Environmental Biotechnology in Water and Wastewater Treatment

Environmental biotechnology “manages microbial communities to provide services to society.” The key services today include detoxifying contaminated water and soil to reclaim lost resources and converting diffuse energy in biomass to forms easily used by society. Two timely examples are the reduction of oxidized water contaminants (e.g., nitrate, perchlorate, selenate, and chlorinated solvents) and the production of methane, hydrogen, and electricity. The key science underlying environmental biotechnology is microbial ecology, which has advanced rapidly in the past 20 or so years through the proliferation of new genomics-based techniques to characterize the communities’ structure and function. The genomic methods provide detailed information that helps us understand what aspects of the microbial community need to be managed to ensure that it provides the desired service. Often, we achieve the management goals through partnering the microorganisms with modern materials and physical/chemical processes. The membrane biofilm reactor and microbial fuel cells offer excellent examples of exciting new technologies that come directly from this kind of partnering.     

Micro-array based whole-genome hybridization for detection of microorganisms in acid mine drainage and bioleaching systems

Micro-array based genomic technology provides the high-throughput advantages for comprehensive understanding of complex microbial communities. In the present study, a community genome array (CGA) has been developed and evaluated for bacterial detection and microbial community analysis of acid mine drainage and bioleaching systems. This array contains 51 probes from pure culture prokaryotic whole genomic DNA. Based on the results of micro-array hybridization, specificity tests with representative pure cultures indicated that the probes on the arrays appeared to be specific to their corresponding target genomic DNA. Cross-hybridization occurred between strains of the same species, but little cross-hybridization was observed among different species. The detection limit was estimated to be approximately 0.2 ng with genomic DNA from a single pure culture bacterial and 5 ng with mixed genomic DNA from mixtures of known amounts of different species' genomic DNA. In addition, strong linear relationships were observed between hybridization signal intensity and the target DNA concentrations for pure cultures and a mixture of DNA template (r² = 0.95 to 0.985). Application of this type of the micro-array revealed the differences in microbial community composition. The results indicate that this technology has potential as a specific, sensitive and quantitative tool for detection and identification of acidophilic microorganism and the microbial community in acid mine drainage and bioleaching systems.     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D

The responses of three different soil microbial communities to the experimental application of 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2,4-D, and one had no prior direct application of any herbicide. Dominant 2,4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2,4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2,4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.     

Monitoring impact of a pesticide treatment on bacterial soil communities by metabolic and genetic fingerprinting in addition to conventional testing procedures

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of "fingerprinting" methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate t test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.     

Nonthermal pasteurization of liquid foods using high-intensity pulsed electric fields

Processing foods with high-intensity pulsed electric fields (PEF) is a new technology to inactivate microorganisms and enzymes with only a small increase in food temperature. The appearance and quality of fresh foods are not altered by the application of PEF, while microbial inactivation is caused by irreversible pore formation and destruction of the semipermeable barrier of the cell membrane. High-intensity PEF provides an excellent alternative to conventional thermal methods, where the inactivation of the microorganisms implies the loss of valuable nutrients and sensory attributes. This article presents recent advances in the PEF technology, including microbial and enzyme inactivation, generation of pulsed high voltage, processing chambers, and batch and continuous systems, as well as the theory and its application to food pasteurization. PEF technology has the potential to improve economical and efficient use of energy, as well as provide consumers with minimally processed, microbiologically safe, nutritious and freshlike food products.     

A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.     

Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 x 10(9) and 2 x 10(11) CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.     

Development of a method to assay the microbial population in heap bioleaching operations

Heap bioleaching is an economically viable approach to the mining of low-grade ores. Oxidation is microbially assisted, involving a consortium of microorganisms that together span the mesophilic to extreme thermophilic range of temperatures (25–80 °C). Temperatures inside the heap are not externally regulated, making the microbial interactions difficult to predict. In order to gain insight into these interactions, a qualitative and quantitative assay of the microorganisms that colonise the ore surface or are present in the liquid phase between the ore clusters at different levels within a heap has been developed. This method was developed using crude ore and liquid samples obtained from the GeoBiotics temperature controlled mesophilic heap operation at the Agnes Gold Mine in Barberton, South Africa, and the high temperature test columns at SGS Lakefield Research, Johannesburg, South Africa. This method of sample analysis may be applied to bioheap leach operations with and without temperature control. Ease of application, reproducibility and turn around time influenced technique design in order to provide a useful assay for commercial bioleaching operations.Following microbial removal from the solid phase using successive washes with detergent and acidified water, the cells were enumerated and genetic DNA was isolated. Microbial identification was achieved via restriction endonuclease analysis of the 16S rRNA genes, as well as 16S rRNA gene sequencing where necessary. Quantification was achieved using species-and genus-specific probes through fluorescent in situ hybridisation (FISH).     

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 delta13C values of palmitic acid (C16:0) with the delta13C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C16:0 with 13C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of 13C in C16:0 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 delta13C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C14:0) was depleted of 13C compared to palmitic acid (C16:0) while octadecanoic acid (C18:0) was enriched. These results are important for the calibration of a new method in which delta13C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.     

