Digestion Of Plant Material Research Articles

Microbial Fermentation in Juvenile and Adult Pond Slider Turtles, Trachemys scripta

Abstract Herbivorous reptiles use microbial gut symbionts to digest plant material. These symbionts ferment cell wall components, producing short-chain fatty acids (SCFA), which the host uses as an energy source. In reptiles, fermentation usually occurs in the large intestine; however, the freshwater Florida Red-Bellied Cooter, Pseudemys nelsoni, has both small and large intestine fermentation. Although small intestine fermentation has not been found in other chelonians, no other freshwater turtles have been examined. We measured SCFA concentrations in the digestive tracts of juvenile and adult Pond Sliders, Trachemys scripta. Like many other turtles, T. scripta experiences an ontogenetic diet shift from carnivory to herbivory, and it is unknown whether juveniles can digest plant material. We determined whether (1) this species harbors small intestine fermentation, (2) juveniles possess SCFA concentrations comparable to other herbivorous reptiles, and (3) a change in relative fermentation chamber capacity...

Pushing the edge: extended activity as an alternative to risky body temperatures in a herbivorous teiid lizard (Cnemidophorus murinus: Squamata)

Summary A combination field and laboratory study tested the hypothesis that a herbivorous lizard, Cnemidophorus murinus, extends activity at high body temperatures to digest plant material. Body temperatures (Tb) of active lizards averaged 37·2 °C and were no higher than those of insect‐eating Cnemidophorus. Near constant Tb was maintained by behavioural means even though habitat temperatures varied greatly. Field‐based behavioural data show that C. murinus extend activity by shifting among microhabitats to increase duration of time available at high Tb for digestion. Laboratory studies revealed large variation in selected temperatures (Tsel), but the overall average was 35·9 °C, which is lower than field Tb. By carefully selecting microhabitats and extending activity, C. murinus maintains constant high body temperatures to digest plant material without risking potentially lethal overheating.

Herbivory Imposes Constraints on Voluntary Hypothermia in Lizards

Animals that are unable to meet their metabolic needs should adopt compensatory behaviors to mitigate their energetic deficits. Voluntary hypothermia is one such energy-saving strategy. Physiological constraints associated with the digestion of plant tissues, however, may limit the thermoregulatory options of herbivorous ectotherms. In this laboratory study, selected body temperatures of Desert Iguanas (Dipsosaurus dorsalis), subjected to two different rations of a carnivorous and a herbivorous diet, were measured in a laboratory thermal gradient. Lizards fed a carnivorous diet and rationed to 50% of their daily energetic requirement selected significantly lower body temperatures than did lizards in the other diet/ration treatments. Lizards fed a herbivorous diet selected higher body temperatures than did the same lizards fed a carnivorous diet regardless of ration. Our study indicates that selected temperatures are influenced by both diet quality and quantity. These results support the hypothesis that herbivorous reptiles require elevated body temperatures to digest plant tissues. Our findings do not fully support the behavioral hypothermia hypothesis, which posits that diurnal lizards will seek cool temperatures at night to reduce their energy expenditure. Thermoregulatory strategies of Desert Iguanas and other herbivorous lizards may be influenced by the need to balance high body temperatures to digest plant material with the high metabolic costs of maintaining high temperatures.

Behavioral preferences for bamboo in a pair of captive giant pandas (Ailuropoda melanoleuca)

Giant pandas (Ailuropoda melanoleuca) are members of the order Carnivora. Their diet, however, consists almost entirely of bamboo. Herbivores are under strong pressure to be selective in what they eat because of the low digestibility of plant material. The purpose of this study was to determine whether two captive giant pandas exhibited preferences among three species of bamboo: black (Phyllostachys nigra), bissetii (Phyllostachys bissetii), and arrow (Pseudosasa japonica). Eighteen classic choice trials were conducted in which the species were randomly paired and placed in one of two predetermined locations in the giant pandas’ indoor enclosures. The pandas preferred leaves to culms or branches for each bamboo species. In the first hour of exposure, both pandas exhibited a preference for arrow bamboo. A comparison of total bamboo consumption over the course of the night indicated a strong preference for arrow bamboo by the male. The female exhibited equal preference for both arrow and bissetii over black bamboo. Further examination of feeding behavior determined that the pandas processed arrow bamboo behaviorally more efficiently than the other two species. This is the first study to experimentally assess bamboo preferences in giant pandas, and may have implications for husbandry and management programs as well as strategies for in situ conservation. Zoo Biol 24:177–183, 2005. � c 2005 Wiley-Liss, Inc.

