Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

Background

Currently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation.

Results

We discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate.

Conclusion

Collectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion.     

Influence of laboratory maintenance,relative humidity and coprophagy on [14C]lignin degradation by Nasutitermes exitiosus

[¹⁴C](lignin)-Acer rubrum L. was produced by infusing stems of A. rubrum with [¹⁴C](3′-side chain)-cinnamic acid. Groups (1g) of Nasutitermes exitiosus (Hill) were placed in sealed flasks which were aspirated over a 2-week period. These released up to 8.3% of the [¹⁴C](lignin)-A. rubrum as ¹⁴CO₂. Termites maintained under standard conditions as 50 g non-breeding groups for 4 months or more showed diminished ability to degrade lignin. Optimal lignin degradation and survival of N. exitiosus was at 90 and 96% relative humidity (r.h.). At 75, 80 and 85% r.h., fungal growth in bioassay flasks was seen, but lignin degradation did not increase. At 100% r.h. where bacterial growth in faeces may have been encouraged due to the development of free water, termite survival was poor and lignin degradation decreased. Starved termites contained much more radioactivity (21.8 and 30.8%) than fed termites (1.6% radioactivity), probably due to greater coprophagy on deprivation of food. However, lignin degradation was only marginally higher in starved termites, suggesting lignin becomes progressively more resistant to termite degradation after passage through the gut.     

Evaluation of angiosperm and fern contributions to soil organic matter using two methods of pyrolysis-gas chromatography-mass spectrometry

Background

Ferns are an important plant group, and older phylogenies of non-polypod ferns contain relatively high concentrations of aliphatic leaf waxes, lignins, and tannins that could contribute to soil organic matter (SOM) biochemistry and stability.

Methods

Pyrolysis gas-chromatography mass-spectrometry (py-GC/MS) analyzes biochemical fragments which can be related to lignin, polysaccharide, lipid, nitrogen (N)-bearing, non-lignin aromatics, and phenol source compounds. Thermochemolysis using tetramethylammonium hydroxide (TMAH) combined with py-GC/MS improves detection of lignin, cutin, and suberin-derived compounds. To examine the advantages and disadvantages of both methods for characterizing plant and soil biochemistry, we characterized non-polypod and polypod fern and angiosperm live tissues, roots and soils from the Kohala Mountains, Hawaii.

Results

Py-GC/MS provided a broad biochemical overview of compound groups including lignin, polysaccharide, lipid, N-bearing, non-lignin aromatics and phenol groups while TMAH-py-GC/MS detailed lignin units and fatty acids at the expense of the other categories. TMAH-py-GC/MS provided more detailed data on lignin, cutin, suberin and tannin-derived compounds. Both methods detected differences in lignin units between species, although p-coumaric and ferulic acids, predominantly found in ferns, were only observed with TMAH-py-GC/MS.

Conclusions

Both py-GC/MS and TMAH-py-GC/MS are methods to detect compound-specific plant biomarkers, but are most useful when combined for their complimentary results.     

Farmers’ perception of termites in agriculture production and their indigenous utilization in Northwest Benin

Background

Although termites are considered as agricultural pests, they play an important role in maintaining the ecosystem. Therefore, it matters to investigate the farmers’ perception of the impacts of the termites on the agriculture and their indigenous utilization.

Methods

A semi-structured questionnaire was used to interview 94 farmers through 10 villages of Atacora department, in the northwestern region of Benin, to obtain information for the development of successful strategies of termite management and conservation. Their perceptions on the importance and management of termites along with the indigenous nomenclature and utilization of termite mounds were assessed. Termite species identified by farmers were collected and preserved in 80% alcohol for identification.

Results

Eight crops were identified by farmers as susceptible to termites with maize, sorghum, and yam as being the most susceptible. According to farmers, the susceptibility to termites of these crops is due to their high-water content and sweet taste. A total of 27 vernacular names of termites were recorded corresponding to 10 species, Amitermes evuncifer, Macrotermes subhyalinus, and Trinervitermes oeconomus being the most damaging termite species. All the names given to termite species had a meaning. The drought was identified by farmers as the main factor favouring termite attacks. Demolition of termite mounds in the fields was the most commonly reported control method. Salt and other pesticides were commonly used by farmers to protect stored farm products. The lack of effective control methods is the main constraint for termite management. In northwestern Benin, farmers reported different purpose utilizations of termite mounds and termites.

