Degradation of cellulose by thermophilic bacteria

Degradation of 1—.10% crystalline cellulose and concentration of:free reducing sugars in the medium, were studied during cultivation of a wild coculture of obligately thermphilic bacteria in 3-L fermentors at 60^°C and pH 7.0 under anaerobic conditions. The coculture was composed of five different species ofBacillus and a single cellulolytic species lof Clostridium. The proportion of degraded substrate was inversely proportional to the initial concentration of cellulose. The higher the initial substrate concentration the lower the proportion of its.degradation. Cellulose at 1 — 2 % concentration is best degraded (98 % in:5.d). The fermentation time increases with increasing cellulose concentration, the level of reducing saccharides increases together with the initial rate of substrate degradation. In the presence of 10 %) cellulose the rate of degradation within a period of a 1-d fermentation is close toV, being 0.455 g L^-1 h^-1withK _m of 12.5 g/L. However, during further cultivation (1—3 d) the rate of degradation of 4—10 % cellulose decreases, probably due to the effect of accumulated reducing saccharides whose levels reach 55—60 mg/L.     

Decomposition of 14C-labeled cellulose substrates in litter and soil from a beechwood on limestone

The decomposition of three different ¹⁴C-labeled cellulose substrates (plant holocellulose, plant cellulose prepared from ¹⁴C-labeled beech wood (Fagus sylvatica) and bacterial cellulose produced by Acetobacter xylinum) in samples from the litter and mineral soil layer of a beechwood on limestone was studied. In a long-term (154 day) experiment, mineralization of cellulose materials, production of ¹⁴C-labeled water-soluble compounds, and incorporation of ¹⁴C in microbial biomass was in the order Acetobacter cellulose > holocellulose > plant cellulose in both litter and soil. In general, mineralization of cellulose, production of ¹⁴C-labeled water-soluble compounds, and incorporation of ¹⁴C in microbial biomass were more pronounced, but microbial biomass ¹⁴C declined more rapidly in litter than in soil. In short-term (14 day) incubations, mineralization of cellulose substrates generally corresponded with cellulase and xylanase activities in litter and soil. Pre-incubation with trace amounts of unlabeled holocellulose significantly increased the decomposition of ¹⁴C-labeled cellulose substrates and increased cellulase activity later in the experiment but did not affect xylanase activity. The sum of ¹⁴CO₂ production, ¹⁴C in microbial biomass, and ¹⁴C in water-soluble compounds is considered to be a sensitive parameter by which to measure cellulolytic activity in soil and litter samples in short-term incubations. Shorter periods than 14 days are preferable in assays using Acetobacter cellulose, because the decomposition of this substrate is more variable than that of holocellulose and plant cellulose.Offprint requests to: S. Scheu.     

Microbial Decomposition of Wood in Streams: Distribution of Microflora and Factors Affecting [14C]Lignocellulose Mineralization

The distribution and lignocellulolytic activity of the microbial community was determined on a large log of Douglas fir (Pseudotsuga menziesii) in a Pacific Northwest stream. Scanning electron microscopy, plate counts, and degradation of [¹⁴C]lignocelluloses prepared from Douglas fir and incubated with samples of wood taken from the surface and within the log revealed that most of the microbial colonization and lignocellulose-degrading activity occurred on the surface. Labeled lignocellulose and surface wood samples were incubated in vitro with nutrient supplements to determine potential limiting factors of [¹⁴C]lignocellulose degradation. Incubations carried out in a nitrogenless mineral salts and trace elements solution were no more favorable to degradation than those carried out in distilled water alone. Incubations supplemented with either (NH₄)₂SO₄ or organic nitrogen sources showed large increases in the rates of mineralization over incubations with mineral salts and trace elements alone, with the greatest effect being observed from an addition of (NH₄)₂SO₄. Subsequent incubations with (NH₄)₂SO₄, KNO₃, and NH₄NO₃ revealed that KNO₃ was the most favorable for lignin degradation, whereas all three supplements were equally favorable for cellulose degradation. Supplementation with glucose repressed both lignin and cellulose mineralization. The results reported in this study indicate that nitrogen limitation of wood decomposition may exist in streams of the Pacific Northwest. The radiotracer technique was shown to be a sensitive and useful tool for assessing relative patterns of lignocellulose decay and microbial activity in wood, along with the importance of thoroughly characterizing the experimental system before its general acceptance.     