Rapid Nitric Acid Digestion of Plant Material with an Open-Vessel Microwave System

Digestion of plant materials in hot (130–140°C) concentrated nitric acid (HNO3) is a common procedure for assessing their nutrient contents. In the conventional HNO3 digestion, desired temperatures are achieved through controlled electrical heating, and digestion occurs within Pyrex test tubes. The main limitations associated with the conventional digestion method may include (1) high labor requirement for monitoring acid levels in the tubes and digest solution transfer at the end of digestion and (2) relatively high background levels, in particular, of trace elements (e.g., Cu, B, Mn, etc.) resulting from the glass matrix or/and repeated use of digestion tubes. The availability of industrial microwave technology provides opportunities for developing improved digestion systems that overcome the above constraints while routinely processing large batches of plant samples. The present article describes a simple, reliable, and rapid digestion procedure for HNO3 with hydrogen peroxide (H2O2) digestion of plant material by using an open-vessel (50 mL polypropylene tubes with caps in which a 3.2 mm diameter ventilation hole is drilled in the center), microwave-digestion system (CEM Mars 5, manufactured by CEM Corp., USA), followed by elemental quantification using an ICP-AES. The proposed method consists of two stages: (1) the predigested (overnight) sample and HNO3 mix is heated at 75°C for 10 min, followed by 109°C for 15 min; (2) after cooling for 10 min, 1 mL of H2O2 is added to each vessel through the ventilation hole and the sample mix is heated at 109°C for a further 15 min. The analytical results were statistically analyzed by using linear regression, linear correlation, and two independent means tests to determine analytical precision and accuracy of the proposed digestion method. The results have demonstrated that this method is suitable for precise and accurate determination of macronutrients calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), sulfur (S), and micronutrients boron (B), copper (Cu), manganese (Mn), and zinc (Zn) in plant materials. The analytical variability (coefficient of variation) was mostly less than 5%, apart from that of iron (Fe) (9%). There were no significant (P ≤ 0.05) differences between the measured and certified concentrations of both macro- and micronutrients in the ASPAC and NIST1515 standard reference materials (SRM), except for Fe in NIST1515 SRM. The recovery rate of Fe in the digest solution varies with plant types, for cereal samples, higher than 90%, but for dicot species (e.g., NIST apple leaves) the recovery rate was as low as 70%. One of the important advantages of this method was the consistently (across samples and different batches) low background reading (mostly under detection limits of the ICP-AES used, for example, the concentrations of B in the blank digests were consistently less than 5 µg/L). The adoption of the present digestion method may result in time saving due to short turn-around time (less than 60 min per 50 samples) and cost saving due to low labor requirement, low acid consumption, and low-cost digestion vessels.

Near Infrared Reflectance Spectroscopy for Quantification of Crop Residue, Green Manure and Catch Crop C and N Fractions Governing Decomposition Dynamics in Soil