Antidotes for arrogance: Training for community psychology.

Advocates that psychologists: broaden definitions of therapeutic activities, expand their definitions for the criteria of competent helpers, become participants in their local communities, and alter time perspectives. It is suggested that existing professional roles are invalid and that ecological concepts are useful in creating new roles. 7 ideas for improving training of community psychologists are presented: (a) field assessment for selection of community psychologists, (b) continuous interagency interaction, (c) developing a longitudinal perspective, (d) mixing theory and practice, (e) taking advantage of community events, (f) identification of community resources, and (g) up-dating the community psychologist. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Microbial degradation of xenobiotics in the environment

Biodegradation of naturally occurring organic compounds follows their synthesis. In contrast, man-made compounds, also known as xenobiotics, are often refractory to degradation. The main reason is that they cannot be recognized by naturally present organisms and therefore do not enter common metabolic pathways. The physical and chemical characteristics of the compounds, as well as environmental factors, may influence their biodegradability. Some compounds may be transformed only in the presence of another compound which appears as a carbon and energy source. Very often compounds are degraded sequentially through the activity of a series of different organisms. The main degraders in nature are microorganisms, mostly bacteria and some fungi. These organisms, due to their rapid rates of multiplication and great metabolic potential, are able to adapt to new substrates. Selection of degradative potent microorganisms and their successive adaptation to a naturally persistent compound might be a powerful means for environmental detoxification. Although numerous laboratory experiments have given positive results, very few are applicable on a large scale. It is necessary to select microorganisms or microbial communities capable of controlled degradation of persistent organic chemicals without their transformation to other, more hazardous compounds. Better understanding of metabolic pathways for the biodegradation of specific organic compounds as well as more thorough knowledge of degrading microorganisms will make purposeful application of biodegradation possible.     

植物-微生物联合修复重金属的研究进展

重金属污染严重威胁到农作物安全和人体健康。植物修复重金属污染方面已有大量研究,其中植物-微生物联合修复被认为是改善重金属含量的一种有效方法。就近年来植物-微生物联合修复重金属的机理进行了评述,总结了近5年植物-微生物联合修复应用于重金属污染的研究。植物与微生物的协同作用,微生物与植物间形成联合体可以在很大程度上减轻环境对植物的影响。此外,微生物通过提高植物的抗性、降低重金属毒性、促进植物生长等方式增强植物修复重金属的能力。由于污染环境的复合性和复杂性,未来植物-微生物-金属相互作用的分子机制研究和开发植物-微生物联合修复的新技术开发将会成为重点。     

Large-scale phenotypic analysis--the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.     

Evaluation of Biolog for identification of members of the family Micrococcaceae

The Biolog Identification System (Biolog, Inc., Hayward, Calif.) was challenged at two separate laboratories with 113 coded isolates, including 33 type strains of staphylococci, 5 strains of Micrococcus spp., and 1 strain of Stomatococcus mucilaginosus. Test parameters between the sites were controlled as much as possible. Discrepancies were arbitrated by using conventional biochemicals. Overall accuracies (correct to the species level) upon initial testing were 47.7 and 59.3%, respectively, at the two laboratories. After repeat testing of isolates generating "no identification" responses or errors, the overall accuracies increased to 69.0 and 74.3% at the two sites, respectively, revealing no significant difference in the final results at the two laboratories (78 of 113 versus 84 of 113; P > 0.05). Error rates were 7.1% at one site and 9.7% at the other. The Biolog is not yet accurate enough to serve as a primary method for identifying staphylococci.     

Effect of Exogenous Rare Earths on Microbial Characteristics in Paddy Soil

Duetoincreasingcropyieldsandimprovingcropquality ,REammoniumhydrogencarbonatecomplexfertilizerwereappliedwidelyinsoils ,whichacceler atetheaccumulationofREinsoilsafteragriculturalapplication[1,2 ] .Previousstudieshavereportedaccu mulationofREsinsoilissignificantafter 3monthsofagriculturalapplication[3] ,andREhardlymigratedownward[4 ] .Itiscertaintythatagriculturalapplica tionofREcanresultREaccumulationinsoilandmayhaveaneffectonsoilmicrobialactivities .Soilmicro bialbiomassisamarkerofthemi…     

Recent Developments in the Bioprocessing of Minerals

Microorganisms are finding increasing use in minerals engineering. Goals include both enhancement of mineral engineering operations and remediation of mineral industry wastes. Some of the applications, such as biologically assisted leaching of sulfide ores and biooxidation of refractory sulfide gold ores. are established commercial processes. Others, such as the use of organisms for the removal of heavy metal ions from dilute aqueous streams, are nearing commercial application. Other uses of microorganisms are potentially possible. These include use of microorganisms in leaching non-sulfide ores, the flocculation or flotation of minerals and remediation of toxic chemicals discharged from mineral engineering operations. Genetic engineering of microorganisms and adaptation of existing strains are important potential tools in many of these applications, as is also the identification of new, novel and useful organisms.     

