each experimental period before (0), and 2, 4, 6, and 8 h after supplements were offered. Data were analyzed using PROC MIXED of SAS. Steers fed PSE had greater (P ≤ 0.041) rumen concentration of acetate, propionate, and butyrate, and lower (P = 0.028) acetate to propionate ratio in comparison with animals fed PS. Rumen acetate concentrations were 61.6 and 58.8 mmol/L and rumen propionate concentrations were 15.2 and 13.7 mmol/L for animals under the PSE and PS, respectively. A quadratic effect (P = 0.048) for carbohydrases enzyme blend was observed in NH3-N ruminal concentration of PES animals, where 4.75 g/day level presented the lower concentrations compared to control (24.13 vs. 32.20 mg/dL). Furthermore, rumen concentrations of acetate (P = 0.002) and propionate (P = 0.021) linearly increased with enzymes inclusion for PSE plan. PSE nutritional strategy positively influence ruminal fermentation of Nellore steers in dry season and its effects can be enhanced with CEB inclusion of 4.75 g/day.

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PENGARUH KONSENTRASI DAN LAMA PERENDAMAN A. pintoi DENGAN URIN TERNAK SAPI TERHADAP PERTUMBUHAN TANAMAN A. pintoi

ZOOTEC ◽

10.35792/zot.37.1.2017.13507 ◽

2016 ◽

Vol 37 (1) ◽

pp. 15

Author(s):

Novalia Lumban Gaol ◽

Ch. L. Kaunang ◽

Rustandi . ◽

F. Dompas

Keyword(s):

Urine Concentration ◽

Plant Roots ◽

Root Weight ◽

Factor B ◽

Soaking Time ◽

Arachis Pintoi ◽

Randomized Design ◽

Cow Urine ◽

Completely Randomized Design ◽

Plant Length

INFLUENCE OF CONCENTRATION AND SOAKING TIME OF A. pintoi IN THE COW URINE ON PLANT GROWTH. Cow urine has an auksin a, auksin b, and IAA (hetero auksin) that can stimulate plant roots. The present study was conducted to determine the influence of concentration and soaking time in the cow urine on Arachis pintoi (A. pinto) growth. This study used Completely Randomized Design (CRD) in Factorial arrangement with 2 factors A and B. Factor A= urine concentration: A1 = urine 75% + water 25% and A2; urine 50% + water 50%; and factor B = soaking time: B1 = 1 hour; B2 = 2 hours; Dan, B3 = 3 hours. Treatments were replicated 4 times. Parameters measured were: leaves amount (sheets), plant length (cm), and root weight (g). Research results showed that mean leaves amount ranged from 41,65 to 46,77 sheets, plant length 35,97-36,67 cm, and root weight 1,95-1,72 g. Utilization of cow urine with a different concentration and soaking time gave a non significant different (P>0.05) on leaves amount, plant length, and root weight. It can be concluded that cow urine concentration and soaking time do not effect leaves amount, plant length, and root weight of Arachis pintoi (A. pinto). Key words: Cow urine concentration, Soaking time, A. pintoi.

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PENGARUH BERBAGAI MACAM RANSUM KOMERSIAL DENGAN MENGGUNAKAN SISTEM KANDANG YANG BERBEDA TERHADAP KUALITAS KARKAS AYAM PEDAGING

ZOOTEC ◽

10.35792/zot.34.0.2014.4795 ◽

2014 ◽

Vol 34 ◽

pp. 76

Author(s):

Anggella T Tombuku ◽

V Rawung ◽

M Montong ◽

Z Poli

Keyword(s):

Abdominal Fat ◽

Carcass Quality ◽

Factor B ◽

Cage System ◽

Randomized Design ◽

Completely Randomized Design

ABSTRACT THE EFFECT OF VARIOUS COMMERCIAL RATIONS COMBINED WITH DIFFERENT CAGE SYSTEM ON CARCASS QUALITY OF BROILERS. The objective of this research was to determine carcass quality of broilers fed with three forms of commercial ration using 2 different cages. Total of 96 day old chicks were used in this study using four animals in each cage. Research was conducted using a completely randomized design divided into 3x2 patterns of treatment with 4 replications. Three forms of commercial ration (Factor A) consisted of A1 = Pokhpand Ration (CP 11 ); A2 = Cargill Ration (Avibro) and A3 = Sierad Ration (BSG 101). Two different cages (Factor B) consisted of B1 = Battery Cage ; B2 = Litter System. The results showed that three forms of commercial ration combined with two different cage systems give the same effect on carcass quality of broilers in terms of carcass percentage and abdominal fat weight. Litter system and animal slaughter at 6 weeks of age reduced the risk of breast blisters. Keywords: Rations, cage, broiler, carcass quality.