For environmental, as well as agronomic reasons, the turnover of carbon (C) and nitrogen (N) from crop residues, catch crops and green manures incorporated into agricultural soils has attracted much attention. It has previously been found that the C and N content in fractions from stepwise chemical digestion of plant materials constitutes an adequate basis for describing a priori the degradability of both C and N in soil. However, the analyses involved are costly and, therefore, unlikely to be used routinely. The aim of the present work was to develop near infrared (NIR) calibrations for C and N fractions governing decomposition dynamics. Within the five Nordic countries, we sampled a uniquely broad-ranged collection representing most of the fresh and mature plant materials that may be incorporated into agricultural soils from temperate regions. The specific objectives of the current study were (1) to produce NIR calibrations with data on C and N in fractions obtained by stepwise chemical digestion (SCD); (2) to validate these calibrations on independent plant samples and (3) to compare the precision and robustness of these broad-based calibrations with calibrations derived from materials within a narrower quality range. According to an internal validation set, plant N, soluble N, cellulose C, holocellulose (hemicellulose + cellulose) C, soluble C and neutral detergent fibre (NDF) dry matter were the parameters best predicted ( r2 = 0.97, 0.95, 0.94, 0.91, 0.90 and 0.94, respectively). However, the calibrations for soluble C and NDF were regarded as unstable, as their validation statistics were substantially poorer than the calibration statistics. The calibrations for all structural N fractions and lignin C were considered poor ( r2 = 0.47–0.70). By comparing our broad-based calibrations for plant N and NDF with similar calibrations for a sample set representing a commercial forage database, it was evident that the broad-based calibrations predicted a narrow-based sample set better than vice versa. For plant N, the residual mean squared error of prediction ( RMSEP), when testing the broad-based calibration with the narrow-based validation set, was substantially smaller than the RMSEP obtained when validating the broad-based calibration internally (1.8 vs 2.7 mg Ng−1 dry matter). Overall, the calibrations that performed best were those concerning the parameters most strongly influencing C and N mineralisation from plant materials.

Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready ® canola in the intestinal, ruminal, and fecal contents of sheep

The stability of transgenic DNA encoding the synthetic cp4 epsps protein in a diet containing Roundup Ready (RR) ® canola meal was determined in duodenal fluid (DF) batch cultures from sheep. A real-time TaqMan ® PCR assay was designed to quantify the degradation of cp4 epsps DNA during incubation in DF at pH 5 or 7. The copy number of cp4 epsps DNA in the diet declined more rapidly ( P < 0.05) in DF at pH 5 as compared to pH 7. The decrease was attributed mainly to microbial activity at pH 7 and perhaps to plant endogenous enzymes at pH 5. The 62-bp fragment of cp4 epsps DNA detected by real-time PCR reached a maximum of approximately 1600 copies in the aqueous phase of DF at pH 7, whereas less than 20 copies were detected during incubations in DF at pH 5. A 1363-bp sequence of cp4 epsps DNA was never detected in the aqueous fraction of DF. Additionally, genomic DNA isolated from RR ® canola seed was used to test the persistence of fragments of free DNA in DF at pH 3.2, 5, and 7, as well as in ruminal fluid and feces. Primers spanning the cp4 epsps DNA coding region amplified sequences ranging in size from 300 to 1363 bp. Free transgenic DNA was least stable in DF at pH 7 where fragments less than 527 bp were detected for up to 2 min and fragments as large as 1363 bp were detected for 0.5 min. This study shows that digestion of plant material and release of transgenic DNA can occur in the ovine small intestine. However, free DNA is rapidly degraded at neutral pH in DF, thus reducing the likelihood that intact transgenic DNA would be available for absorption through the Peyer’s Patches in the distal ileum.

Spectrophotometric flow-injection determination of zinc in plant digests based on a spot test

A spot test was implemented in a flow-injection system for the spectrophotometric determination of zinc in digests of plant materials. It is based on the influence of Zn2+ on the oxidation rate of 1-naphthylethylenediamine (NED) by hexacyanoferrate(III) under acidic conditions. In order to control the precipitate formation and to maintain the resulting suspension, a micellar medium was established by adding Triton X-100. The proposed system handles about 65 samples per hour, meaning 72μg NED and 9.0mg K3[Fe(CN)6] per determination. Baseline drift is usually <0.01 absorbance per hour and the analytical signals for 0.5–2.5mgl−1 Zn range within ca. 0.07–0.45 absorbance. Linearity of the analytical curve is fair (r>0.999, n=6) and detection limit was estimated as 0.2mgl−1 Zn. Results are precise (R.S.D.<1%, n=10) and in agreement with flame atomic absorption spectrometry and with certified values of standard reference materials.