Conclusions

The study has shown that farmers perceived termites as pests of several agricultural crops and apply various indigenous control practices whose efficiency need to be verified. Utilization of termites and termite mound soil as food and medicinal resources underlines the need for a more focused approach to termite control for the conservation of non-pest termite species. The sensitization of farmers on the importance of termites as well as the development of an integrated control method to combat termite pests proved necessary.

     

Structural characterization of alkaline hydrogen peroxide pretreated grasses exhibiting diverse lignin phenotypes

Background

For cellulosic biofuels processes, suitable characterization of the lignin remaining within the cell wall and correlation of quantified properties of lignin to cell wall polysaccharide enzymatic deconstruction is underrepresented in the literature. This is particularly true for grasses which represent a number of promising bioenergy feedstocks where quantification of grass lignins is particularly problematic due to the high fraction of p-hydroxycinnamates. The main focus of this work is to use grasses with a diverse range of lignin properties, and applying multiple lignin characterization platforms, attempt to correlate the differences in these lignin properties to the susceptibility to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic deconstruction.

Results

We were able to determine that the enzymatic hydrolysis of cellulose to to glucose (i.e. digestibility) of four grasses with relatively diverse lignin phenotypes could be correlated to total lignin content and the content of p-hydroxycinnamates, while S/G ratios did not appear to contribute to the enzymatic digestibility or delignification. The lignins of the brown midrib corn stovers tested were significantly more condensed than a typical commercial corn stover and a significant finding was that pretreatment with alkaline hydrogen peroxide increases the fraction of lignins involved in condensed linkages from 88?C95% to ~99% for all the corn stovers tested, which is much more than has been reported in the literature for other pretreatments. This indicates significant scission of ??-O-4 bonds by pretreatment and/or induction of lignin condensation reactions. The S/G ratios in grasses determined by analytical pyrolysis are significantly lower than values obtained using either thioacidolysis or 2DHSQC NMR due to presumed interference by ferulates.

Conclusions

It was found that grass cell wall polysaccharide hydrolysis by cellulolytic enzymes for grasses exhibiting a diversity of lignin structures and compositions could be linked to quantifiable changes in the composition of the cell wall and properties of the lignin including apparent content of the p-hydroxycinnamates while the limitations of S/G estimation in grasses is highlighted.     

Characterization and genomic analysis of kraft lignin biodegradation by the beta-proteobacterium Cupriavidus basilensis B-8

Background

Lignin materials are abundant and among the most important potential sources for biofuel production. Development of an efficient lignin degradation process has considerable potential for the production of a variety of chemicals, including bioethanol. However, lignin degradation using current methods is inefficient. Given their immense environmental adaptability and biochemical versatility, bacterial could be used as a valuable tool for the rapid degradation of lignin. Kraft lignin (KL) is a polymer by-product of the pulp and paper industry resulting from alkaline sulfide treatment of lignocellulose, and it has been widely used for lignin-related studies.

Results

Beta-proteobacterium Cupriavidus basilensis B-8 isolated from erosive bamboo slips displayed substantial KL degradation capability. With initial concentrations of 0.5–6 g L^-1, at least 31.3% KL could be degraded in 7 days. The maximum degradation rate was 44.4% at the initial concentration of 2 g L^-1. The optimum pH and temperature for KL degradation were 7.0 and 30°C, respectively. Manganese peroxidase (MnP) and laccase (Lac) demonstrated their greatest level of activity, 1685.3 U L^-1 and 815.6 U L^-1, at the third and fourth days, respectively. Many small molecule intermediates were formed during the process of KL degradation, as determined using GC-MS analysis. In order to perform metabolic reconstruction of lignin degradation in this bacterium, a draft genome sequence for C. basilensis B-8 was generated. Genomic analysis focused on the catabolic potential of this bacterium against several lignin-derived compounds. These analyses together with sequence comparisons predicted the existence of three major metabolic pathways: β-ketoadipate, phenol degradation, and gentisate pathways.

Conclusion

These results confirmed the capability of C. basilensis B-8 to promote KL degradation. Whole genomic sequencing and systematic analysis of the C. basilensis B-8 genome identified degradation steps and intermediates from this bacterial-mediated KL degradation method. Our findings provide a theoretical basis for research into the mechanisms of lignin degradation as well as a practical basis for biofuel production using lignin materials.     

