Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora

The importance of the gut microorganisms in the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding them with antibiotics. With N. exitiosus, antibiotics which killed both the bacteria and the spirochaetes (ampicillin, kanamycin, chloramphenicol, erythromycin, cephaloridine, tetracycline) reduced the life span of the termite from 250 days to about 13 days, whereas antibiotics which had little effect on the flora (penicillin, methicillin) did not greatly reduce the life span of the termite. The essential role of the spirochaetes in N. exitiosus was shown by feeding metronidazole, or exposing the termites to pure oxygen. Both treatments killed the spirochaetes, but not the bacteria, resulting in a life span for the termite of 13–22 days. Acid fuchsin did not kill the spirochaetes. Fungi were not essential for N. exitiosus. In C. lacteus all treatments, except that with acid fuchsin, killed the protozoa, thereby reducing the life span of the termite from 69 days to 6–29 days.     

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

Background

Termites are highly effective at degrading lignocelluloses, and thus can be used as a model for studying plant cell-wall degradation in biological systems. However, the process of lignin deconstruction and/or degradation in termites is still not well understood.

Methods

We investigated the associated structural modification caused by termites in the lignin biomolecular assembly in softwood tissues crucial for cell-wall degradation. We conducted comparative studies on the termite-digested (i.e. termite feces) and native (control) softwood tissues with the aid of advanced analytical techniques: ¹³C crosspolarization magic angle spinning and nuclear magnetic resonance (CP-MAS-NMR) spectroscopy, flash pyrolysis with gas chromatography mass spectrometry (Py-GC/MS), and Py-GC-MS in the presence of tetramethylammonium hydroxide (Py-TMAH)-GC/MS.

Results

The ¹³C CP/MAS NMR spectroscopic analysis revealed an increased level of guaiacyl-derived (G unit) polymeric framework in the termite-digested softwood (feces), while providing specific evidence of cellulose degradation. The Py-GC/MS data were in agreement with the ¹³C CP/MAS NMR spectroscopic studies, thus indicating dehydroxylation and modification of selective intermonomer side-chain linkages in the lignin in the termite feces. Moreover, Py-TMAH-GC/MS analysis showed significant differences in the product distribution between control and termite feces. This strongly suggests that the structural modification in lignin could be associated with the formation of additional condensed interunit linkages.

Conclusion

Collectively, these data further establish: 1) that the major β-O-4' (β-aryl ether) was conserved, albeit with substructure degeneracy, and 2) that the nature of the resulting polymer in termite feces retained most of its original aromatic moieties (G unit-derived). Overall, these results provide insight into lignin-unlocking mechanisms for understanding plant cell-wall deconstruction, which could be useful in development of new enzymatic pretreatment processes mimicking the termite system for biochemical conversion of lignocellulosic biomass to fuels and chemicals.     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

The function of the pentose phosphate pathway in Phaseolus mungo hypocotyls

The metabolism of d-gluconate-[1-¹⁴C] and -[6-¹⁴C] by segments from etiolated hypocotyls of Phaseolus mungo has been studied. The release of ¹⁴CO₂ from gluconate-[1-¹⁴C] was greater than that from gluconate-[6-¹⁴C] in all parts of hypocotyls examined. Incorporation of the radioactivity from gluconate-[6-¹⁴C] into RNA, lignin and aromatic amino acid fractions was greater in the upper (younger) part of the hypocotyls. Incorporation into sugars was greater in the lower (more mature) parts.     

Proton NMR Spectra of 2,4-Dinitrophenyl Derivatives of Amino Acids

During the decay of wood by the typical white rot fungus Coriolus versicolor, Laccase III was the most abundantly secreted phenol oxidase. In this study, we proposed a possibility of the intermediate degradation steps of polymeric lignin by a purified Laccase III using synthetic [β-¹³C] and [β-¹⁴C]lignin (DHP). When the [β-¹⁴C]DHP was incubated with Laccase III, the water-soluble degradation product formed was about 8% of the applied [β-¹⁴C]DHP. The enzymic attack of Laccase III catalyzed the cleavage of the intermonomer linkages in the side chain structure of the polymeric lignin. In polymeric lignin metabolism by this fungus, laccase activity was closely related to the accumulation of water-soluble degradation products.     

The Selection of an Isolate of the Hyphomycete Fungus,Metarhizium anisopliae,for Control of Termites in Australia

Ninety-three isolates ofMetarhizium anisopliae,mostly derived from a survey of termite material, were screened for activity againstNasutitermes exitiosusandCoptotermes frenchiorC. acinaciformisusing a grooming assay technique. Twenty-six of the most promising isolates were further evaluated by bioassay againstN. exitiosusandC. acinaciformis.All isolates were pathogenic withCoptotermesspp. being more susceptible thanN. exitiosus.A group of nine isolates, chosen for their level of pathogenicity for one or other genus of termites and to represent a genetically diverse group, was finally compared in a minicolony test using termite colonies in 1 liter jars. The isolate, code-named FI-610 (derived from nest-mound material ofC. lacteusin SE New South Wales), was one of the most effective isolates against termites from both of the two colonies tested. This isolate also grew relatively well on agar plates at 36°C. FI-610 was thus selected for field trials and was found to be effective in killing colonies ofC. acinaciformiswhen 10 g (=3 × 10¹¹conidia) or more of conidial powder was blown into the center of the large mound colonies.     