Population dynamics of rumen microbes using modern techniques in rumen enhanced solid incubation

The microbial ecology of the rumen is very complex. Different species of bacteria, protozoa, and fungi are involved in digestion of plant material in ruminants. In spite of complicated interrelationships among the various groups of microorganisms in the rumen ecosystem, Bacteria and Archaea are believed to play a major role because of their numerical predominance and metabolic diversity. In this work we are presenting the results for microbial population dynamics of rumen microbes during two-stage anaerobic digestion of grass. The reactors were inoculated with fresh rumen content. Fluorescent in situ hybridization, confocal laser scanning microscopy and epifluorescence microscopy were employed for microbial investigation. It was observed that Bacteria dominated in the hydrolytic reactor (1st stage) whereas Archaea were predominant in the methanogenic reactor (2nd stage). The stability of the methanogenic reactor was result of the dominance of Methanosaeta species (mainly the filamentous type).

Effect of acid concentration on closed-vessel microwave-assisted digestion of plant materials

The efficiency of microwave-assisted acid digestion of plants using different concentrations of nitric acid (2.0, 3.0, 5.0, 7.0 and 14 mol l −1) with hydrogen peroxide (30% v/v) was evaluated by measuring the residual carbon content (RCC) using inductively coupled plasma optical emission spectrometry (ICP-OES) with axial viewing. Certified reference materials were used for evaluating the accuracy attained when 2 mol l −1 HNO 3 was employed for digestion. Under all experimental conditions RCC values were always lower than 13% w/v, and even the highest concentration did not cause any interference with element recovery. It seems that the high pressure reached for closed-vessel operation improved the oxidative action of nitric acid due to consequent temperature increase, even when this reagent was not used at high concentrations. According to acid–base titration data, residual acid in the digestates varied from 1.2 to 4.0 mol l −1, depending on the acid concentration initially added. It can be concluded that for plant materials, microwave-assisted acid digestion can be carried out under mild conditions, which implies that digestates do not need extensive dilution before introduction by pneumatic nebulization to ICP-OES. An additional advantage is the lower amount of residue generated when working with less concentrated acid solutions.

The microbial flora of the different gut regions of the variegated grasshopper Zonocerus variegatus (L) (Orthoptera :Pyrgomorphidae)

The microbial flora of the gut regions and gut contents of the variegated grasshopper Zonocerus variegatus instars was studied using the pour plate technique. The gut sections (Fore-, mid-, and hind-gut) harboured a variety organisms mainly bacteria, fungi and mould. Yeasts species isolated were Candida, Saccharomyces and Pichia spp. Aspergilus, Penicillium, Fusarium and Rhizopus spp. Both gram positive and gram negative bacteria were isolated. The gram negative bacteria isolated were mainly rods and members of the family Enterobacteriaceae including Proteus, Alcaligenes, Escherichia and Serratia. These species were more widely distributed in and more frequently isolated from the gut regions and their contents than the gram positive bacteria which were represented by Sreptococcus, Lactobacillus and Staphylococcus species. Gram positive bacteria were isolated from the gut extracts of the 3rd and 4th instars but were found in some of the gut regions of the 6th and adult instars. Mould population decreased gradually as the grasshopper was moulting from the 3rd to adult instars. Ranking according to microbial load was adult> 6th >4th >3rd, a reflection of their gut capacity since the microbes were part of the diet. The number and types of microbes in the gut regions of these instars are similar but significantly different in terms of total microbial load (P>=0.05). The results of this study could provide a lead into the proper understanding of the physiological processes involved in the digestion of plant materials by the insect. Keywords: Z.variegatus, microbial, bacteria, mould, yeast ,gut. (Global Journal of Pure and Applied Sciences: 2002 8(4): 447-454)

Boron determination: A comparison between ICP-AES and 3He+-activation

The analytical potential of ICP-AES (ICP) and 3He+-activation for the determination of boron was examined. The paper compares ICP-levels of B, obtained from aqueous solutions containing digested plant material, with minimum detectable limits attained in solid matrices that were subjected to prompt gamma-ray analysis using 2 MeV 3He+ ions. The analytically useful gamma-rays originated mainly from (3He,p) and (3He,a) reactions on 11B and Coulomb excitation of 10B. The numerical capabilities of the two techniques for such measurements are discussed, and detailed methodologies and possible difficulties are presented.