Effects of pH on Lignin and Cellulose Degradation by Streptomyces viridosporus

Lignocellulose degradation by Streptomyces viridosporus results in the oxidative depolymerization of lignin and the production of a water-soluble lignin polymer, acid-precipitable polymeric lignin (APPL). The effects of the culture pH on lignin and cellulose metabolism and APPL production by S. viridosporus are reported. Dry, ground, hot-water-extracted corn (Zea mays) lignocellulose was autoclaved in 1-liter reagent bottles (5 g per bottle) and inoculated with 50-ml volumes of S. viridosporus cells suspended in buffers of specific pH (pH 6.0 to 9.2 at 0.4 pH unit intervals). Four replicates of inoculated cultures and of uninoculated controls at each pH were incubated as solid-state fermentations at 37°C. After 6 weeks of incubation the percent loss of lignocellulose, lignin, and carbohydrate and the amount of APPL produced were determined for each replicate. Optimal lignocellulose degradation, as shown by substrate weight loss, was observed in the pH range of 8.4 to 8.8. Only minor differences were seen in the Klason lignin, carbohydrate, protein, and ash contents of the APPLS produced by cultures at each pH. The effects of pH on the degradation of a spruce (Picea pungens) [¹⁴C-lignin]lignocellulose and a Douglas fir (Pseudotsuga menziesii) [¹⁴C-glucan]-lignocellulose were also determined at pH values between 6.5 and 9.5 (0.5 pH unit intervals). The incubations were carried out for 3 weeks at 37°C with bubbler-tube cultures. The percentage of initial ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-labeled water-soluble products, and [¹⁴C]APPL was then determined. The mineralization of lignin and cellulose to CO₂ was optimal at pHs 6.5 and 7.0, respectively. However, the optimum for lignin and cellulose solubilization was pH 8.5, which correlated with the pH 8.5 optimum for APPL production. Overall, the data show that, whereas lignin mineralization is optimal at neutral to slightly acidic pHs, lignocellulose degradation with lignin solubilization and APPL production is promoted by alkaline pHs. These findings indicate that lignin-solubilizing actinomycetes may play an important role in the metabolism of lignin in neutral to alkaline soils in which ligninolytic fungi are not highly competitive.     

Tracking the cellulolytic activity of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> biofilms

Background

Microbial cellulose conversion by Clostridium thermocellum 27405 occurs predominantly through the activity of substrate-adherent bacteria organized in thin, primarily single cell-layered biofilms. The importance of cellulosic surface exposure to microbial hydrolysis has received little attention despite its implied impact on conversion kinetics.

Results

We showed the spatial heterogeneity of fiber distribution in pure cellulosic sheets, which made direct measurements of biofilm colonization and surface penetration impossible. Therefore, we utilized on-line measurements of carbon dioxide (CO₂) production in continuous-flow reactors, in conjunction with confocal imaging, to observe patterns of biofilm invasion and to indirectly estimate microbial accessibility to the substrate’s surface and the resulting limitations on conversion kinetics. A strong positive correlation was found between cellulose consumption and CO₂ production (R²?=?0.996) and between surface area and maximum biofilm activity (R²?=?0.981). We observed an initial biofilm development rate (0.46 h^-1, 0.34 h^-1 and 0.33 h^-1) on Whatman sheets (#1, #598 and #3, respectively) that stabilized when the accessible surface was maximally colonized. The results suggest that cellulose conversion kinetics is initially subject to a microbial limitation period where the substrate is in excess, followed by a substrate limitation period where cellular mass, in the form of biofilms, is not limiting. Accessible surface area acts as an important determinant of the respective lengths of these two distinct periods. At end-point fermentation, all sheets were digested predominantly under substrate accessibility limitations (e.g., up to 81% of total CO₂ production for Whatman #1). Integration of CO₂ production rates over time showed Whatman #3 underwent the fastest conversion efficiency under microbial limitation, suggestive of best biofilm penetration, while Whatman #1 exhibited the least recalcitrance and the faster degradation during the substrate limitation period.

