Microplastic surface properties affect bacterial colonization in freshwater.

Microplastics are a global concern in aquatic ecology and are readily colonized by bacteria in the environment. There is a lack of information on bacterial colonization of eroded and un-eroded microplastics in freshwater. In this study, six types of microplastics were incubated for 8 weeks in microcosms with water from Lake Erie. Microcosms were inoculated with one of three species: Acinetobacter (A.) calcoaceticus, Burkholderia (B.) cepacia, and Escherichia (E.) coli. These bacterial species are ubiquitous in water bodies associated with human populations. Bacterial surface coverage was determined using electron and fluorescent microscopy. Quantifications of EPS and surface roughness were performed by confocal microscopy and measuring contact angles (θw ) of water droplets on microplastics, respectively. Analyses revealed surface coverage differed among bacterial species and plastic types after 8 weeks. As the study progressed, E. coli remained the most abundant while A. calcoaceticus gradually decreased on most surfaces. Analyses of microcosms revealed polypropylene disks had lower bacterial abundance. Conversely, eroded polypropylene disks had highest bacterial abundance, indicating importance of surface roughness (lower θw values) and surface physicochemical properties of microplastics in bacterial colonization. Our results demonstrated that bacterial colonization of microplastics is affected by both the physicochemical properties of microplastics and the physiological properties of colonizing bacteria.

Initial nitrogen enrichment conditions determines variations in nitrogen substrate utilization by heterotrophic bacterial isolates.

BACKGROUND: The nitrogen (N) cycle consists of complex microbe-mediated transformations driven by a variety of factors, including diversity and concentrations of N compounds. In this study, we examined taxonomic diversity and N substrate utilization by heterotrophic bacteria isolated from streams under complex and simple N-enrichment conditions. RESULTS: Diversity estimates differed among isolates from the enrichments, but no significant composition were detected. Substrate utilization and substrate range of bacterial assemblages differed within and among enrichments types, and not simply between simple and complex N-enrichments. CONCLUSIONS: N substrate use patterns differed between isolates from some complex and simple N-enrichments while others were unexpectedly similar. Taxonomic composition of isolates did not differ among enrichments and was unrelated to N use suggesting strong functional redundancy. Ultimately, our results imply that the available N pool influences physiology and selects for bacteria with various abilities that are unrelated to their taxonomic affiliation.

Impacts of labile organic carbon concentration on organic and inorganic nitrogen utilization by a stream biofilm bacterial community.

In aquatic ecosystems, carbon (C) availability strongly influences nitrogen (N) dynamics. One manifestation of this linkage is the importance in the dissolved organic matter (DOM) pool of dissolved organic nitrogen (DON), which can serve as both a C and an N source, yet our knowledge of how specific properties of DOM influence N dynamics are limited. To empirically examine the impact of labile DOM on the responses of bacteria to DON and dissolved inorganic nitrogen (DIN), bacterial abundance and community composition were examined in controlled laboratory microcosms subjected to various combinations of dissolved organic carbon (DOC), DON, and DIN treatments. Bacterial communities that had colonized glass beads incubated in a stream were treated with various glucose concentrations and combinations of inorganic and organic N (derived from algal exudate, bacterial protein, and humic matter). The results revealed a strong influence of C availability on bacterial utilization of DON and DIN, with preferential uptake of DON under low C concentrations. Bacterial DON uptake was affected by the concentration and by its chemical nature (labile versus recalcitrant). Labile organic N sources (algal exudate and bacterial protein) were utilized equally well as DIN as an N source, but this was not the case for the recalcitrant humic matter DON treatment. Clear differences in bacterial community composition among treatments were observed based on terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. C, DIN, and DON treatments likely drove changes in bacterial community composition that in turn affected the rates of DON and DIN utilization under various C concentrations.

Detecting antibiotic resistance genes in anthropogenically impacted streams and rivers.