Transition metal analysis: A comparison between ICP-AES and deuteron activation

The analytical capabilities of ICP-AES (ICP) and deuteron activation (associated with prompt gamma-rays and delayed X-rays) for the determination of certain transition metals, were investigated. The sensitivities of the three methods are given, and the general applicability of each technique is evaluated. In the case of ICP, numerical data were obtained from aqueous solutions containing digested plant material. These experimental results were assessed against minimum detectable limits attained in relevant solid matrices that were subjected to prompt gamma-ray and delayed X-ray spectrometry using 5 MeV 2H+ ions. The analytically useful lines originated mainly from (d,p) and (d,n) reactions. The potential of the three techniques for routine analysis is discussed and detailed methodologies are presented.

Spectrophotometric determination of total nitrogen in plant materials using a flow-injection system with an AgCl (s) reactor

An improved flow-injection system with an AgCl (s) reactor is proposed for the spectrophotometric determination of total nitrogen in Kjeldahl digests of plant materials. After sample injection, the established sample plug is alkalinised and allowed to flow through the mini-column where Ag + ions are displaced as the soluble [Ag(NH 3) 2] + complex. Bromopyrogallol red and o-phenanthroline are then added and a coloured ternary complex is formed under neutral conditions. The system is very stable, and baseline drift is usually <0.01 absorbance after 4 h operation periods. Precise results (R.S.D.<2%) are obtained at a rate of 100 h −1. The analytical curve is linear up to 6.00% (w/w) N (dry basis), regression coefficient has been estimated as >0.999 ( n=6) and detection limit is 0.2% (w/w) N. Accuracy was confirmed by running standard reference materials and typical samples already assayed by conductimetry.

Analytical methods and quality assurance

Microwave‐oven digestion of plant materials for the determination of plant‐nutrient concentrations is an aggressive and effective dissolution procedure. Commercial ovens are expensive, however, and ovens designed for home use may provide an inexpensive alternative. We evaluated three microwave ovens: Panasonic NN5510, Sharp Carousel II, and a commercial unit CEM MDS‐2000; and two Teflon vessels: Savillex 571R2 and CEM 327011 for their ability to digest batches of six or twelve plant samples and to compare their analyses with those from conventional block‐heater digestions. For the microwave‐oven digestions, we placed in each vessel 0.1 g National Institute of Standards and Technology (NIST) standard reference material (SRM) 1547 peach leaves, 1.0 mL conc. HNO3, and 0.6 mL 30% w/v H2O2. The digestates were stirred, soaked for 15 min., heated in a rotating carousel as described below, cooled 15 min, added 8.4 mL deionized‐distilled water to make 10 mL volume, filtered, and analyzed with ICP‐AES. The block‐heater method used a nitric/peroxide digestion, unsealed glass tubes, 0.5 g samples and a 25 mL dilution. There were no differences between Ca, K, S, P, Zn, Cu, and Fe concentrations of NIST SRM 1547 when samples were digested using commercial or domestic microwave ovens (P>0.05) and recoveries ranged from 84 to 95% of NIST certified values. Recoveries were greater from the block heater digestions than with the microwave digestions from four of the seven elements analyzed (P<0.05). Variability was greater from the microwave oven digestions than the block heater. We recommend home‐use microwave ovens used for the digestion of plant materials be set to 300 to 350 watts and heated for ≥30 min. for six‐vessel digestions and 45 min. for twelve‐vessel batches. Our procedure produces low vessel pressures (≥410 kPa) with little chance of relief valves rupturing and the loss of samples. The domestic ovens may be useful for those requiring occasional analyses or those who analyze samples of similar nature negating the need for frequent method modification. Laboratory personnel must adhere to our recommended sample sizes and reagent volumes to prevent explosions.

Turbidimetric determination of orthophosphate in digested plant material by flow-injection analysis

A simple, fast, and low-cost flow-injection system is proposed for the determination of orthophosphate in digested plant material. The determination is based on the precipitation reaction of orthophosphate with 0.05 mol L−1 zinc solution in 0.05 mol L−1 triethanolamine buffer (TEA/TEAH+, pH 6.0) solution containing 0.025% (w/v) poly-(vinylalcohol). The zinc orthophosphate suspension produced was directly measured at 410 nm. Under the best experimental conditions, orthophosphate was determinated in the concentration range from 5 to 60 mg P L−1, and the relative standard deviations were less than 1.6% for 10 and 20 mg P L−1 solutions (n = 9). The analytical frequency was 180 h−1. The results obtained using the proposed FIA procedure and those obtained using a standard spectrophotometric procedure agree at the 95% confidence level. © 2000 John Wiley & Sons, Inc. Lab Robotics and Automation 12:236–240, 2000