The biosynthesis of C55 polyprenols by a cell-free preparation of Lactobacillus plantarum

A cell-free supernatant of lysates of Lactobacillus plantarum catalyses the synthesis of lipids from [2-¹⁴C]mevalonate. Of the added mevalonate, 7.5% is incorporated into lipids, which were fractionated into five components. About 4% of the radioactivity in these lipids co-chromatographs with compounds shown by mass spectrometry, n.m.r. and i.r. spectroscopy to be C₅₅ polyprenols, and about 2% co-chromatographs with a hexamer. The rest of the radioactivity is in more complex fractions. Analysis by mass spectrometry, n.m.r. and i.r. spectroscopy shows that the major C₅₅ polyprenol is undecaprenol, accompanied by an isomer containing one reduced isoprene unit. A Kuhn–Roth degradation of [¹⁴C]polyprenols indicates that the supernatant catalyses synthesis of these compounds de novo.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)]

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 × 10⁴ cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH₃ was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by ¹⁴CO₂ evolution from [2-¹⁴C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut × h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites

The redox potential and pH of the gut of the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding the insects with redox dyes and pH indicators. For N. exitiosus the E′₀ (pH 7.0) of the foregut was above +200 mV; the midgut was about +100 to +150 mV and the hindgut was in the region of ?20 to +30 mV. For C. lacteus the fore- and midgut were about +30 to +50 mV and the hindgut about ?20 to +20 mV. The colours of the ingested dyes indicated that the gut was aerobic in both termites. The pH of the whole gut ranged from 6.5 to 7.5 for N. exitiosus and 6.0 to 7.0 for C. lacteus.     

Methane Oxidation in Termite Hindguts: Absence of Evidence and Evidence of Absence

A steep oxygen gradient and the presence of methane render the hindgut internal periphery of termites a potential habitat for aerobic methane-oxidizing bacteria. However, methane emissions of various termites increased, if at all, only slightly when termites were exposed to an anoxic (nitrogen) atmosphere, and ¹⁴CH₄ added to the air headspace over live termites was not converted to ¹⁴CO₂. Evidence for the absence of methane oxidation in living termites was corroborated by the failure to detect pmoA, the marker gene for particulate methane monooxygenase, in hindgut DNA extracts of all termites investigated. This adds robustness to our concept of the degradation network in the termite hindgut and eliminates the gut itself as a potential sink of this important greenhouse gas.     

Aromatic compound degradation by the wood-feeding termite Coptotermes formosanus (Shiraki)

Wood-feeding termites (WFT) have proven to be highly efficient for wood digestion. There is evidence to support the hypothesis that there are ligninolytic enzymes existing in the gut of WFT responsible for wood pretreatment toward cellulose utilization. Elucidating the mechanism of biomass pretreatment through lignin modification in termites will help to develop more efficient lignocellulosic biofuel production processes. The in-vivo degradation of aromatic compounds with different substructures, including dyes, lignin model monomers and dimers, and lignin sulfonate, by Coptotermes formosanus (Shiraki) was investigated. The degradation of aromatic compounds was determined using pyrolysis-gas chromatography/mass spectrometry. The results revealed that WFT were able to metabolize the conjugated aromatic structures and that the degradation efficiency is higher in the foregut and midgut regions than in the hindgut. This is the first time that evidence has been provided to show different aromatic compound degradation in the separate gut segments of a termite. This study provides information on the C. formosanus (Shiraki) lignin modification phenomenon, and it demonstrates that phenomenon’s potential in the breakdown of the plant cell wall. Understanding this lignin modification could contribute to technology that will supplant current harsh pretreatment protocols for plant cell walls and thereby better facilitate the conversion of cellulose and hemicellulose.     

Bacteria from the Gut of Australian Termites

The major gut bacteria of the worker caste of nine species of Australian termites, belonging to four families, were isolated and identified to generic level. All species were either facultative anaerobes or strict aerobes. A correlation appears to exist between the major gut bacterium and the family to which the termite belongs. The major bacterium from the two lowest termites, Mastotermes darwiniensis (family Mastotermitidae) and Cryptotermes primus (family Kalotermitidae), was Streptococcus; from four species belonging to the Rhinotermitidae (Heterotermes ferox, Coptotermes acinaciformis, C. lacteus, Schedorhinotermes intermedius intermedius) it was Enterobacter; and from three species of the Termitidae (Nasutitermes exitiosus, N. graveolus, N. walkeri) it was Staphylococcus. Enterobacter was a minor symbiont of M. darwiniensis, C. primus, and N. graveolus; Streptococcus was a minor symbiont of H. ferox, C. lacteus, S. intermedius intermedius, and N. exitiosus; and Bacillus was a minor symbiont of C. acinaciformis and S. intermedius intermedius. M. darwiniensis possessed another minor symbiont tentatively identified as Flavobacterium. C. acinaciformis from three widely separated locations possessed a similar microbiota, indicating some form of control on the composition of the gut bacteria. Bacteria, capable of growth on N-free medium in the presence of nitrogen gas, were isolated from all termites, except N. exitiosus and N. walkeri, and were identified as Enterobacter. No cellulose-degrading bacteria were isolated.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens

Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-¹⁴C]adenine to N⁶-[¹⁴C](Δ²-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N⁶-(Δ²-isopentenyl)adenine and N⁶-(Δ²-isopentenyl)adenosine monophosphate.