Nitrogen and Phosphorus Phytoextraction by Cattail (Typha spp.) during Wetland-based Phytoremediation of an End-of-Life Municipal Lagoon.

Spreading biosolids on farmland can be an effective and beneficial option for managing end-of-life municipal lagoons. Where the spreading of biosolids on farmland is restricted or unavailable, in situ phytoremediation could be a sustainable alternative. This study examined nitrogen (N) and phosphorus (P) phytoextraction by cattail ( spp.) from biosolids in a wetland constructed within a lagoon cell previously used for primary treatment of municipal wastewater. The effect of harvesting season as well as harvest frequency on N and P removal were evaluated. Forty-eight 4-m plots within the constructed wetland were used to determine the effect of cattail harvest frequency on plant N and P phytoextraction. Harvesting twice per season resulted in a 50 to 60% decrease in phytoextraction of N and P relative to a single harvest per season, which produced biomass yields of 0.58 to 0.6 kg m per year and accumulated 36.7 g N m and 5.6 g P m over the 4-yr period. Compared with August, harvesting cattails in November or April reduced N and P phytoextraction by 63 to 85%. These results demonstrate that phytoextraction of nutrients is more effective with a single harvest compared with two harvests per season. Additionally, we found that while harvesting in November and April is appealing logistically (since the wetland is frozen and provides easier access to harvest equipment), nutrient removal rates are significantly reduced.

Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46.

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36 g L-1 h-1 under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14 h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14 h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

Lipid and carotenoid synthesis by Rhodosporidium diobovatum, grown on glucose versus glycerol, and its biodiesel properties.

Relationships between lipid and carotenoid synthesis by Rhodosporidium diobovatum were investigated for cell cultures in nitrogen-limited medium (GMY) containing equimolar amounts of carbon of glucose or glycerol. The cultures were also supplemented with additional substrate at 120 h postinoculation (pi) and during a fed-batch experiment. Growth of R. diobovatum on glucose resulted in higher yields of triacyglycerides (TAGs) and carotenoid than when grown on glycerol, even though the cultures contained equimolar amounts of carbon. After the addition of fresh substrate at 120 h pi, total carotenoid concentrations were significantly different from the concentrations measured at 120 h pi in both glucose and glycerol cultures, with no concomitant increase in lipid concentrations, suggesting that carotenoid synthesis is linked to exponential-phase growth, while lipid synthesis is linked to stationary phase. We also compared the calculated properties of biodiesel that could be made with TAGs derived from R. diobovatum with properties of biodiesel made from TAGs of other oleaginous yeasts, microalgae, vegetable oils, and animal fats. This study shows that R. diobovatum can be an effective strain for production of neutral lipids containing high percentages of oleic acid, palmitic acid, and linoleic acid, as well as carotenoids.

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis.

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.

Transcriptomic and proteomic analyses of core metabolism in Clostridium termitidis CT1112 during growth on α-cellulose, xylan, cellobiose and xylose.

BACKGROUND: Clostridium termitidis CT1112 is an anaerobic, Gram-positive, mesophilic, spore-forming, cellulolytic bacterium, originally isolated from the gut of a wood feeding termite Nasusitermes lujae. It has the ability to hydrolyze both cellulose and hemicellulose, and ferment the degradation products to acetate, formate, ethanol, lactate, H2, and CO2. It is therefore ges in gene and gene product expression during growth of C. termitidis on cellobiose, xylose, xylan, and α-cellulose. RESULTS: Correlation of transcriptome and proteome data with growth and fermentation profiles identified putative carbon-catabolism pathways in C. termitidis. The majority of the proteins associated with central metabolism were detected in high abundance. While major differences were not observed in gene and gene-product expression for enzymes associated with metabolic pathways under the different substrate conditions, xylulokinase and xylose isomerase of the pentose phosphate pathway were found to be highly up-regulated on five carbon sugars compared to hexoses. In addition, genes and gene-products associated with a variety of cellulosome and non-cellulosome associated CAZymes were found to be differentially expressed. Specifically, genes for cellulosomal enzymes and components were highly expressed on α-cellulose, while xylanases and glucosidases were up-regulated on 5 carbon sugars with respect to cellobiose. Chitinase and cellobiophosphorylases were the predominant CAZymes expressed on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also demonstrated. CONCLUSION: There are little changes in core-metabolic pathways under the different carbon sources compared. The most significant differences were found to be associated with the CAZymes, as well as specific up regulation of some key components of the pentose phosphate pathway in the presence of xylose and xylan. This study has enhanced our understanding of the physiology and metabolism of C. termitidis, and provides a foundation for future studies on metabolic engineering to optimize biofuel production from natural biomass.