Conclusion

The results showed that the specific biofilm development rate of cellulolytic bacteria such as C. thermocellum has a notable effect on overall reactor kinetics during the period of microbial limitation, when ca. 20% of cellulose conversion occurs. The study further demonstrated the utility of on-line CO₂ measurements as a method to assess biofilm development and substrate digestibility pertaining to microbial solubilization of cellulose, which is relevant when considering feedstock pre-treatment options.

     

Elevated CO2 increases microbial carbon substrate use and nitrogen cycling in Mojave Desert soils

Identifying soil microbial responses to anthropogenically driven environmental changes is critically important as concerns intensify over the potential degradation of ecosystem function. We assessed the effects of elevated atmospheric CO₂ on microbial carbon (C) and nitrogen (N) cycling in Mojave Desert soils using extracellular enzyme activities (EEAs), community‐level physiological profiles (CLPPs), and gross N transformation rates. Soils were collected from unvegetated interspaces between plants and under the dominant shrub (Larrea tridentata) during the 2004–2005 growing season, an above‐average rainfall year. Because most measured variables responded strongly to soil water availability, all significant effects of soil water content were used as covariates to remove potential confounding effects of water availability on microbial responses to experimental treatment effects of cover type, CO₂, and sampling date. Microbial C and N activities were lower in interspace soils compared with soils under Larrea, and responses to date and CO₂ treatments were cover specific. Over the growing season, EEAs involved in cellulose (cellobiohydrolase) and orthophosphate (alkaline phosphatase) degradation decreased under ambient CO₂, but increased under elevated CO₂. Microbial C use and substrate use diversity in CLPPs decreased over time, and elevated CO₂ positively affected both. Elevated CO₂ also altered microbial C use patterns, suggesting changes in the quantity and/or quality of soil C inputs. In contrast, microbial biomass N was higher in interspace soils than soils under Larrea, and was lower in soils exposed to elevated CO₂. Gross rates of NH₄⁺ transformations increased over the growing season, and late‐season NH₄⁺ fluxes were negatively affected by elevated CO₂. Gross NO₃⁻ fluxes decreased over time, with early season interspace soils positively affected by elevated CO₂. General increases in microbial activities under elevated CO₂ are likely attributable to greater microbial biomass in interspace soils, and to increased microbial turnover rates and/or metabolic levels rather than pool size in soils under Larrea. Because soil water content and plant cover type dominates microbial C and N responses to CO₂, the ability of desert landscapes to mitigate or intensify the impacts of global change will ultimately depend on how changes in precipitation and increasing atmospheric CO₂ shift the spatial distribution of Mojave Desert plant communities.     

Patterns of hemolymph osmoregulation in three desert arthropods

Summary Hemolymph osmoregulation was examined in three species of desert arthropods: a tenebrionid beetle,Eleodes hispilabris, a vejovid scorpion,Paruroctonus aquilonalis, and a spirostreptid millipede,Orthoporus ornatus. During desiccation, beetles regulated hemolymph osmolality and scorpions tolerated increasing osmolality. Millipedes displayed both osmotic regulation and tolerance patterns depending on sex and on duration of desiccation. Rehydration after desiccation depressed blood osmolality of male scorpions and beetles below levels found for freshly collected specimens. This was not the case in millipedes. Seasonal osmolality changes were studied and recorded among field-collected scorpions and beetles. Patterns of regulation and tolerance of hemolymph osmolality appear to vary among different kinds of desert arthropods.     

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10⁹ cells per g of substrate, were evident after 4 and 5 days of incubation at 30°C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO₂ production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10⁷ cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10⁹ cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.     

Microbial Dynamics Associated with Multiphasic Decomposition of 14C-Labeled Cellulose in Soil

