Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Bacteria associated with deep,alkaline, anaerobic groundwaters in Southeast Washington

The microbial diversity in two deep, confined aquifers, the Grande Ronde (1270 m) and the Priest Rapids (316 m), Hanford Reservation, Washington, USA, was investigated by sampling from artesian wells. These basaltic aquifers were alkaline (pH 8.5 to 10.5) and anaerobic (Eh –200 to –450 mV). The wells were allowed to free-flow until pH and Eh stabilized, then the microflora was sampled with water filtration and flow-through sandtrap methods. Direct microscopic counts showed 7.6 × 10⁵ and 3.6 × 10³ bacteria ml^–1 in water from the Grande Ronde and Priest Rapids aquifers, respectively. The sand filter method yielded 5.7 × 10⁸ and 1.1 × 10⁵ cells g^–1 wet weight of sand. The numbers of bacteria did not decrease as increasing volumes of water were flushed out. The heterotrophic diversity of these bacterial populations was assessed using enrichments for 20 functional groups. These groups were defined by their ability to grow in a matrix of five different electron acceptors (O₂, Fe(III), NO₃ ^–, SO₄ ^2–, HCO₃ ^–) and four groups of electron donors (fermentation products, monomers, polymers, aromatics) in a mineral salts medium at pH 9.5. Growth was assessed by protein production. Culture media were subsequently analyzed to determine substrate utilization patterns. Substrate utilization patterns proved to be more reliable indicators of the presence of a particular physiological group than was protein production. The sand-trap method obtained a greater diversity of bacteria than did water filtration, presumably by enriching the proportion of normally sessile bacteria relative to planktonic bacteria. Substrate utilization patterns were different for microflora from the two aquifers and corresponded to their different geochemistries. Activities in the filtered water enrichments more closely matched those predicted by aquifer geochemistry than did the sand-trap enrichments. The greatest activities were found in Fe(III)-reducing enrichments from both wells, SO₄-reducing enrichments from the Grande Ronde aquifer, and methanogenic enrichments from the Priest Rapids aquifer. Organisms from these aquifers may be useful for high-pH bioremediation applications as well as production of biotechnological products. These organisms may also be useful for modeling potential reactions near buried concrete, as might be found in subsurface waste depositories. Offprint requests to: T. O. Stevens.     

Properties of 16S acetylcholinesterase from rat motor nerve and skeletal muscle

Rat obturator nerve 16S acetylcholinesterase (16S AChE) was separated by sucrose gradient velocity sedimentation and compared to the 16S form of AChE similarly derived from endplate regions of anterior gracilis muscles. The 16S AChE from both tissues could only be extracted in high ionic strength buffer; as it aggregated under low ionic strength conditions. Treatment of nerve and muscle 16S AChE with purified collagenase, in the presence of calcium, caused an identical shift in the enzyme's sedimentation coefficient to 17.5S. Other properties which were also equivalent for 16S AChE from both tissue sources included: an excess substrate inhibition above 2×10^–3 M acetylcholine andK _m of 1.6×10^–4 M, relative sensitivity to the specific inhibitors BW284C51 (I₅₀ of 5×10^–8 M) and Iso-OMPA (I₅₀ of 5×10^–4 M), and a half maximal thermal inactivation at 62.5°C. These and additional results indicate that the 16S forms of AChE in both tissues are analogous molecules, which have a highly asymmetric conformation probably containing a collagen-like domain. The present findings are also consistent with the view that motor neurons provide at least a fraction of the 16S AChE present at the neuromuscular junction.     

Factors influencing the species composition,distribution and abundance of benthic invertebrates in the profundal zone of a eutrophic northern lake

Factors influencing the species composition, distribution and abundance of benthic invertebrates were determined in a eutrophic subarctic lake from April 1978 to April 1979. Collections were made at five stations located at depths of 4 to 13 m. The largest populations of up to 5 × 10³ animals m^–2 were found in the deepest part of the lake. of the 24 species recorded in this area, the chironomidsProcladius denticulatus, Dicrotendipes modestus, Chironomus decorus andGlyptotendipes barbipes were most common. The strong development of benthos in the profundal zone was attributed to a consistently large supply of food and warm (4 °C) winter temperatures on bottom. Slightly smaller populations (up to 4 × 10³ animals, m^–2), composed of 19–23 species, occurred in shallower water, a reflection of lower (1.5 °C) winter temperatures. In the anoxic northern part of the lake, only 4–8 species were found in low numbers (400–1 000 animals m^–2). This was likely due to low (<5% saturation) oxygen levels in water and high organic content (18.5%) of the sediments.     