Streams and rivers are widely impacted by human activities ranging from hydrological modifications to point and nonpoint pollution. Among the pollutants that enter lotic ecosystems are pharmaceuticals and personal care products, including antibiotics, that may play a role in the occurrence of antibiotic resistance genes (ARGs). Oftentimes, ARGs are detected based on culturing of bacteria or by using quantitative polymerase chain reaction; the limitations of these methods create barriers to our understanding. Use of more exhaustive methods, such as metagenomics, may overcome some of these barriers. The public health and ecological impacts of ARGs may be profound but are largely understudied. Antibiotic resistance is a growing concern for public health.

Effect of modeled reduced gravity conditions on bacterial morphology and physiology.

BACKGROUND: Bacterial phenotypes result from responses to environmental conditions under which these organisms grow; reduced gravity has been demonstrated in many studies as an environmental condition that profoundly influences microorganisms. In this study, we focused on low-shear stress, modeled reduced gravity (MRG) conditions and examined, for Escherichia coli and Staphlyococcus aureus, a suite of bacterial responses (including total protein concentrations, biovolume, membrane potential and membrane integrity) in rich and dilute media and at exponential and stationary phases for growth. The parameters selected have not been studied in E. coli and S. aureus under MRG conditions and provide critical information about bacterial viability and potential for population growth. RESULTS: With the exception of S. aureus in dilute Luria Bertani (LB) broth, specific growth rates (based on optical density) of the bacteria were not significantly different between normal gravity (NG) and MRG conditions. However, significantly higher bacterial yields were observed for both bacteria under MRG than NG, irrespective of the medium with the exception of E. coli grown in LB. Also, enumeration of cells after staining with 4',6-diamidino-2-phenylindole showed that significantly higher numbers were achieved under MRG conditions during stationary phase for E. coli and S. aureus grown in M9 and dilute LB, respectively. In addition, with the exception of smaller S. aureus volume under MRG conditions at exponential phase in dilute LB, biovolume and protein concentrations per cell did not significantly differ between MRG and NG treatments. Both E. coli and S. aureus had higher average membrane potential and integrity under MRG than NG conditions; however, these responses varied with growth medium and growth phase. CONCLUSIONS: Overall, our data provides novel information about E. coli and S. aureus membrane potential and integrity and suggest that bacteria are physiologically more active and a larger percentage are viable under MRG as compared to NG conditions. In addition, these results demonstrate that bacterial physiological responses to MRG conditions vary with growth medium and growth phase demonstrating that nutrient resources are a modulator of response.

Cultivable bacteria from ancient algal mats from the McMurdo Dry Valleys, Antarctica.

The McMurdo Dry Valleys in Antarctica are a favorable location for preservation of dormant microbes due to their persistent cold and dry climate. In this study, we examined cultivable bacteria in a series of algal mat samples ranging from 8 to 26539 years old. Cultivable bacteria were found in all samples except one (12303 years old), but abundance and diversity of cultivable bacteria decreased with increasing sample age. Only members of the Actinobacteria, Bacteroidetes, and Firmicutes were found in the ancient samples, whereas bacteria in the 8-year-old sample also included Cyanobacteria, Proteobacteria, and Deinococcus-Thermus. Isolates of the Gram-positive spore-forming bacterium Sporosarcina were found in 5 of 8 samples. The growth of these isolates at different temperatures was related to the phylogenetic distance among genotypes measured by BOX-PCR. These findings suggest that adaptation to growth at different temperatures had occurred among Sporosarcina genotypes in the Dry Valleys, causing the existence of physiologically distinct but closely related genotypes. Additionally, fully psychrophilic isolates (that grew at 15°C, but not 25°C) were found in ancient samples, but not in the modern sample. The preservation of viable bacteria in the Dry Valleys could potentially represent a legacy of bacteria that impacts on current microbial communities of this environment.

Effects of remediation on the bacterial community of an acid mine drainage impacted stream.