Abstract Recent emphasis on residue management in sustainable agriculture highlights the importance of elucidating the mechanisms of microbial degradation of cellulose. Cellulose decomposition and its associated microbial dynamics in soil were investigated in incubation experiments. Population dynamics of actinomycetes, bacteria, and fungi were monitored by direct counts. Populations of oligotrophic bacteria in cellulose-amended soil were determined by plate count using a low C medium containing 4 mg C liter⁻¹ agar, and copiotrophs using a high C medium. Cumulative ¹⁴CO₂ evolution from ¹⁴C-labeled cellulose was best described by a multiphasic curve in a 28-day incubation experiment. The initial phase of decomposition was attributed mainly to the activity of bacterial populations with a low oligotroph-to-copiotroph ratio, and the second phase mainly to fungal populations. An increase in oligotroph-to-copiotroph ratio coincided with the emergence of a rapid ¹⁴CO₂ evolution stage. Streptomycin reduced ¹⁴CO₂ evolution during the first phase and prompted earlier emergence of the second phase, compared to the control. Cycloheximide initially promoted ¹⁴CO₂ evolution but subsequently had a lasting negative effect on ¹⁴CO₂ evolution. Cycloheximide addition significantly increased bacterial biomass and resulted in substantially stronger oscillation of active bacterial populations, whereas it initially reduced, and then stimulated, active fungal biomass. The observed changes in ¹⁴CO₂ evolution could not be explained by observed shifts in fungal and bacterial biomass, probably because functional groups of fungi and bacteria could not be distinguished. However, it was suggested that oligotrophic bacteria prompted activation of cellulolytic enzumes in fungi and played an important role in leading to fungal dominance of cellulose decomposition. Received: 2 October 1995; Accepted: 10 February 1996     

Feeding-season production in the desert millipede Orthoporus ornatus (Girard) (Diplopoda)

Summary Production during an assumed 131-day feeding season in 1974 was estimated for Orthoporus ornatus between 4.0 and 12.0 mm in midsegment width at Tornillo Flat, Big Bend National Park, Texas.A conservative density estimate in 1973 of 1,302 millipedes ha^-1 involved daily specimen removal from three, 929-m² plots for a month. Each plot typified a different aspect of local desert vegetation; most specimens came from the plot with greatest plant diversity and relatively high (20%) cover.Production calculations using 1973 density estimates were based on increase in size-class specific dry weight (minus gut contents) between 14 May and 21 September, 1974. Production ha^-1 of cuticle and tissue was estimated at 0.85 kg (1972 kcal), while that of tissue alone came to 0.29 kg (1971 kcal). Orthoporus ornatus from Albuquerque, New Mexico increased in dry weight during 92 days in 1974 more rapidly and to a greater extent than comparable size classes at Tornillo Flat.An estimated feeding-season energy budget based on ash-free values of shrub food eaten at Tornillo Flat indicated ingestion ha^-1 of 3,434 g (13,712 kcal) and defecation of 3,181 g (9,187 kcal). An independent estimate of ingestion based on known ingestion rates was 8,851 g ha^-1.Considering probable net primary production at Tornillo Flat, local O. ornatus exert a trophic impact similar to that of other large invertebrate detritivores elsewhere.     

Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded cellulose faster with organic nitrogen sources than with NH₄Cl. Simple and complex nitrogen sources added at the time of inoculation to N-limited cultures of P. chrysosporium, with glucose as carbon/energy source, transiently stimulated degradation of synthetic [¹⁴C]lignin to ¹⁴CO₂. The same nitrogen sources added 5 days after inoculation, when the cultures were entering secondary metabolism, delayed ¹⁴CO₂ production. The various N sources affected synthetic lignin degradation in defined medium differently than lignin degradation in aspen wood.     

Effects of heat treatment on hydrogen production potential and microbial community of thermophilic compost enrichment cultures

Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70 °C. Highest cellulose degradation and highest H₂ yield were 57% and 1.4 mol-H₂ mol-hexose⁻¹ (2.4 mol-H₂ mol-hexose-degraded⁻¹), respectively, obtained at 52 °C with the heat-treated (80 °C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation.     

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.     

Ratios of Carbon Isotopes in Microbial Lipids as an Indicator of Substrate Usage

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of δ¹³C values of palmitic acid (C_16:0) with the δ¹³C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C_16:0 with ¹³C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of ¹³C in C_16:0 while incorporating the carbons of glucose, and most of them were enriched with ¹³C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in ¹³C compared to the phospholipid fractions. The δ¹³C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C_14:0) was depleted of ¹³C compared to palmitic acid (C_16:0) while octadecanoic acid (C_18:0) was enriched. These results are important for the calibration of a new method in which δ¹³C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Characterization of Clostridium thermocellum JW20