Culturable Bacteria in Subglacial Sediments and Ice from Two Southern Hemisphere Glaciers

Viable prokaryotes have been detected in basal sediments beneath the few Northern Hemisphere glaciers that have been sampled for microbial communities. However, parallel studies have not previously been conducted in the Southern Hemisphere, and subglacial environments in general are a new and underexplored niche for microbes. Unfrozen subglacial sediments and overlying glacier ice samples collected aseptically from the Fox Glacier and Franz Josef Glacier in the Southern Alps of New Zealand now have been shown to harbor viable microbial populations. Total direct counts of 2–7 × 10⁶ cells g^–1 dry weight sediment were observed, whereas culturable aerobic heterotrophs ranged from 6–9 × 10⁵ colony-forming units g^–1 dry weight. Viable counts in the glacier ice typically were 3–4 orders of magnitude smaller than in sediment. Nitrate-reducing and ferric iron–reducing bacteria were detected in sediment samples from both glaciers, but were few or below detection limits in the ice samples. Nitrogen-fixing bacteria were detected only in the Fox Glacier sediment. Restriction fragment analysis of 16S rDNA amplified from 37 pure cultures of aerobic heterotrophs capable of growth at 4°C yielded 23 distinct groups, of which 11 were identified as -Proteobacteria. 16S rDNA sequences from representatives of these 11 groups were analyzed phylogenetically and shown to cluster with bacteria such as Polaromonas vacuolata and Rhodoferax antarcticus, or with clones obtained from permanently cold environments. Chemical analysis of sediment and ice samples revealed a dilute environment for microbial life. Nevertheless, both the sediment samples and one ice sample demonstrated substantial aerobic mineralization of ¹⁴C-acetate at 8°C, indicating that sufficient nutrients and viable psychrotolerant microbes were present to support metabolism. Unfrozen subglacial sediments may represent a significant global reservoir of biological activity with the potential to influence glacier meltwater chemistry.     

Analysis of the genetic diversity of Bemisia tabaci populations in Shanxi province, China

Five different primer combinations were used for the analysis of 152 B biotype Bemisia tabaci (Gennadius) individuals and five Trialeurodes vaporairiorum individuals collected from 19 counties and seven host plants in Shanxi province in China, respectively. The main objective of the present study was to use AFLP markers to determine the genetic diversity of B. tabaci populations collected from Shanxi Province. The use of these primer combinations allowed the identification of 127 polymorphic bands (52.26%) from 60 to 500 bp. The average number of polymorphic bands per primer was 25.4 while the range for the five primers was 20–32. The average degree of heterozygosity was 0.251, while the range for the five primers was 0.204–0.289. The results suggested definite genetic diversity among different B. tabaci populations. Cluster analysis showed that B. tabaci populations were firstly scattered to three genetic groups according to the regions, then every genetic group was scattered to several subgroups according to the host plants, which revealed the genetic variability of B biotype B. tabaci populations has been not only among different regions, but also among different host plants in Shanxi Province.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Bacterial Population Association with Phytoplankton Cultured in a Bivalve Hatchery

Bacterial populations association with phytoplankton cultures used as food for bivalve larvae were enumerated and identified from their partial 16S rDNA gene sequences. Microalgae were provided from different European hatcheries during the larval production season. Average concentration (direct counts) of bacteria ranged from 1.3 × 10⁵ to 5.3 × 10⁸ mL^–1 while culturable bacteria represented from 10% to >60% of total bacteria. In most cases, three to six representatives of each type of colony were collected on solid medium. The identity of isolates from the same colony type was checked by two different randomly amplified polymorphic DNA (RAPD) typing methods, after which the 16S rDNA gene of one to three isolates by colony type were partially sequenced. Algae harbored a large spectrum of bacteria belonging to the -Proteobacteria, -Proteobacteria, -Proteobacteria, Cytophaga– Flavobacterium– Bacteroides (CFB) group, Actinobacteria, and Bacillus. Members of the Roseobacter clade and CFB group were the most abundant. In the majority of cases one strain constituted 50% or more of the culturable bacterial flora. About half of the isolates were common to two hatcheries or at least two microalgal cultures. Several isolates were closely related to bacteria associated with harmful dinoflagellates in culture. Thus, the algal cultures seemed to favor certain bacterial species which belonged to distantly separated groups. As some of them could disturb the development of bivalve larvae, the control of bacterial populations would undoubtedly make it possible to reduce larval losses in bivalve rearing.     

