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Humic substances play a key role in the global carbon cycling and the sequestration of micropollutants in soil. The transformation of these substances by earthworms, the dominant soil macroinvertebrates of many terrestrial ecosystems, and the mechanisms involved are still obscure. We prepared two chemically identical humic model compounds that were specifically 14 C-labeled either in the aromatic or the proteinaceous component, and added them to soil incubated with the geophagous earthworm species Metaphire guillelmi (anecic) and Amynthas corrugatus (endogeic). In the absence of the earthworms, both the aromatic and the proteinaceous components were mineralized at similarly low rates (5−8% after 9 days of incubation). In the presence of the earthworms, mineralization rate of the proteinaceous component was strongly stimulated (2-fold by M. guillelmi and 1.4-fold by A. corrugatus ). The mineralization rate of the aromatic component was (slightly) stimulated (1.2-fold; P A. corrugatus . In all cases, the stimulated mineralization was accompanied by a transformation of radiolabeled humic acids to fulvic acids within the earthworm guts and by an incorporation of radiolabel into the earthworm tissues. Digestion of the proteinaceous component of humic acids by the earthworms was corroborated also by a decrease of extractable humic acids in fresh cast and a stimulated mineralization of soil nitrogen; in the case of M. guillelmi , the fresh cast contained sixfold more NH 4 + than the non-ingested soil. Our study provides direct evidence for the selective digestion of humic components by earthworms. Considering the ubiquity of geophagous earthworms and their large biomass, the alteration of the chemical structure of humic substances by the earthworms through their selective digestion of peptidic components may have significant impacts on the stability of humic substances and the bioavailability of micropollutants in soil.

Previous feeding experiments have shown that soil-feeding termites (Termitidae: Termitinae) preferentially mineralize the peptidic component of synthetic humic acids, but nothing was known about the mechanism involved in digestion. Here, we studied the hydrolysis of humus-stabilized peptides in gut extracts of Cubitermes orthognathus by measuring the release of radiolabel from 14C-peptide-labeled synthetic humic acids. Gut extracts exhibited proteolytic activity over a wide pH range (from 4 to 12) with a maximum at about pH 8. The highest activity was located in the gut section containing the midgut and the extremely alkaline (up to pH 12) mixed segment. Chemical hydrolysis at in situ pH (up to pH 12) was negligible. Proteolytic activity in the hindgut fluid was generally relatively low, but alkaline proteases dominated in the anterior hindgut. When compared to other alkaline proteases, the proteolytic activity of gut extracts had a higher alkali-stability and tolerance to humic acids than subtilisin and an alkaline protease of Streptomyces griseus. Gut extracts also hydrolyzed the peptidic component of synthetic humic acids more efficiently than the commercial enzymes. Together with previous results, this study strongly supports the hypothesis that soil-feeding termites mobilize and digest the peptidic component of organic nitrogen in soil humic substances by a combination of proteolytic activities and extreme alkalinity in their intestinal tract.

In order to identify the potential nutrient and energy sources of humivorous beetle larvae, we carried out feeding trials with soil supplemented with specifically 14C-labeled model humic acids synthesized by peroxidase-initiated radical polymerization, using the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model organism. Ingestion of soil by the larvae significantly increased the mineralization of humic acids labeled in their peptide (HA-*peptide) or polysaccharide components (HA-*peptidoglycan and HA-*chitin), whereas the mineralization of humic acids labeled in the aromatic components (HA-*catechol) did not increase significantly. Mineralization was accompanied by a reduction of residual radiolabel in the acid-soluble fraction and an increase in the humic acid and humin fractions of the fecal pellets. During the gut passage, the residual label in peptide or polysaccharide components was transformed into acid-soluble products, especially in the alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by large changes in the molecular weight of the residual material. The amount of radiolabel derived from the peptide and polysaccharide components recovered from the larval body and hemolymph was significantly higher than that derived from the aromatic component, which supports the hypothesis that humivorous beetle larvae selectively digest the peptide and polysaccharide components of humic substances, whereas the aromatic components of humic substances are not an important source of nutrients and energy. This is also the first experimental evidence that also chitin and peptidoglycan, the major structural polymers in fungal and bacterial biomass, can be protected from microbial degradation in soil by a copolymerization with phenols and might contribute substantially to the refractory nitrogen pool in soil organic matter.

In order to investigate whether microbial biomass and its residues are nutrient and energy sources for humivorous beetle larvae, we carried out feeding trials using soil supplemented with 14 C-labeled fungal biomass ( Penicillium chrysogenum ), bacterial biomass ( Bacillus megaterium ), fungal or bacterial structural polysaccharide (chitin, peptidoglycan), bacterial protein, or cellulose, taking the larva of the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model of a humus-feeding beetle larva with a highly alkaline gut. The results showed that gut passage strongly stimulated the mineralization of the structural polymers. The stimulatory effect correlated positively with the recalcitrance of the preparation in the control soil, and was accompanied by a transformation of the residual radiolabel to alkali-soluble and acid-soluble products. The solubility increase was highest in the extremely alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by reciprocal changes in the molecular weight of the residual material and that the residual material in the fecal pellets was more humified than in the control soil. The amount of radiolabel recovered from the body and hemolymph of the larvae indicated that microbial biomass and its structural components were assimilated more efficiently than cellulose, which supports the hypothesis that microorganisms and the nitrogenous components of humus are an important dietary resource for humivorous soil macroinvertebrates.