Acid mine drainage (AMD) represents a global threat to water resources, and as such, remediation of AMD-impacted streams is a common practice. During this study, we examined bacterial community structure and environmental conditions in a low-order AMD-impacted stream before, during, and after remediation. Bacterial community structure was examined via polymerase chain reaction amplification of 16S rRNA genes followed by denaturing gradient gel electrophoresis. Also, bacterial abundance and physicochemical data (including metal concentrations) were collected and relationships to bacterial community structure were determined using BIO-ENV analysis. Remediation of the study stream altered environmental conditions, including pH and concentrations of some metals, and consequently, the bacterial community changed. However, remediation did not necessarily restore the stream to conditions found in the unimpacted reference stream; for example, bacterial abundances and concentrations of some elements, such as sulfur, magnesium, and manganese, were different in the remediated stream than in the reference stream. BIO-ENV analysis revealed that changes in pH and iron concentration, associated with remediation, primarily explained temporal alterations in bacterial community structure. Although the sites sampled in the remediated stream were in relatively close proximity to each other, spatial variation in community composition suggests that differences in local environmental conditions may have large impacts on the microbial assemblage.

Placing the effects of leaf litter diversity on saprotrophic microorganisms in the context of leaf type and habitat.

Because of conflicting results in previous studies, it is unclear whether litter diversity has a predictable impact on microbial communities or ecosystem processes. We examined whether effects of litter diversity depend on factors that could confound comparisons among previous studies, including leaf type, habitat type, identity of other leaves in the mixture, and spatial covariance at two scales within habitats. We also examined how litter diversity affects the saprotrophic microbial community using terminal restriction fragment length polymorphism to profile bacterial and fungal community composition, direct microscopy to quantify bacterial biomass, and ergosterol extraction to quantify fungal biomass. We found that leaf mixture diversity was rarely significant as a main effect (only for fungal biomass), but was often significant as an interaction with leaf type (for ash-free dry mass recovered, carbon-to-nitrogen ratio, fungal biomass, and bacterial community composition). Leaf type and habitat were significant as main effects for all response variables. The majority of variance in leaf ash-free dry mass and C/N ratio was explained after accounting for treatment effects and spatial covariation at the meter (block) and centimeter (litterbag) scales. However, a substantial amount of variability in microbial communities was left unexplained and must be driven by factors at other spatial scales or more complex spatiotemporal dynamics. We conclude that litter diversity effects are primarily dependent on leaf type, rather than habitat type or identity of surrounding leaves, which can guide the search for mechanisms underlying effects of litter diversity on ecosystem processes.

The effects of N:P ratio and nitrogen form on four major freshwater bacterial taxa in biofilms.

Bacteria in freshwater systems play an important role in nutrient cycling through both assimilatory and dissimilatory processes. Biotic and abiotic components of the environment affect these transformations as does the stoichiometry of the nutrients. We examined responses of four major taxa of bacteria in biofilms subjected to various N:P molar ratios using either nitrate or ammonium as a nitrogen source. Fluorescent in situ hybridization was used to enumerate the Domain bacteria as well as the alpha-, beta-, and gamma-proteobacteria, and the Cytophaga-Flavobacteria cluster. Generally, bacterial responses to the treatments were limited. However, the Cytophaga-Flavobacteria and beta-proteobacteria both responded more to the ammonium additions than nitrate, whereas, the alpha-proteobacteria responded more to nitrate additions. The beta-proteobacteria also exhibited peak relative abundance at the highest N:P ratio. Nutrient concentrations were significantly different after the incubation period, and there were distinct changes in the stoichiometry of the microcosms with ammonium. We demonstrated that bacteria may play an important role in nutrient uptake, and transformation, and can have a dramatic effect on the nutrient stoichiometry of the surrounding water. However, although some taxa exhibited differences in response to ammonium and nitrate, the impact of nutrient stoichiometry on the abundance of the taxa examined was limited.