Density-dependent regulation of natural and laboratory rotifer populations

Density-dependent regulation of abundance is fundamentally important in the dynamics of most animal populations. Density effects, however, have rarely been quantified in natural populations, so population models typically have a large uncertainty in their predictions. We used models generated from time series analysis to explore the form and strength of density-dependence in several natural rotifer populations. Population growth rate (r) decreased linearly or non-linearly with increased population density, depending on the rotifer species. Density effects in natural populations reduced r to 0 at densities of 1–10 l^–1 for 8 of the 9 rotifer species investigated. The sensitivities of these species to density effects appeared normally distributed, with a mean r=0 density of 2.3 l^–1 and a standard deviation of 1.9. Brachionus rotundiformis was the outlier with 10–100× higher density tolerance. Density effects in laboratory rotifer populations reduced r to 0 at population densities of 10–100 ml^–1, which is 10⁴ higher than densities in natural populations. Density effects in laboratory populations are due to food limitation, autotoxicity or to their combined effects. Experiments with B. rotundiformis demonstrated the absence of autotoxicity at densities as high as 865 ml^–1, a much higher density than observed in natural populations. It is, therefore, likely that food limitation rather than autotoxicity plays a major role in regulating natural rotifer populations.     

Costs of Caregiving: Weight Loss in Captive Adult Male Cotton-Top Tamarins (Saguinus oedipus) Following the Birth of Infants

We examined changes in weight for 10 captive adult male cotton-top tamarins (Saguinus oedipus) from before the birth of infants through the first 16 weeks of infant life. Compared to before birth, males weighed significantly less in Weeks 1–4, 5–8, and 9–12 following the birth. Weights in Weeks 13–16 did not differ significantly from prebirth weights. Maximum weight loss for individual males ranged from 1.3 to 10.8% of prebirth body weight. Males in groups with fewer helpers lost significantly more weight than ones in groups with more helpers. For the 3 males that had no helper other than their mates, weight loss was particularly striking, ranging from 10.0 to 10.8% of their prebirth body weight. These results suggest that caring for infants is energetically costly, and that in this cooperatively breeding species, the presence of more individuals to share the burden of infant carrying reduces the cost to individual caregivers.     

Larviculture of the fairy shrimp,Streptocephalus proboscideus (Crustacea: Anostraca): effect of food concentration and physical and chemical properties of the culture medium

Food concentration (0.5 × 10³ – 5 × 10⁵ Scenedesmus cells m1^–1) significant influenced the somatic growth, maturation and survivorship ofS. proboscideus larvae. A density of 5 × 10⁴ cells m1^–1 was optimal for early larval stages. Of four temperature tested (20–35 °C), 30 °C resulted in the best growth and survival. Maturation time was inversely related to temperature, and was size- rather that age-dependent. Larvae were tolerant of a wide conductivity range, but optimal growth and survival were observed at 260 µS cm^–1. Nitrate-Nitrogen (NO₂-N) caused a larval mortality of 50% after 24 h at 0.58 mg1^–1.     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.     

Colonization potential of bacteria in the rhizosphere

The effect of inoculum density on growth and steady-state populations of aPseudomonas sp., aMycoplana sp., and aCurtobacterium sp. in the rhizosphere if gnotobiotic barley plants was studied. Inoculation of sterile barley seedling at concentrations of about 1×10³, 1×10⁵ and 1×10⁷ viable cells (mg dry wt root)^–1 resulted in rapid colonization; maximum populations of about 5×10⁷ viable cells (mg dry wt root)^–1 developed in each case. We define this maximum population as the colonization potential. Measurement of growth of known rhizosphere bacteria might be a useful index of the amount of available carbon and energy lost by growing roots.     

The maintenance ofBdellovibrio at low prey density

A mathematical model for the interaction ofBdellovibrio and its prey predicted that a relatively high prey density (7×10⁵ cells ml^–1) would be required for the establishment of an equilibrium in a mixed population [8]. The present report shows thatBdellovibrio can be maintained in a continuous culture when the prey cell density is much lower (2–5×10⁴ cells ml^–1), and closer to that of naturally occurring bacterial populations in sea waters.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

Bacterial heterogeneity in deep subsurface tunnels at Rainier Mesa,Nevada test site

To characterize the deep subsurface environment of Rainier Mesa, Nevada Test Site, rock samples were taken from tunnels U 12b, U12g, U12p, and U 12n, which varied in depth from 50 m to 450 m and in gravimetric moisture content from 4% to 27%. Values for total count, viable count, biomass, Simpson diversity, equitability, similarity coefficient, and number of distinct colony types indicated microbiological variability between samples. Viable counts ranged from less than 1 × 10¹ to 2.4 × 10⁵ CFU g dry wt^–1 of rock. Direct counts and enumeration based on phospholipid determination indicated larger numbers of cells g dry wt-1 of rock than viable counts. Simpson diversity indices, equitability, and numbers of distinct colony types varied from 3.00 to 8.05, 0.21 to 0.89, and 7 to 19, respectively, and indicated heterogeneity between samples. Each distinct morphotype was purified and characterized. Gram reaction, morphology, metal and antibiotic resistances, and metabolic activities of each isolate confirmed spatial variability among microbiota isolated from different locations. Most probable numbers of nitrifying, sulfur oxidizing, and sulfur-reducing bacteria were below the limit of detection in all samples, while the numbers of nitrogen fixing bacteria ranged from below the level of detection to 7.8 × 10² cells g dry wt^–1 of rock sample, and the numbers of dentrifying bacteria ranged from below the level of detection to greater than 1.6 × 10³ cells g dry wt^–1 of rock sample. Offprint requests to: P. S. Amy.     

