Genetic organization of a plasmid from an industrial wastewater bioreactor

Pseudomonas strain CT14 was isolated from activated sludge. Strain CT14 contained a 55, 216 bp plasmid that was characterized by sequence analysis. The plasmid had a modular structure with 51 open reading frames (ORFs) that were distributed between two clearly demarcated domains. Domain I primarily contained genes for plasmid-related functions and a novel origin of replication. Domain II bore evidence of extensive transposition and recombination. Domain II contained several genes from a meta-cleavage pathway for aromatic rings. These genes appeared to have been recruited from different hosts. This observation suggests that sequencing pCT14 may have revealed an intermediate stage in the evolution of a new assemblage of meta-cleavage pathway genes.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Insight into the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to antimicrobial drugs analysed by the 454-pyrosequencing technology

Wastewater treatment plants (WWTPs) are a reservoir for bacteria harbouring antibiotic resistance plasmids. To get a comprehensive overview on the plasmid metagenome of WWTP bacteria showing reduced susceptibility to certain antimicrobial drugs an ultrafast sequencing approach applying the 454-technology was carried out. One run on the GS 20 System yielded 346,427 reads with an average read length of 104 bases resulting in a total of 36,071,493 bases sequence data. The obtained plasmid metagenome was analysed and functionally annotated by means of the Sequence Analysis and Management System (SAMS) software package. Known plasmid genes could be identified within the WWTP plasmid metagenome data set by BLAST searches using the NCBI Plasmid Database. Most abundant hits represent genes involved in plasmid replication, stability, mobility and transposition. Mapping of plasmid metagenome reads to completely sequenced plasmids revealed that many sequences could be assigned to the cryptic pAsa plasmids previously identified in Aeromonas salmonicida subsp. salmonicida and to the accessory modules of the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas punctata. Matches of sequence reads to antibiotic resistance genes indicate that plasmids from WWTP bacteria encode resistances to all major classes of antimicrobial drugs. Plasmid metagenome sequence reads could be assembled into 605 contigs with a minimum length of 500 bases. Contigs predominantly encode plasmid survival functions and transposition enzymes.     

Biodegradation potential and bacterial diversity of a petrochemical wastewater treatment plant in Iran

An activated sludge treatment was evaluated for its effectiveness in cleaning up a petrochemical wastewater in Iran. For assessing biodegradation potential of activated sludge, seven characteristics of wastewater (temperature, pH, dissolved oxygen, chemical oxygen demand, concentrations of ethylene dichloride, vinyl chloride, and total hydrocarbons) were monitored during six months. It was shown that dominant pollutants in order of magnitude were normal-alkanes (C(10)-C(21)), aromatics, and polycyclic hydrocarbons. The activated sludge treatment revealed maximum reduction of 89%, 99%, 92%, and 80% in COD, ethylene dichloride, vinyl chloride and total hydrocarbons concentrations, respectively. Preliminary screening of culturable petrochemical-degrading microorganisms of the activated sludge resulted in the collection of 67 bacterial and one mold species. Bacterial strains mainly belonged to Pseudomonas, Flavobacterium, Comamonas, Cytophaga, Acidovorax, Sphingomonas, Bacillus and Acinetobacter genera. The isolated mold was identified as Trichoderma sp.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomyces: Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.     

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.     

工业废水中阿特拉津降解细菌的遗传和生理多样性

【目的】通过遗传学和生理学实验,揭示分离自工业废水的阿特拉津降解细菌具有遗传和生理多样性,为阐明阿特拉津生物降解的分子机理和阿特拉津降解细菌在污染环境生物修复中的应用提供新见解。【方法】用普通PCR方法检测菌株的阿特拉津降解基因,分析其降解基因组成;用基因组重复序列PCR技术(rep-PCR)分析降解菌株的基因组类型;用Western blot方法检测菌株阿特拉津降解途径的第一个酶三嗪水解酶(TrzN);用不同氮源(阿特拉津、莠灭净、扑草净、西玛津、氰草净、阿特拉通和氰尿酸)和碳源(蔗糖、葡萄糖、麦芽糖、乳糖、柠檬酸钠、乙酸钠和琥珀酸钠)培养降解菌株,通过检测培养液的OD600值,证明菌株能够利用的氮源和碳源种类。【结果】对分离自工业废水的27个阿特拉津降解菌株所进行的阿特拉津降解基因PCR检测表明,其降解基因组成分别为trzN-atzBC、trzN-atzABC和atzADEF;通过rep-PCR实验将27个阿特拉津降解菌株分为7个群;Western blot结果表明,27个菌株中有24个含有三嗪水解酶TrzN;氮源利用实验表明,2个菌株能够利用所有7种氮源生长,其余25个菌株只能利用其中的2-6种;碳源利用实验表明,10个菌株能够利用所有7种碳源生长,其余17个菌株只能利用其中的3-6种。【结论】分离自某工业废水的27株阿特拉津降解功能菌存在相当广泛的遗传和生理学上的多样性,trzN-atzABC降解基因组成为首次发现。     