Distribution and co-occurrence of antibiotic and metal resistance genes in biofilms of an anthropogenically impacted stream.

Urban stream biofilms are potential hotspots for resistomes and antibiotic resistance genes (ARGs). Biofilm communities that harbor resistance genes may be influenced by contaminant input (e.g., metals and antibiotics) from urban drainage (i.e., Wastewater Treatment Plant effluent and stormwater runoff); understanding the ecology of these communities and their resistome is needed. Given the potential importance of the co-occurrence of ARGs and metal resistance genes (MRGs), we investigated the spatial and temporal distribution of three ARGs (tetracycline [tetW] and sulfonamides [sulI and sulII]), four MRGs (lead [pbrT], copper [copA], and cadmium/cobalt/zinc [czcA and czcC]) via quantitative PCR and biofilm bacterial community composition via MiSeq 16S sequencing at four time points along an urbanization gradient (i.e., developed, agriculture, and forested sites) in a stream's watershed. Our results revealed that ARG and MRG abundances were significantly affected by land use-time interaction, with greater resistance abundances occurring in more urban locations during particular times of the year. It was also observed that changes in ARG and MRG profiles were influenced by differences in community composition among land use types, and that these differences were in response to changes in stream physicochemical parameters (pH, redox, temperature, nutrient availability, and metal concentration) that were driven by sub-watershed land use. Moreover, the dynamics between ARGs and MRGs within these communities correlated strongly and positively with one another. Taken altogether, our results demonstrate that changes in environmental properties due to human activity may drive the ARG-MRG profiles of biofilm communities by modulating community structure over time and space.

Sediment bacteria in an urban stream: Spatiotemporal patterns in community composition.

Sediment bacterial communities play a critical role in biogeochemical cycling in lotic ecosystems. Despite their ecological significance, the effects of urban discharge on spatiotemporal distribution of bacterial communities are understudied. In this study, we examined the effect of urban discharge on the spatiotemporal distribution of stream sediment bacteria in a northeast Ohio stream. Water and sediment samples were collected after large storm events (discharge > 100 m) from sites along a highly impacted stream (Tinkers Creek, Cuyahoga River watershed, Ohio, USA) and two reference streams. Although alpha (α) diversity was relatively constant spatially, multivariate analysis of bacterial community 16S rDNA profiles revealed significant spatial and temporal effects on beta (ß) diversity and community composition and identified a number of significant correlative abiotic parameters. Clustering of upstream and reference sites from downstream sites of Tinkers Creek combined with the dominant families observed in specific locales suggests that environmentally-induced species sorting had a strong impact on the composition of sediment bacterial communities. Distinct groupings of bacterial families that are often associated with nutrient pollution (i.e., Comamonadaceae, Rhodobacteraceae, and Pirellulaceae) and other contaminants (i.e., Sphingomonadaceae and Phyllobacteriaceae) were more prominent at sites experiencing higher degrees of discharge associated with urbanization. Additionally, there were marked seasonal changes in community composition, with individual taxa exhibiting different seasonal abundance patterns. However, spatiotemporal variation in stream conditions did not affect bacterial community functional profiles. Together, these results suggest that local environmental drivers and niche filtering from discharge events associated with urbanization shape the bacterial community structure. However, dispersal limitations and interactions among other species likely play a role as well.

Bacterial communities of leachate from tire monofill disposal sites.

