Shining a Light on Wastewater Treatment with Microalgae.

Microalgae can produce biofuels, nutriceuticals, pigments and many other products, but commercialization has been limited by the cost of growing, harvesting and processing algal biomass. Nutrients, chiefly nitrogen and phosphorus, are a key cost for growing microalgae, but these nutrients are present in abundance in municipal wastewater where they pose environmental problems if not removed. This is not a traditional review article; rather, it is a fact-based set of suggestions that will have to be investigated by scientists and engineers. It is suggested that if microalgae were grown as biofilms rather than as planktonic cells, and if internal illumination rather than external illumination were employed, then the use of microalgae may provide useful improvements to the wastewater treatment process. The use of microalgae to remove nutrients from wastewater has been demonstrated, but has not yet been widely implemented due to cost, and because microalgae derived from wastewater treatment has not yet been demonstrated as a commercial source for value-added products. Future facilities are likely to be called Municipal Resource Recovery Facilities as wastewater will increasingly be viewed as a resource for water, biofuels, fertilizer, monitoring public health and value-added products. Advances in photonics will accelerate this transition.

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution.

In previous work from our laboratories a synthetic gene encoding a peptide ("Sulpeptide 1" or "S1") with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells' overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.

Biodesulfurization: a model system for microbial physiology research.

Biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) by the 4S pathway is a model system for an enviromentally benign way to lower the sulfur content of petroleum. Despite a large amount of effort the efficiency of the 4S pathway is still too low for a commercial oil biodesulfurization process, but the 4S pathway could potentially be used now for commercial processes to produce surfactants, antibiotics, polythioesters and other chemicals and for the detoxification of some chemical warfare agents. Proteins containing disulfide bonds are resistant to temperature, pH, and solvents, but the production of disulfide-rich proteins in microbial hosts is challenging. The study of the 4S pathway can provide insights as to how to maximize the production of disulfide-rich proteins. Engineering of the operon encoding the 4S pathway to contain a greater content of methionine and cysteine may be able to link use of DBT as a sole sulfur source to increasing 4S pathway activity by increasing the nutritional demand for sulfur. This strategy could result in the development of biocatalysts suitable for use in an oil biodesulfurization process, but the study of the 4S pathway can also lead to a better understanding of microbial physiology to optimize activity of a mult-step co-factor-requiring pathway, as well as the production of highly stable industrially relevant enzymes for numerous applications.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

OBJECTIVE: To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. RESULTS: A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. CONCLUSION: The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Characterization of the dszABC genes of Gordonia amicalis F.5.25.8 and identification of conserved protein and DNA sequences.

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355-362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.

Microbial biocatalyst developments to upgrade fossil fuels.

Steady increases in the average sulfur content of petroleum and stricter environmental regulations concerning the sulfur content have promoted studies of bioprocessing to upgrade fossil fuels. Bioprocesses can potentially provide a solution to the need for improved and expanded fuel upgrading worldwide, because bioprocesses for fuel upgrading do not require hydrogen and produce far less carbon dioxide than thermochemical processes. Recent advances have demonstrated that biodesulfurization is capable of removing sulfur from hydrotreated diesel to yield a product with an ultra-low sulfur concentration that meets current environmental regulations. However, the technology has not yet progressed beyond laboratory-scale testing, as more efficient biocatalysts are needed. Genetic studies to obtain improved biocatalysts for the selective removal of sulfur and nitrogen from petroleum provide the focus of current research efforts.

Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S-23S rDNA spacer region and denaturing gradient gel electrophoresis.

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS-PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic "fingerprints" of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS-PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.

New method to characterize microbial diversity using flow cytometry.

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.

Heterologous gene expression in Thermus thermophilus: beta-galactosidase, dibenzothiophene monooxygenase, PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase, and chloramphenicol acetyl transferase.

Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus, the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile ( bglA from Pyrococcus woesei), a cofactor-requiring enzyme ( dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme ( carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.

Characterization of microbial communities in gas industry pipelines.

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria. The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales: Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion.

Effect of aeration and agitation on growth rate of Thermus thermophilus in batch mode.

The growth kinetics of Thermus thermophilus HB27 was investigated in rich medium (Thermus medium) under batch cultivation at 65 degrees C in 3-l fermentors. The growth and oxygen consumption rates were highly dependent on the aeration and agitation rates. Volumetric mass transfer coefficient (K(L)a, h(-1)) and hence oxygen transfer rate (OTR, mol m (-3) h(-1)) into the fermentation broth increased with increased aeration and/or agitation rates. A K(L)a and OTR of 175.4 h(-1) and 31.7 mol m(-3)h(-1) respectively, corresponding to 500 rpm agitation and 2 vvm aeration with a mixture of air and oxygen, were required to avoid oxygen limitation. The maximum growth rate (mu(max), h(-1)), doubling time (t(D), h), and dry cell weight determined for T. thermophilus HB27 growing under these conditions were 0.27 h(-1), 2.67 h, and 3g/l respectively. This cell yield is higher than any previously published reports for growth of Thermus cultures, including studies that employed pressurized bioreactors.

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum.

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.