Energy from vascular plant wastewater treatment systems

Water hyacinth (Eichhornia crassipes), duckweed (Spirodela sp. andLemna sp.), water pennywort (Hydrocotyle ranunculoides), and kudzu (Pueraria lobata) were anaerobically fermented using an anaerobic filter technique that reduced the total digestion time from 90 d to an average of 23 d and produced 0.14-0.22 m³ CH₄/kg (dry weight) (2.3-3.6 ft³/lb) from mature filters for the 3 aquatic species. Kudzu required an average digestion time of 33 d and produced an average of 0.21 m³ CH₄/kg (dry weight) (3.4 ft³/lb). The anaerobic filter provided a large surface area for the anaerobic bacteria to establish and maintain an optimal balance of facultative, acid-forming, and methane-producing bacteria. Consequently the efficiency of the process was greatly improved over prior batch fermentations.     

Characterization of bacterial diversity in two aerated lagoons of a wastewater treatment plant using PCR-DGGE analysis

Aerated lagoons are commonly used for domestic and industrial wastewater treatment due to their low cost and minimal need of operational requirements. However, little information is known regarding microbial communities that inhabit these ecosystems. In this study, a 16S-DGGE approach was used to estimate bacterial diversity and to monitor community changes in two aerated lagoons from a wastewater treatment plant receiving urban and industrial effluents. Pronounced shifts between bacterial communities collected in winter–spring and summer–autumn months were detected. Temperature, dissolved oxygen (DO) and pH were the variables that most influenced the bacterial communities. Phylogenetic affiliation of predominant members was assessed by the determination of the 16S rDNA sequence of correspondent bands. Affiliations to Cytophaga–Flexibacter–Bacteroides (CFB) group, Firmicutes, and β- and ε-proteobacteria were found.     

Clade composition of a plant community indicates its phylogenetic diversity

Phylogenetic diversity quantification is based on indices computed from phylogenetic distances among species, which are derived from phylogenetic trees. This approach requires phylogenetic expertise and available molecular data, or a fully sampled synthesis‐based phylogeny. Here, we propose and evaluate a simpler alternative approach based on taxonomic coding. We developed metrics, the clade indices, based on information about clade proportions in communities and species richness of a community or a clade, which do not require phylogenies. Using vegetation records from herbaceous plots from Central Europe and simulated vegetation plots based on a megaphylogeny of vascular plants, we examined fit accuracy of our proposed indices for all dimensions of phylogenetic diversity (richness, divergence, and regularity). For real vegetation data, the clade indices fitted phylogeny‐based metrics very accurately (explanatory power was usually higher than 80% for phylogenetic richness, almost always higher than 90% for phylogenetic divergence, and often higher than 70% for phylogenetic regularity). For phylogenetic regularity, fit accuracy was habitat and species richness dependent. For phylogenetic richness and divergence, the clade indices performed consistently. In simulated datasets, fit accuracy of all clade indices increased with increasing species richness, suggesting better precision in species‐rich habitats and at larger spatial scales. Fit accuracy for phylogenetic divergence and regularity was unreliable at large phylogenetic scales, suggesting inadvisability of our method in habitats including many distantly related lineages. The clade indices are promising alternative measures for all projects with a phylogenetic framework, which can trade‐off a little precision for a significant speed‐up and simplification, such as macroecological analyses or where phylogenetic data is incomplete.     

Genetic composition,genetic diversity,and small-scale environmental variation matter for the experimental reintroduction of a rare plant

Aims Reintroduction has become an important tool for the management of endangered plant species. We tested the little-explored effects of small-scale environmental variation, genotypic composition (i.e. identity of genotypes) and genotypic diversity on the population survival of the regionally rare clonal plant Ranunculus reptans. For this species of periodically inundated lakeshores, genetic differentiation had been reported between populations and between short-flooded and long-flooded microsites within populations.Methods We established 306 experimental test populations at a previously unoccupied lakeshore, comprising either monocultures of 32 genotypes, mixtures of genotypes within populations or mixtures of genotypes between populations. In 2000, three years after planting out at the experimental site, a long-lasting flood caused the death of half of the experimental populations. In 2003, an extreme drought resulted in the lowest summer water levels ever measured.Important findings Despite these climatic extremes, 27% of the established populations survived in December 2003. The success of experimental populations largely differed between microsites. Moreover, the success of genotype monocultures depended on genotype and source population. Genetic differentiation between microsites played a minor role for the success of reintroduction. After the flood, populations planted with genotypes from different source populations increased in abundance, whereas populations with genotypes from single source populations and genotype monocultures decreased. In 2014, several small patches of Ranunculus clones were still present, but plants were strongly intermingled, which precluded their assignment to the original treatments. We conclude that sources for reintroductions need to be selected carefully. Moreover, mixtures of plants from different populations appear to be the best choice for successful reintroduction, at least in unpredictably varying environments.     