Photosynthetic bacteria in meromictic lakes and stratified fjords of the Vestfold Hills,Antarctica

Thirteen meromictic lakes and two permanently stratified fjords in the Vestfold Hills, Antarctica, were surveyed in 1983 for photosynthetic bacteria. Burton Lake and Ellis Fjord were sampled throughout the year to determine seasonal variations. Physical and chemical parameters were recorded and related to the species present. The dominant species in waters with salinities of 100.7 g kg^–1 were Chlorobium vibrioforme and Chlorobium limicola with populations at the O₂–H₂S interface in the range 0.3 to 6.7 × 10⁶ ml^–1. Neither of these species was found at higher salinities. Thiocapsa roseopersicina and a Chromatium sp. were found in low numbers (< 10⁵ ml^–1) in most of the same waters as the Chlorobium spp. These bacterial phototrophs developed in a narrow band below the O₂–H₂S interface where both light and H₂S were available. Very low numbers (< 10² ml^–1) of Rhodopseudomonas palustris were found in both oxic and anoxic waters having salinity 148 g kg^–1. The dominance of the Chlorobium spp. is ascribed to their more efficient maintenance metabolism during the darkness, their faster growth at low light intensities (< 1 µE m^–2 s^–1) and the lack of selective filtering of incident light. The Chlorobium spp. grew well at –2 °C, but not –5°C in hypersaline waters. The concentration of H₂S had no apparent effect on the development of the bacterial flora. Viable cells were found to depths of 100 m in Ellis Fjord indicating that viability in total darkness could have been maintained for periods of the order of 1700 days.     

In situ measurements of uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. in an acid stream,Kashiranashigawa, Japan

In situ uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. was measured in an acid stream, Kashiranashigawa, Japan. The uptake activities were similar in the both species. The activities were highest at the tip of shoots, and decreased gradually towards the base. Carbon uptake at the tip in the light was 10.4 × 10^–4 for J. vulcanicola and 8.1 × 10^–4 g C g dry wt^–1 h^–1 1 for S. undulata. Ammonium was effectively incorporated into the shoots, and the uptake activity at the tip was between 1.9 × 10^–5 and 5.8 × 10^–5 g N g dry wt^–1 h^–1. Nitrate uptake was smaller than ammonium uptake. The ratio of dark to light uptake in ammonium uptake experiments was larger than that in carbon uptake experiments. These results suggest that the liverworts use ammonium as a major nitrogen source, and that ammonium uptake was less dependent on light than carbon uptake.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

Microbial diversity of Minnesota peatlands

Microbial diversity, numbers, and metabolic activities in Minnesota peatlands were investigated using a variety of microbial enrichment and enumeration procedures together with radioisotopic measurements of microbial degradative processes. Minnesota peatlands were shown to contain large microbial populations of wide metabolic diversity. Direct counts of bacteria using epifluorescence microscopy indicated bacterial populations of about 10⁸ ml^–1 of peatland water, irrespective of depth. Radioisotopic most-probable-number (MPN) counts of heterotrophs able to mineralize¹⁴C-labeled substrates to¹⁴CO₂ showed significant populations of glucose degraders (10⁴–10⁶ ml^–1) as well as degraders of benzoate (10²–10³ ml^–1), 2,4-dichlorophenoxyacetate (10²–10⁵ ml^–1), and sphagnum (10³–10⁷ ml^–1) in the various peatlands examined. The MPNs of NO₃ ^– reducers varied from 10³–10⁶ ml^–1, SO₄ ^– reducers from 10²–10³ ml^–1, methanogenic bacteria from 10³–10⁶ ml^–1, and methane oxidizers from 10³–10⁴ ml^–1, depending on sampling site and depth. Eighty pure cultures of aerobic bacteria and fungi were isolated from Minnesota peats. Most of those cultures tested were able to grow on at least 20 organic compounds (carbohydrates, aromatic molecules, hydrocarbons, etc.) as sole sources of carbon and energy. One isolate, aBacillus, was able to fix atmospheric N₂. Several of the isolates were able to mineralize¹⁴C-labeled lignin.