Proteinases in the humus-feeding larva of Pachnoda ephippiata were partially purified and characterized. Proteinase activity from the midgut was alkali-stable and its pH optimum for activity was about pH 12. Nine proteolytic bands were visible on zymogram gels containing gelatin; one band of 19 kDa was dominant. P. ephippiata mainly employed serine proteinases for digestive proteolysis. The combination of strong midgut alkalinity and midgut proteinases with pronounced alkali stability, a high proteolytic activity on model humic acids, and a large tolerance to high humic acid concentrations would enable the P. ephippiata to gain nutrients from soil by digesting the proteinaceous component of soil humic substances.

The diet of soil-feeding termites contains large amounts of redox-active humic substances and inorganic compounds such as nitrate, sulfate, and iron minerals, which are potential mediators and/or electron acceptors for the mineralization of organic matter. We have shown previously that the intestinal tract of Cubitermes spp. (Isoptera, Termitidae) is characterized by strong changes in oxygen and hydrogen partial pressures and an extreme alkalinity of the anterior hindgut. Microelectrode measurements performed in this study indicated that the intestinal redox potential is controlled not only by the presence of oxygen or hydrogen and the prevailing pH in the different gut compartments, but also by other electroactive components. Speciation of the acid-extractable iron showed that parent soil and nest material contained mostly iron(III), whereas the gut contained mainly iron(II). Also, the humic acids in the individual gut compartments were more reduced than those in parent soil or feces. Together, these findings indicate that humic acid reduction and ferric iron reduction are important processes for the mineralization of soil organic matter in the gut of soil-feeding termites.

The feeding activity of soil-feeding termites is considered an important factor in the turnover of soil organic matter in tropical ecosystems, but the nature of the components exploited as carbon and energy source and the consequences of the gut passage for the residual fraction are still largely obscure. In order to determine mineralization rates of specific components in feeding experiments, we prepared 14C-labeled synthetic humic model compounds by peroxidase-initiated radical polymerization of a mixture of phenolic compounds, peptides, amino acids, and carbohydrates. A general characterization of the polymerization products showed that they resembled natural humic substances prepared from tropical soil used in feeding trials with soil-feeding termites in their elemental content, infrared spectra, and molecular weight distribution. Solubility-based fractionation of chemically identical preparations, arising from selective labeling of different precursors, revealed that almost all of the label stemming from the protein precursors and approximately two-thirds stemming from the peptone and catechol precursors were recovered in the humic acid fraction. In contrast, the label of glucose or glycine was recovered almost completely in the acid-soluble fraction. High-performance gel-permeation chromatography (HP-GPC) of the humic and fulvic acid fractions combined with online radiotracer analysis showed large differences in the size distribution of the radiolabel, which depended strongly on the chemical nature of labeled precursor.

The majority of termite species are considered to be humivorous, but the exact nature of their carbon and energy source, the mechanisms involved in digestion and the impact of this feeding habit on the humification of soil organic matter are largely obscure. We performed feeding trials with soil-feeding termites (Termitidae: Termitinae), using 14 C-labeled humic model compounds as substrates. In the case of Cubitermes orthognathus, the components of chemically identical synthetic humic acids (HA), labeled either in their proteinaceous or aromatic building blocks, and synthetic fulvic acids (FA), labeled in the aromatic building blocks, all had similarly low mineralization rates (2.5, 2.2 and 2.7%, respectively) when incubated in soil for 10 days in the absence of termites. When termites were present, the mineralization rate of the aromatic component of HA and FA increased only slightly but significantly (2.4 and 3.1%, respectively; P < 0.05), whereas that of the proteinaceous component increased more than 10-fold (30.8%). Similar results were obtained when the protein was peptonized prior to polymerization (9.7 vs. 27.4% mineralization). Mineralization of HA was accompanied by a transformation of the residual peptide label to FA, whereas the aromatic label of HA and FA was partly transformed to humin during gut passage. High-performance gel permeation chromatography showed a strong shift in the size-class distribution of peptide label towards low-molecular-weight products, especially of the material ingested by the termites; the smallest molecules were recovered from the termite bodies. Similar results were obtained in feeding trials with Cubitermes umbratus and Thoracotermes macrothorax. Together with previous findings, the current results provide strong evidence that during gut passage, the combined action of extreme alkalinity in the anterior hindgut, autoxidative processes, and probably also proteolytic activities, renders a large reservoir of potential substrates accessible. While peptidic components of humic substances are selectively digested, aromatic components are apparently not an important food source for soil-feeding termites. These findings have important implications for the mobilization of organic nitrogen in tropical soils. 7 2000 Elsevier Science Ltd. All rights reserved.