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THE EFFECT OF DURATION TIME ON ONGGOK FERMENTATION WITH ENRICHMENT N, S, P BY Trichoderma reesei ON NUTRIEN VALUE

Bulletin of Applied Animal Research ◽

10.36423/baar.v2i1.226 ◽

2020 ◽

Vol 2 (1) ◽

pp. 27-32

Author(s):

Nila Cahyaning Febriyani ◽

Agung - Subrata

Keyword(s):

Data Analysis ◽

Trichoderma Reesei ◽

Incubation Time ◽

Crude Protein ◽

P Element ◽

Nutrient Value ◽

Randomized Design ◽

Nitrogen Free Extract ◽

Completely Randomized Design ◽

Range Test

The aim of this research was to study the effect of incubation time on onggok fermentation with enrichment N, S, P by Trichoderma reesei on nutrient value. The materials of this research were onggok, T. Reesei and N, S, P element. This research used completely randomized design with 4 treatments different incubation time with 5 replication; T0: 0 day, T1: 2 days, T2: 4 days and T3: 6 days. The parameters observed were crude protein, crude fat, crude fiber, and nitrogen free extract of fermented onggok. Data analysis was used by Analysis of Variance (ANOVA) and continued by Duncan’s Multiple Range test if they were significantly different. Results of this research showed that there were haven’t effect (P>0.05) of the treatment on nutrient value. The conclusion of this research was incubation time on onggok fermentation with enrichment N, S, P by Trichoderma reesei have not been able to decrease crude fiber.Keywords : Onggok, fermentation, T.reesei

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Biomass production, B accumulation and Ca/B ratio in Eucalyptus under various conditions of water availability and B doses

CERNE ◽

10.1590/s0104-77602013000200013 ◽

2013 ◽

Vol 19 (2) ◽

pp. 289-295 ◽

Cited By ~ 1

Author(s):

Sílvio Júnio Ramos ◽

Valdemar Faquin ◽

Fabrício William Ávila ◽

Rose Myrian Alves Ferreira ◽

Josinaldo Lopes Araújo

Keyword(s):

Biomass Production ◽

Water Availability ◽

Boron Deficiency ◽

Natural Conditions ◽

Randomized Design ◽

Growth And Nutrition ◽

Dry Biomass ◽

Completely Randomized Design ◽

Red Latosol ◽

Water Tension

Soils destined for forest crops are often limiting with respect to boron (B) and in several eucalyptus crops boron deficiency has been found to be a common occurrence. The objective of this study is to evaluate biomass production, B accumulation and Ca/B ratio in eucalyptus cultivated under different conditions of soil, water availability and doses of B. To that end, an experiment was conducted using a completely randomized design with a 5 x 2 x 2 factorial arrangement consisting of five doses of B (0.00, 0.25, 0.75, 2.25 and 6.25 mg kg-1), two types of soil, namely a Dark Red Latosol (LE) and a Red-Yellow Latosol (LV), and two water tension levels (-0.033 and -0.010 MPa), with four replicates. After plants were grown, determinations were made of Ca and B contents as well as Ca/B ratios in different portions of the plants. Accumulation of B was obtained from the ratio of its contents to dry biomass in each plant portion. It was found that, under natural conditions, both Dark Red Latosol and Red-Yellow Latosol fail to meet the nutritional requirements for B in eucalyptus, potentially being limiting to that species. Overall, soil moisture influenced dry biomass production and accumulation of B only in the case of the more sandy texture soil (Red-Yellow Latosol), denoting that this factor is conditional on soil texture. Ca/B ratio results showed that application of B to the soil without a suitable supply of Ca can lead to an imbalance between these nutrients, with possible implications for plant growth and nutrition.