Characterization of Lignins Isolated with Alkaline Ethanol from the Hydrothermal Pretreated Tamarix ramosissima

The objective of this study was to characterize the changes in lignin structure during hydrothermal pretreatment of shrub Tamarix ramosissima. Lignins in residual wood meal were isolated with alkaline ethanol solution and recovered with acid precipitation. A comparison between the recovered lignin fractions with milled wood lignin has been made in terms of yield, purity, gel permeation chromatography, Fourier transform infrared spectroscopy, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), 1D ¹³C and 2D heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopic techniques. Semiquantitative HSQC NMR showed that the relative amounts of β-O-4′ (around 76 % side chains) and resinol type substructures (16 %) of lignins were significantly modified during hydrothermal pretreatment. Py-GC/MS analyses brought direct evidences of these lignin samples with high S/G ratios ranging from 1.7 to 2.6. Moreover, the results indicated that an increase in the severity of the hydrothermal pretreatment enhanced the degradation of lignin unit side chains and the condensation of lignin and decreased the molecular weight of the recovered lignin fractions. This study demonstrated that the combination of autohydrolysis and alkaline ethanol process could potentially turn the recovered lignin fractions into value added products being in accordance with the “biorefinery” concept.     

Genomics and biochemistry investigation on the metabolic pathway of milled wood and alkali lignin-derived aromatic metabolites of <Emphasis Type="Italic">Comamonas serinivorans</Emphasis> SP-35

Background

The efficient depolymerization and utilization of lignin are one of the most important goals for the renewable use of lignocelluloses. The degradation and complete mineralization of lignin by bacteria represent a key step for carbon recycling in land ecosystems as well. However, many aspects of this process remain unclear, for example, the complex network of metabolic pathways involved in the degradation of lignin and the catabolic pathway of intermediate aromatic metabolites. To address these subjects, we characterized the deconstruction and mineralization of lignin with milled wood lignin (MWL, the most representative molecule of lignin in its native state) and alkali lignin (AL), and elucidated metabolic pathways of their intermediate metabolites by a bacterium named Comamonas serinivorans SP-35.

Results

The degradation rate of MWL reached 30.9%, and its particle size range was decreased from 6 to 30 µm to 2–4 µm—when cultured with C. serinivorans SP35 over 7 days. FTIR analysis showed that the C–C and C–O–C bonds between the phenyl propane structures of lignin were oxidized and cleaved and the side chain structure was modified. More than twenty intermediate aromatic metabolites were identified in the MWL and AL cultures based on GC–MS analysis. Through genome sequencing and annotation, and from GC–MS analysis, 93 genes encoding 33 enzymes and 5 regulatory factors that may be involved in lignin degradation were identified and more than nine metabolic pathways of lignin and its intermediates were predicted. Of particular note is that the metabolic pathway to form the powerful antioxidant 3,4-dihydroxyphenylglycol is described for the first time in bacteria.

Conclusion

Elucidation of the β-aryl ether cleavage pathway in the strain SP-35 indicates that the β-aryl ether catabolic system is not only present in the family of Sphingomonadaceae, but also other species of bacteria kingdom. These newly elucidated catabolic pathways of lignin in strain SP-35 and the enzymes responsible for them provide exciting biotechnological opportunities for lignin valorization in future.

     

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.     

The influence of subterranean termites on the hydrological characteristics of a Chihuahuan desert ecosystem

Summary Rainfall simulation at an average intensity of 124 mm·h^-1 was used to compare infiltration and run off on arid areas where subterranean termites had been eliminated four years prior to the initiation of the study (termite free) with adjacent areas populated by subterranean termites (termites present). Infiltration rates on termite free plots with less than 5% perennial plant cover were significantly lower 51.3±6.8 mm·h^-1 than rates on comparable termites present plots 88.4±5.6 mm·h^-1. On plots centered on Larrea tridentata shrubs, there were no differences in infiltration rates with or without termites. Plots with shrub cover had the highest infiltration rates 101±6 mm·h^-1. Highest run-off volumes were recorded from termite free <5% grass cover plots and the lowest from plots with shrubs. There were no differences in suspended sediment concentrations from termites present and termite free plots. Average bed load concentration was more than three times greater from termite free, <5% cover plots than from termites present, <5% cover plots.The reduction in infiltration, high run-off volumes and high bedloads from termite free areas without shrub cover is related to increased soil bulk density resulting from the collapse of subterranean galleries of the termites that provide avenues of bulk flow into the soil. Subterranean termites affect the hydrology of Chihuahuan desert systems by enhancing water infiltration and retention of top soil. The presence of a shrub canopy and litter layer cancels any effect of subterranean termites on hydrological parameters. Since approximately 2/3 of the area is not under shrub canopies, subterranean termites are considered to be essential for the maintenance of the soil water characteristics that support the present vegetation.     