A comparison of visual- and response-generated cues in a spatial DMTS task for rats

This study focused on the capacity of finished compost, often used as packing material in biofiltration units, to support microbial biodegradation of trichloroethylene (TCE). Finished compost was enriched with methane or propane (10% head space) to stimulate cometabolic biodegradation of gaseous TCE. Successful hydrocarbon enrichment, as indicated by rapid depletion of hydrocarbon gas and measurable growth of hydrocarbon-utilizing micro-organisms, occurred within a week. Within batch reactor flasks, approximately 75% of head space TCE (1-40 ppmv) was rapidly sorbed onto compost material. Up to 99% of the remaining head space TCE was removed via biodegradation in compost enriched with either hydrocarbon. Hydrocarbon enrichment with methane or propane corresponded to 10-fold increases in methanotrophic or propanotrophic populations, respectively. Based on growth assessment under different nutritional regimes, there appeared to be complex metabolic interactions within the microbial community in enriched compost. Five separate bacterial cultures were derived from the hydrocarbon-enriched compost and assayed for the ability to degrade TCE.     

Statistical design of REACT (Rapid Early Action for Coronary Treatment), a multisite community trial with continual data collection

Unusual problems in statistical design were faced by Rapid Early Action for Coronary Treatment (REACT), a multisite trial testing a community intervention to reduce the delay between onset of symptoms of acute myocardial infarction (MI) and patients' arrival at a hospital emergency department. In 20 pair-matched U.S. communities, hospital staff members recorded delay time throughout a 4-month baseline period and the subsequent 18-month intervention period, during which one randomly selected community of each pair received a campaign of public and professional education. To exploit the continual nature of its data-collection protocol, REACT estimated the trend of delay time separately in each community by linear regression, adjusting for age, sex, and history of MI, and compared the ten adjusted slopes from intervention communities with those from control communities by a paired t-test. Power calculations based on the analytical model showed that with K=600-800 cases per community, REACT would have 80% power to demonstrate a differential reduction of 30 min in mean delay time between intervention and control communities, as well as effects on a variety of secondary outcomes. Sensitivity analysis confirmed that the number of communities was optimal within constraints of funding and that the detectable effect depended weakly on the effectiveness of matching but strongly on K, helping the investigators set operational priorities. The methodologic strategy developed for REACT should prove useful in the design of similar trials in the future.     

The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term

PROBLEM: Subclinical microbial invasion of the amniotic cavity occurs in 18.8% of women with term labor and intact membranes and in 34% of patients with term PROM and is a risk factor for the development of puerperal infection related morbidity. Although amniotic fluid white blood cell count, interleukin-6 determination, and Gram stain examination have been used for the diagnosis of intrauterine infection in patients with preterm labor and preterm premature rupture of membranes, no information is available about the accuracy and specific cut-off values for these tests in patients at term. The purpose of this study was to compare the performance of the amniotic fluid Gram stain examination, white blood cell count, and interleukin-6 determination in the identification of microbial invasion of the amniotic cavity in patients at term with and without PROM. METHOD: Amniotic fluid was retrieved from 148 patients with term gestations (90 patients with spontaneous labor and intact membranes and 58 patients with PROM). Samples were cultured for bacteria and Mycoplasma species. Amniotic fluid Gram stain, white blood cell count, and interleukin-6 determinations (ELISA, sensitivity: 43 pg/ml) were performed in all samples. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Analysis was conducted using Mann-Whitney U test, Fisher's exact test, receiver operating characteristic curves and logistic regression. RESULTS: Patients with spontaneous labor and intact membranes: The prevalence of microbial invasion of amniotic cavity in this group was 15.6% (14/90). The most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determination (sensitivity for: interleukin-6 > or = 5.7 ng/ml = 86%, white blood cell count > or = 20 cells/mm3 = 64%, Gram stain = 28%). The most specific test was the Gram stain of the amniotic fluid (specificity for: Gram stain = 84%, interleukin-6 = 79% and white blood cell count = 63%). Multiple logistic regression demonstrated that amniotic fluid interleukin-6 concentration was the only covariate that retained statistical significance when intrauterine infection was used as outcome variable. Patients with PROM: The prevalence of a positive amniotic fluid culture in this group was 39.7% (23/58). Logistic regression demonstrated that only interleukin-6 retained a significant relationship with the results of amniotic culture when all variables were entered simultaneously into a model to predict amniotic fluid culture results. The most sensitive tests for the detection of intrauterine infection were interleukin-6 determination and white blood cell count (sensitivity for interleukin-6 > or = 3.4 ng/ml and white blood cell count > or = 20 cells/mm3 = 69.6% for both). The most specific test was Gram stain (97.1%). CONCLUSIONS: Amniotic fluid interleukin-6 determination is the best rapid test for the detection of microbial invasion of the amniotic cavity in patients at term with and without PROM. When this test is not available, amniotic fluid Gram stain and white blood cell count represent valid diagnostic tools to assess the microbial state of amniotic cavity.