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Winter Species Promote Phytoremediation of Soil Contaminated with Protox-Inhibiting Herbicides

Planta Daninha ◽

10.1590/s0100-83582019370100020 ◽

2019 ◽

Vol 37 ◽

Cited By ~ 1

Author(s):

C. ALVES ◽

L. GALON ◽

F.L. WINTER ◽

F.J.M. BASSO ◽

C.M. HOLZ ◽

...

Keyword(s):

Experimental Design ◽

Dry Matter ◽

The Other ◽

Bioindicator Species ◽

Factor B ◽

Inorganic Substances ◽

Randomized Design ◽

White Lupine ◽

Completely Randomized Design ◽

Phytoremediator Species

ABSTRACT: Phytoremediation comprises one of the main forms of decontamination of organic and inorganic substances in the soil, being economically viable and with a low environmental impact. The aim of this study was to verify the efficiency of winter plant species in the phytoremediation of soil contaminated with fomesafen and sulfentrazone using cucumber as an indicator species to the presence of residue. The experimental design was a completely randomized design arranged in a 6 x 4 factorial scheme with four replications for each herbicide. Factor A consisted of the phytoremediator species black oats, garden vetch, radish, bird’s-foot trefoil, white lupine, and a treatment without prior cultivation. Factor B, on the other hand, consisted of the doses of fomesafen (0.000, 0.125, 0.250, and 0.500 kg h-1) or sulfentrazone (0.000, 0.300, 0.600 and 1,200 kg ha-1) applied in crop pre-emergence. At 45 days after sowing, the phytoremediator species were cut close to the soil. Subsequently, the bioindicator species of herbicide residues in the soil (cucumber) was sown in the pot. Phytotoxicity of herbicides to cucumber plants was assessed at 7, 14, 21, and 28 days after emergence (DAE). At 28 DAE, leaf area, height, and dry matter were determined in the bioindicator plant. Fomesafen and sulfentrazone doses interfered negatively with the assessed variables of cucumber when cultivated in succession to phytoremediator species. Cucumber phytotoxicity increased for all potential phytoremediator species as fomesafen and sulfentrazone doses increased. Sulfentrazone residues promoted the highest toxic effects on the bioindicator plant when compared to fomesafen. In general, black oats, radish, and white lupine were the species with the highest capacity to phytoremediate soil contaminated with fomesafen and sulfentrazone when applying the dose and twice the recommended doses of the herbicides.

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Production and physiological quality of Pennisetum glaucum after zinc (Zn) application

Australian Journal of Crop Science ◽

10.21475/ajcs.19.13.08.p1385 ◽

2019 ◽

pp. 1223-1231

Author(s):

Rilner Alves Flores ◽

Leonardo Rodrigues Barros ◽

Raiane Ferreira de Miranda ◽

Fernanda Rodrigues dos Santos ◽

Phaulo Maciel Santos ◽

...

Keyword(s):

Photosystem Ii ◽

Biomass Production ◽

Pennisetum Glaucum ◽

Photochemical Efficiency ◽

Nutrient Utilization ◽

Randomized Design ◽

Dry Biomass ◽

Physiological Quality ◽

Completely Randomized Design

The purpose of this study was to evaluate the dry biomass production and the physiological quality of millet (Pennisetum glaucum) based on the application of zinc (Zn) on the soil. The study was conducted in a completely randomized design consisting of five Zn doses: 0; 3; 6; 9 and 12 kg ha-1, with four replications. The following biometrics such as physiological quality, biomass production and nutritional efficiency as for the use of Zn by plants were evaluated. The results showed that doses around 9 kg ha-1 are the ones promoting the highest observed rates. However, higher Zn supply in the soil negatively affected the physiological quality of the crop, as for the evaluated variables, except for the photochemical efficiency of photosystem II. However, Pennisetum glaucum is responsive to the production of dry biomass up to the Zn dose of 9 kg ha-1, without reducing the efficiency of the nutrient utilization by the crop.

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