The disposal of used automobile tires is problematic due to the large number generated each year. Newer methods of tire disposal have been developed in which tires are shredded and used for construction materials or buried in dedicated tire monofill sites. In this study, the bacterial assemblage associated with aqueous leachate collected from tire monofill sites was examined and compared to the bacterial assemblage of reference streams. Leachate from two tire monofill sites in Ohio (USA) was collected four times during the course of one year and analyzed for total bacterial number, culturable bacterial number, and number of the bacterium Acinetobacter calcoaceticus. The numbers of culturable bacteria (CFU/ml), total bacteria (cells/ml), and A. calcoaceticus (cells/ml) from the leachate at both monofill sites were generally lower or equal to numbers from the reference streams. Exceptions occurred during February when culturable bacteria at one site and total bacteria at the other site were more abundant in the leachate compared to reference streams; correspondingly the leachate temperature in February was higher than the surface waters. Culturable bacteria, total bacteria, and A. calcoaceticus numbers in the leachate ranged from 5.7 x 10(2)-1.8 x 10(4), 5.3 x 10(5)-3.8 x 10(6), and 9.9 x 10(1)-1.8 x 10(4) respectively. Twenty isolates were selected for species identification using FAME analysis. Nine of the 17 identified isolates belonged to the genus Pseudomonas and two isolates each belonged to the Flavobacterium and Aeromonas genera. The culturable bacterial assemblage of tire leachate was found to be similar to natural surface waters.

Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens.

Mg(2+) can potentially influence bacterial adhesion directly through effects on electrostatic interactions and indirectly by affecting physiology-dependent attachment processes. However, the effects of Mg(2+) on biofilm structure are largely unknown. In this study, Pseudomonas fluorescens was used to investigate the influence of Mg(2+) concentration (0, 0.1 and 1.0mM MgCl(2)) on biofilm growth. Planktonic and attached cells were enumerated (based on DAPI staining) while biofilm structures were examined via confocal laser scanning microscopy and three-dimensional structures were reconstructed. Mg(2+) concentration had no influence on growth of planktonic cells but, during biofilm formation, Mg(2+) increased the abundance of attached cells. For attached cells, the influence of Mg(2+) concentration changed over time, suggesting that the role of Mg(2+) in bacterial attachment is complex and dynamic. Biofilm structures were heterogeneous and surface colonization and depth increased with increasing Mg(2+) concentrations. Overall, for P. fluorescens, Mg(2+) increased initial attachment and altered subsequent biofilm formation and structure.

Identification and characterization of bacterial isolates from the Mir space station.

Twenty bacterial isolates (supplied by NASA) from the Mir space station water system were identified using Vitek GNI+ test card, API 20NE, and 16S rRNA gene sequencing. The identification of only one isolate agreed among the three techniques. The utility of the API 20NE and Vitek GNI+ test card approaches for identifying these isolates was Limited. Although 16S rRNA gene sequencing effectively identified many of the bacteria to the genus level, 74% of the isolates could not be identified to the species level. Isolates were also characterized based on motility and hydrophobicity. About 40% of the isolates were motile and four isolates were hydrophobic, suggesting that many of the bacteria have the potential to colonize surfaces and form biofilms. These findings demonstrate the difficulties in identifying bacteria from some environments to the species level and have implications for determining the risks of contamination in water systems of space shuttles and stations.

Hydrogeochemical controls on the organic matter and bacterial ecology of a small freshwater wetland in the New Jersey Pine Barrens.

This study investigated the effects of variable ground-water/surface-water exchange and photoinduced processes on longitudinal patterns in dissolved organic matter (DOM) and bacterial communities in a small first-order stream in the New Jersey Pine Barrens. DOM concentration, along with DOM weight average molecular weight (Mw) and absorptivity (epsilon280, an estimator of aromaticity), and bacterial cell counts all decreased from the stream and hyporheic zone into the shallow aquifer in a ground-water recharge zone. Further downstream, influx of ground water into the stream resulted in a lower Mw DOM pool and was accompanied by decreased cell counts. The observed effect of this ground-water discharge on bacterial numbers may be direct, if discharge temporarily dilutes cell counts, or indirect, if changes in DOM concentration and properties control the bacterial community. In either case, this study suggested the importance of considering ground-water-surface-water exchange in studies of longitudinal changes in the bacterial communities of streams.

Escherichia coli growth under modeled reduced gravity.

Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

Denitrification in agriculturally impacted streams: seasonal changes in structure and function of the bacterial community.

Denitrifiers remove fixed nitrogen from aquatic environments and hydrologic conditions are one potential driver of denitrification rate and denitrifier community composition. In this study, two agriculturally impacted streams in the Sugar Creek watershed in Indiana, USA with different hydrologic regimes were examined; one stream is seasonally ephemeral because of its source (tile drainage), whereas the other stream has permanent flow. Additionally, a simulated flooding experiment was performed on the riparian benches of the ephemeral stream during a dry period. Denitrification activity was assayed using the chloramphenicol amended acetylene block method and bacterial communities were examined based on quantitative PCR and terminal restriction length polymorphisms of the nitrous oxide reductase (nosZ) and 16S rRNA genes. In the stream channel, hydrology had a substantial impact on denitrification rates, likely by significantly lowering water potential in sediments. Clear patterns in denitrification rates were observed among pre-drying, dry, and post-drying dates; however, a less clear scenario was apparent when analyzing bacterial community structure suggesting that denitrifier community structure and denitrification rate were not strongly coupled. This implies that the nature of the response to short-term hydrologic changes was physiological rather than increases in abundance of denitrifiers or changes in composition of the denitrifier community. Flooding of riparian bench soils had a short-term, transient effect on denitrification rate. Our results imply that brief flooding of riparian zones is unlikely to contribute substantially to removal of nitrate (NO3-) and that seasonal drying of stream channels has a negative impact on NO3- removal, particularly because of the time lag required for denitrification to rebound. This time lag is presumably attributable to the time required for the denitrifiers to respond physiologically rather than a change in abundance or community composition.

Measurement of bacterial volume by transmission-through-dye imaging.

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.

Nutrients and other abiotic factors affecting bacterial communities in an Ohio River (USA).

Nitrogen and phosphorus additions from anthropogenic sources can alter the nutrient pool of aquatic systems, both through increased nutrient concentrations and changes in stoichiometry. Because bacteria are important in nutrient cycling and aquatic food webs, information about how nutrients affect bacterial communities enhances our understanding of how changes in nutrient concentrations and stoichiometry potentially affect aquatic ecosystems as a whole. In this study, bacterial communities were examined in biofilms from cobbles collected across seasons at three sites along the Mahoning River (Ohio) with differing levels of inorganic nutrient inputs. Members of the alpha-, beta-, and gamma-proteobacteria, the Cytophaga-Flavobacteria cluster, and the Domain Bacteria were enumerated using fluorescent in situ hybridization. Detrended canonical correspondence analysis (DCCA) revealed that stoichiometric ratios, especially the dissolved inorganic nitrogen (DIN):soluble reactive phosphorus (SRP) molar ratio (NO(2)/NO(3) + NH(4):soluble reactive phosphorus), were correlated with abundance of the various bacterial taxa. However, the patterns were complicated by correlations with single nutrient concentrations and seasonal changes in temperature. Seasonal cycles appeared to play an important role in structuring the community, as there were distinct winter communities and temperature was negatively correlated with abundance of both alpha-proteobacteria and Cytophaga-Flavobacteria. However, nutrients and stoichiometry also appeared to affect the community. Numbers of cells hybridizing the Domain Bacteria probe were correlated with the DOC:DIN ratio, the beta-proteobacteria had a negative correlation with soluble reactive phosphorus concentrations and a positive correlation with the DIN:SRP ratio, and the Cytophaga-Flavobacteria had a significant negative partial correlation with the DIN:SRP ratio. Abundances of the alpha- or gamma-proteobacteria were not directly correlated to nutrient concentrations or stoichiometry. It appears that nutrient stoichiometry may be an important factor structuring bacterial communities; however, it is one of many factors, such as temperature, that are interlinked and must be considered together when studying environmental bacteria.

Diversity of fungi, bacteria, and actinomycetes on leaves decomposing in a stream.

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.