Transformation of N-phenylpiperazine by mixed cultures from a municipal wastewater treatment plant

Samples from a wastewater treatment plant were used as inocula for mixed cultures dosed with N-phenylpiperazine (NPP), a model compound containing the piperazine ring found in many fluoroquinolones. Chemical analyses showed that NPP (50 mg liter(-1)) disappeared in 12 days, with the appearance of a transient metabolite and two nitrosated compounds.     

Environmental performance of a municipal wastewater treatment plant

Goal, Scope and Background  Nowadays, every strategy must be developed taking into account the global impact on the environment; if this aspect is forgotten, a change of environmental loads or their effect will be caused and no reduction will be attained. For instance, a wastewater treatment plant (WWIP), which is considereda priori as an ecological treatment system, gives rise to an environmental impact due to its energy consumption, use of chemical compounds, emissions to the atmosphere and sludge production, the post-treatment of which will also have diverse environmental effects. The goal of this study is to evaluate the potential environmental impact corresponding to a municipal WW1P and to identify the hot spots associated with the process. Methods  In this study, the Centre of Environmental Science (CML) of Leiden University methodology has been considered to quantify the potential environmental impact associated with the system under study. A comprehensive analysis of the WWTP was evaluated for the physico-chemical characterisation of the wastewaters as well as the inventory of all the inputs (energy, chemical compounds, ...) and outputs (emissions to air, water, soil and solid waste generation) associated with the global process. Regarding Life Cycle Inventory Assessment, SimaPro 5.0 was used and in particular CML factors (updated in 2002) were chosen for characterisation and normalisation stages. Results and Discussion  A comprehensive inventory of empirical data from water, sludge and gas flows during 2000 and 2001 was obtained. Two impact categories arise due to their significance: eutrophication and terrestrial ecotoxicity. Consequently, the aspects to be minimised in order to reduce the environmental impact of the system are the pollutant load at the watercourse discharge (mainly NH₃, PO₄ ^[3- and COD, even when all of them are below legal limits) and the emissions to soil (mainly Cr, Hg and Zn, even when they are present in low concentrations) when the sludge is used for agricultural application. Conclusions  As far as the environmental impact is concerned, differentiation between humid and dry season is not required as results are practically equal for both situations. Water discharge and sludge application to land have turned out to be the main contributors in the environmental performance of a WWTP. Regarding the former, the removal of nitrogen by means of a nitrification-denitrification system coupled to conventional biological aerobic treatment implies a high environmental impact reduction and, as for the latter, bearing in mind the proposed legislation, heavy metals as well as pathogens are supposed to be the key parameters to define the most adequate treatment strategies for the generated sludge. Recommendations and Outlook  This study can serve as a basis for future studies that can apply a similar policy to a great number of wastewater facilities. Besides, features such as different treatment systems and capacities can provide additional information with the final aim of including the environmental vector in the decision-making process when the operation of a WWTP is intended to be optimised. Moreover, sludge must also be a focus of attention due to the expected increase and its major contribution to the global environmental impact of a WWTP, which can determine other treatment alternatives.     

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.     

R-plasmid transfer in a wastewater treatment plant.

Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents.     

Restoring plant species diversity and community composition in a ponderosa pine-bunchgrass ecosystem

Monitoring of ecological restoration treatments often focuses on changes in community structure and function. We suggest that long-term changes in community composition also need to be explicitly considered when evaluating the success of restoration treatments. In 1992, we initiated an experiment in a ponderosa pine-bunchgrass ecosystem to evaluate responses to restoration treatments: (a) thinning the overstory vegetation (‘thinning’), (b) thinning plus forest floor manipulation with periodic prescribed burning (‘composite’), and (c) untreated ‘control.’ Treatments were further stratified by forest patch type: presettlement tree clumps (trees that established prior to the onset of fire exclusion in 1876), patches of retained postsettlement trees, patches where all postsettlement trees were removed, and remnant grass openings. Species richness did not differ among treatments for 10 years, but was highest in the composite treatment in 11th and 12th year after initial treatment. Community composition diverged among treatments 5 years after initial treatment, and compositional changes were greatest in the composite treatment. Species richness and composition differed among patch types prior to treatment. Remnant grass patches were the most diverse and presettlement patches were the least diverse. Following treatment, species richness in the postsettlement removed and retained patches, gradually approached levels found in remnant grass patches. Compositional differences among patch types changed a little by 2005. Species richness at the 2 m² scale increased only where the overstory was thinned and the understory was burned. However, these changes may not be detectable for many years, and can vary temporally in response to events such as severe droughts. Nonnative species establishment may be reduced by scheduling longer burn intervals or by refraining from burning where fuel loads are not hazardous, though these options may hinder goals of increasing diversity. Restoring species diversity and community composition continues to be more difficult than restoring ecosystem structure and function.