Digesta retention time and recovery rates of ants and termites in Chinese pangolins (Manis pentadactyla)

Pangolins are myrmecophagous mammals whose biology and ecology remain poorly studied. Termite mandibles and ant head capsules are the two primary remains found in pangolin feces. Determining the retention time of insect cuticles is important for understanding the digestive physiology of pangolins, while determining the recovery rate of termites and ants in feces is required to estimate the number of these prey items that are consumed by pangolins. In this study, the authors conducted feeding trials with captive Chinese pangolins (Manis pentadactyla). Sixty grams of the fungus-growing termite Odontotermes formosanus (18,816 individuals) and 15–20 g of the yellow crazy ant Anoplolepis gracilipes (14,400–19,200 individuals) were fed to each pangolin. After feeding, pangolin feces were collected daily for 1 week. The authors also assessed the accuracy of using chromium (III) oxide powder (Cr₂O₃) as a proxy for determining gut passage time, as has been done in previous studies. The results showed that remaining termite mandibles and ant head capsules in feces peaked at 66 and 90 hr after feeding and their recovery rates were 0.35 ± 0.10 and 0.65 ± 0.04, respectively. In both feeding trials, the retention time of Cr₂O₃ was much shorter than that of the termite mandibles and ant head capsules, indicating that Cr₂O₃ is not an appropriate indicator for estimating food retention time of myrmecophagous animals. Our results revealed that the ant head capsules were preserved better in feces compared with the termite mandibles, suggesting that termites may be considerably underestimated in the feces of wild pangolins.     

Biodegradation of hardwood lignocellulosics by the western poplar clearwing borer, Paranthrene robiniae (Hy. Edwards)

Lignin in plant cell wall is a source of useful chemicals and also the major barrier for saccharification of lignocellulosic biomass for producing biofuel and bioproducts. Enzymatic lignin degradation/modification process could bypass the need for chemical pretreatment and thereby facilitate bioprocess consolidation. Herein, we reveal our new discovery in elucidating the process of hardwood lignin modification/degradation by clearwing borer, Paranthrene robiniae . The wood-boring clearwing borer, P. robiniae , effectively tunnels hardwood structures during the larval stage; its digestion products from wood components, however, has not yet been investigated. A series of analysis conducted in this study on tunnel walls and frass produced provided evidence of structural alterations and lignin degradation during such hardwood digestion process. The analysis included solid state (13)C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflectance Fourier transform infrared (ATR-FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermogravimetric (TG) analysis; the results strongly suggest that the structural alteration of lignin primarily involved a preferential degradation of syringyl units accompanied by oxidation on the side chains of lignin guaiacyl moieties. This study also further indicated that unlike the wood-feeding termite the clearwing borer does not target cellulose as an energy source, and thus its lignin degradation ability should provide potential information on how to disassemble and utilize hardwood lignin. Overall, this biological model with an efficient lignin disruption system will provide the new insight into novel enzyme system required for effective plant cell wall disintegration for enhanced cellulose accessibility by enzymes and production of value-added lignin derived products.     

Influence of lignin in Reticulitermes santonensis: symbiotic interactions investigated through proteomics

The gut of lower termites is populated by numerous microbial species belonging to prokaryotes, fungi, yeasts and protists. These micro-organisms are organized in a complex symbiotic system, interacting together and with the insect host. Their likely ability to degrade ligno-cellulosic compounds could lead to improvements in second generation biofuels production. Lignin elimination represents a critical point as this polymer significantly interferes with industrial process of cellulose. Although host produces its own lignin-degrading enzymes, some symbionts may participate in digestion of lignin and its degradation products in termite gut. Here, we compared gut proteomes from R. santonensis after rearing on artificial diets composed of cellulose with and without lignin. The effect of lignin in artificial diets on different parts of the digestive tract was compared through liquid chromatography associated with tandem mass spectrometry (LC-MS/MS) experiments. Enzymatic assays were performed to characterize activities present in R. santonensis digestive tract after feeding on artificial diets. Microscopic observations of microbial communities provided some information on population balances after feeding experiment.     