Clostridium thermocellum JW20 (ATCC 31549), which was isolated from a Louisiana cotton bale, grew on cellulose, cellobiose, and xylooligomers and, after adaptation, on glucose, fructose, and xylose in the pH range of 7.5 to 6.1 with T_opt of 60°C, T_max of 69°C, and T_min of above 28°C. Doubling times during growth on cellulose and cellobiose were 6.5 and 2.5 h, respectively. The G+C content of the DNA was 40 mol% (chemical analysis). Growth on cellulose as substrate was totally inhibited in the presence of more than 125 mM sodium sulfate, 300 mM sodium chloride, 250 mM potassium chloride, 200 mM calcium chloride, 125 mM magnesium chloride, 40 mM lactate, or 250 mM acetate. The ratio of the fermentation products ethanol to acetate plus H₂ decreased when the culture was agitated. Agitation otherwise increased the rate of cellulose degradation in a growing culture but not under nongrowth conditions or with cell-free culture supernatant containing the extracellular cellulase. Shaking lowered the concentration of H₂ in the culture broth and thus minimized inhibition by the H₂ formed. Externally added H₂ caused an increased formation of ethanol during growth on cellulose or cellobiose. However, at an atmospheric pressure as high as 355 kPa (50 lb/in²), H₂ did not cause significant growth inhibition beyond an increasing lag phase (up to 24 h). Several criteria to specifically prove the purity of C. thermocellum cultures were suggested.     

Cellulose Fermentation: Effect of Substrate Pretreatment on Microbial Growth

The effects of chemical, physical, and enzymatic treatments of rice straw and sugarcane bagasse on the microbial digestibility of cellulose have been investigated. Treatment with 4% NaOH for 15 min at 100 C increased the digestibility of cellulose from 29.4 to 73%. Treatment with 5.2% NH₃ could increase digestibility to 57.0% Treatments with sulfuric acid and crude cellulase preparation solubilized cellulose but did not increase the digestibility. Grinding or high-pressure cooking of the substrate had little effect on increasing the digestibility of cellulosic substrates by the Cellulomonas species.     

Transformation of14C labelled plant components in soil in relation to immobilization and remineralization of15N fertilizer

Summary Uniformly¹⁴C labelled glucose, cellulose and wheat straw and specifically¹⁴C labelled lignin component in corn stalks were aerobically incubated for 12 weeks in a chernozem soil alongwith¹⁵N labelled ammonium sulphate. Glucose was most readily decomposed, followed in order by cellulose, wheat straw and corn stalk lignins labelled at methoxyl-, side chain 2-and ring-C. More than 50% of¹⁴C applied as glucose, cellulose and wheat straw evolved as CO₂ during the first week. Lignin however, decomposed relatively slowly. A higher proportion of¹⁴C was transformed into microbial biomass whereas lignins contributed a little to this fraction.After 12 weeks of incubation nearly 60% of the lignin¹⁴C was found in humic compounds of which more than 70% was resistant to hydrolysis with 6N HCl. Maximum incorporation of¹⁵N in humic compounds was observed in cellulose amended soil. However, in this case more than 80% of the¹⁵N was in hydrolysable forms.Immobilization-remineralization of applied¹⁵N was most rapid in glucose treated soil and a complete immobilization followed by remineralization was observed after 3 days. The process was much slow in soil treated with cellulose, wheat straw or corn stalks. More than 70% of the newly immobilized N was in hydrolysable forms mainly reepresenting the microbial component.Serial hydrolysis of soil at different incubation intervals showed a greater proportion of 6N HCl hydrolysable¹⁴C and¹⁵N in fractions representing microbial material.¹⁴C from lignin carbons was relatively more uniformly distributed in different fractions as compared to glucose, cellulose and wheat straw where a major portion of¹⁴C was in easily hydrolysable fractions.     

Microbial Population Dynamics Associated with Crude-Oil Biodegradation in Diverse Soils

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-¹⁴C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [¹⁴C]hexadecane was converted to ¹⁴CO₂. However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.     

Requirement for a Growth Substrate During Lignin Decomposition by Two Wood-Rotting Fungi

Decomposition of ¹⁴C-labeled lignin to ¹⁴CO₂ by the lignin-decomposing fungi Phanerochaete chrysosporium and Coriolus versicolor required a growth substrate such as cellulose or glucose. Growth with lignin as sole carbon addition to an otherwise complete medium was negligible.