Use of zincon to study uptake and accumulation of zinc by zinc tolerant and hyperaccumulating plants

A quick and cheap method to digest plant material using 2% sulphosalicylic acid and to analyze the extract for zinc (Zn) using the colorimetric agent zincon is described. It is shown that the recovery of Zn is excellent, within 90% of that determined by nitric acid digestion and atomic absorption spectrophotometry(AAS). The sensitivity is similar to the AAS. Its usefulness for investigating Zn uptake by the Zn hyperaccumulator Cardaminopsis halleri is investigated. Almost 50% of the variation between samples is due to between‐plant variance, with between‐leaf variation making up a further 37%. Some of the between‐leaf variance is due to age: young leaves contain less Zn than old leaves. In roots, root tips contain about twice as much Zn as root bases.

Viruses and virus-like particles identified in ostrich gut contents.

Samples of either gut content from ostriches showing symptoms of enteritis, or allantoic fluid of eggs inoculated with ostrich isolates, were examined for the presence of viral agents by direct negative-contrast electron microscopy. Only a few virus types could be identified with certainty, namely a circovirus (1 sample), a coronavirus (1 sample), a member of either the toga- or bunyaviridae (1 sample), enterovirus (16 samples) and paramyxovirus (26 samples). A large number of samples contained structures resembling myxovirus particles that were interpreted as fringed membranous particles of non-viral origin. An unusual observation of probable single-strand nucleocapsid helices, possibly originating from digested plant material and which were identified in a number of small intestine samples, is reported. This is the 1st report of a spectrum of viruses and virus-like particles occurring in enteric samples from ostriches in South Africa. The low incidence and variety of viruses reported here contribute to the multifactorial origin and complexity of enteric disease in ostriches as well as in other birds and mammalian species.

Multicommutation in flow analysis. Part 6. Binary sampling for wide concentration range turbidimetric determination of sulphate in plant digests

An automatic continuous flow procedure for turbidimetric determination of sulphate in acidic digests of plant materials, employing a flow network based on multicommutation and binary sampling is described. The method is based on the precipitation of sulphate anions with barium ions followed by photometric monitoring of the resulting suspension. A single pumping channel was used, handling both samples and reagents by means of computer-controlled three-way solenoid valves. In order to extend the determination concentration range up to 500 mg l −1 SO 2− 4 without any modification in the structure of the flow network two analytical curves are generated. The first curve comprized the concentration range from 10–150 mg l −1 SO 2− 4 and the second one, that from 100–500 mg l −1 SO 2− 4. Results presented good agreement at the 95% confidence level with those obtained by the usual FIA system. Other relevant features such as a throughput of 100 determinations per hour, a relative standard deviation of 2% ( n=11) and a reagent consumption of 7 mg BaCl 2.2H 2O plus 1.5 mg EDTA per determination were also achieved.

The distribution and function of mucous cells and their secretions in the alimentary tract of Arrhamphus sclerolepis krefftii

The mucosa of the mouth, pharynx, oesophagus and rectum of Arrhamphus sclerolepis krefftii contain saccular mucous cells and the lining of the intestinal mucosa contains goblet mucous cells. Saccular mucous cells in the buccal epithelium are present in relatively low densities and contain acidic and neutral glycoprotein‐secreting cells in an approximately 1:1 ratio. The saccular mucous cells in the mucosa of the pharynx, oesophagus and rectum are abundant and contain acidic glycoprotein which consists principally of sialomucin with traces of sulphomucin distributed around the periphery of the mucous vacuoles. Goblet cells in the intestinal mucosa contain neutral glycoprotein. Mechanically digested plant material within the lumen of the gut is bound by a sheath of acidic glycoprotein which is in contact with the intestinal mucosa. From these observations and with information on the known properties of acidic glycoproteins, a novel mechanism for the involvement of mucus in the extraction of nutrients from plant material mechanically digested by fish is proposed.