Termite ecology in a dry evergreen forest in Thailand in terms of stable (δ 13C and δ 15N) and radio (14C, 137Cs and 210Pb) isotopes

Stable (¹³C and ¹⁵N) and radio- (¹⁴C, ¹³⁷Cs and ²¹⁰Pb) isotopes were determined for termites that have been sampled from a dry evergreen forest in Thailand. A wood-feeding termite, Microcerotermes crassus, was separated from soil-feeders: Termes propinquus, Termes comis and Dicuspiditermes makhamensis by ¹³C and ¹⁵N values. The Termes group in Thailand had less diverse values in ¹³C and ¹⁵N than those in Australia, where the feeding habits of the Termes group are more diverse. Other soil-feeding termites produced similar ¹³C values, but a larger range in ¹⁵N values. ¹⁴C-percent modern carbon (pMC) values suggest that the soil-feeding termites used younger carbon than the wood-feeding termites, and this was consistent with the termites from Cameroon, central Africa. Values of ¹³C and ¹⁴C-pMC indicate that surface soil was used by a soil-feeding termite, D. makhamensis, in making the nest mounds, and deeper soil (10–30 cm) by a fungus-growing termite, Macrotermes carbonarius. ²¹⁰Pb and ¹³⁷Cs were scarcely incorporated into the termites, although ²¹⁴Pb was recovered from the workers. The results suggest that stable- and radioisotopes are useful in the study of detritivorous animals, organic matter decomposition and ecosystem engineering.Takuya Abe - deceased.     

Caste- and development-associated gene expression in a lower termite

Background  

Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes.     

Reactivity improvement of cellulolytic enzyme lignin via mild hydrothermal modification

Isolated by the cellulolytic enzyme lignin (CEL) process, water-alcohol (1:1, v/v) was introduced as co-solvent in the process of the hydrothermal treatment. The modification parameters such as reaction temperature and time, solid-to-liquid ratio, and catalysts (NaOH and NaOAlO₂) have been investigated in terms of the specific lignin properties, such as the phenolic hydroxyl content (OH_phen), DPPH free radical scavenging rate, and formaldehyde value. The CELs were also characterized by GPC, FT-IR and ¹ H NMR spectroscopy, and Py-GC/MS. The key data are under optimal lignin modification conditions (solid-to-liquid ratio of 1:10 (w/v) and a temperature of 250 °C for 60 min) are: OH_phen content: 2.50 mmol/g; half maximal inhibitory concentration (IC₅₀) towards DPPH free radicals: 88.2 mg/L; formaldehyde value: 446.9 g/kg). Both base catalysts decrease the residue rate, but phenol reactivities of the products were also detracted. Py-GC/MS results revealed that modified lignin had a higher phenolic composition than the CEL did, especially the modified lignin without catalyst (ML), which represented 74.51% phenolic content.     

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.     

Calcium carbonate in termite galleries – biomineralization or upward transport?

Termites and soil calcium carbonate are major factors in the global carbon cycle: termites by their role in decomposition of organic matter and methane production, and soil calcium carbonate by its storage of atmospheric carbon dioxide. In arid and semiarid soils, these two factors potentially come together by means of biomineralization of calcium carbonate by termites. In this study, we evaluated this possibility by testing two hypotheses. Hypothesis 1 states that termites biomineralize calcium carbonate internally and use it as a cementing agent for building aboveground galleries. Hypothesis 2 states that termites transport calcium carbonate particles from subsoil horizons to aboveground termite galleries where the carbonate detritus becomes part of the gallery construction. These hypotheses were tested by using (1) field documentation that determined if carbonate-containing galleries only occurred on soils containing calcic horizons, (2) ¹³C/¹²C ratios, (3) X-ray diffraction, (4) petrographic thin sections, (5) scanning electron microscopy, and (6) X-ray mapping. Four study sites were evaluated: a C₄-grassland site with no calcic horizons in the underlying soil, a C₄-grassland site with calcic horizons, a C₃-shrubland site with no calcic horizons, and a C₃-shrubland site with calcic horizons. The results revealed that carbonate is not ubiquitously present in termite galleries. It only occurs in galleries if subsoil carbonate exists within a depth of 100 cm. ¹³C/¹²C ratios of carbonate in termite galleries typically matched ¹³C/¹²C ratios of subsoil carbonate. X-ray diffraction revealed that the carbonate mineralogy is calcite in all galleries, in all soils, and in the termites themselves. Thin sections, scanning electron microscopy, and X-ray mapping revealed that carbonate exists in the termite gut along with other soil particles and plant opal. Each test argued against the biomineralization hypothesis and for the upward-transport hypothesis. We conclude, therefore, that the gallery carbonate originated from upward transport and that this CaCO₃ plays a less active role in short-term carbon sequestration than it would have otherwise played if it had been biomineralized directly by the termites.