Cell immobilization for microbial production of 1,3-propanediol.

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum.

Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

Biomass, Nutrient, and Trace Element Accumulation and Partitioning in Cattail ( L.) during Wetland Phytoremediation of Municipal Biosolids.

Biomass and contaminant accumulation and partitioning in plants determine the harvest stage for optimum contaminant uptake during phytoremediation of municipal biosolids. This wetland microcosm bioassay characterized accumulation and partitioning of biomass, nutrients (N and P), and trace elements (Zn, Cu, Cr, and Cd) in cattail ( L.) in a growth room. Four cattail seedlings were transplanted into each 20-L plastic pail containing 3.9 kg (dry wt.) biosolids from an end-of-life municipal lagoon. A 10-cm-deep water column was maintained above the 12-cm-thick biosolids layer. Plants were harvested every 14 d over a period of 126 d for determination of aboveground biomass (AGB) and belowground biomass (BGB) yields, along with contaminant concentrations in these plant tissues. Logistic model fits to biomass yield data indicated no significant difference in asymptotic yield between AGB and BGB. Aboveground biomass accumulated significantly greater amounts of N and P and lower amounts of trace elements than BGB. Maximum N accumulation in AGB occurred 83 d after transplanting (DAT), and peak P uptake occurred at 86 DAT. Harvesting at maximum aboveground accumulation removed (percent of the initial element concentration in the biosolids) 4% N, 3% P, 0.05% Zn, 0.6% Cu, 0.1% Cd, and 0.2% Cr. Therefore, under the conditions of this study, phytoremediation would be most effective if cattail is harvested at 86 DAT. These results contribute toward the identification of the harvest stage that will optimize contaminant uptake and enhance in situ phytoremediation of biosolids using cattail.

Optimization of influential nutrients during direct cellulose fermentation into hydrogen by Clostridium thermocellum.

Combinatorial effects of influential growth nutrients were investigated in order to enhance hydrogen (H2) production during direct conversion of cellulose by Clostridium thermocellum DSM 1237. A central composite face-centered design and response surface methodology (RSM) were applied to optimize concentrations of cellulose, yeast extract (YE), and magnesium chloride (Mg) in culture. The overall optimum composition generated by the desirability function resulted in 57.28 mmol H2/L-culture with 1.30 mol H2/mol glucose and 7.48 mmol/(g·cell·h) when cultures contained 25 g/L cellulose, 2 g/L YE, and 1.75 g/L Mg. Compared with the unaltered medium, the optimized medium produced approximately 3.2-fold more H2 within the same time-frame with 50% higher specific productivity, which are also better than previously reported values from similar studies. Nutrient composition that diverted carbon and electron flux away from H2 promoting ethanol production was also determined. This study represents the first investigation dealing with multifactor optimization with RSM for H2 production during direct cellulose fermentation.

Role of transcription and enzyme activities in redistribution of carbon and electron flux in response to N2 and H2 sparging of open-batch cultures of Clostridium thermocellum ATCC 27405.

Growth, end-product synthesis, enzyme activities, and transcription of select genes associated with the "malate shunt," pyruvate catabolism, H2 synthesis, and ethanol production were studied in the cellulolytic anaerobe, Clostridium thermocellum ATCC 27405, during open-batch fermentation of cellobiose to determine the effect of elevated N2 and H2 gas sparging on metabolism using a 14-L fermenter with a 7-L working volume. The metabolic shift from acetate, H2, and CO2 to ethanol and formate in response to high H2 versus high N2 sparging (20 mL s(-1)) was accompanied by (a) a 2-fold increase in nicotinamide adenine dinucleotide (NADH)-dependent alcohol dehydrogenase (Adh) activity, (b) a 10-fold increase in adhE transcription, and (c) a 3-fold decrease in adhZ transcription. A similar, but less pronounced, metabolic shift was also observed when the rate of N2 sparging was decreased from 20 to 2 mL s(-1), during which (a) NADH-dependent ADH and pyruvate: ferredoxin oxidoreductase (PFOR) activities increased by ∼1.5-fold, (b) adhY transcription increased 6-fold, and (c) transcription of selected pfor genes increased 2-fold. Here we demonstrate that transcription of genes involved in ethanol metabolism is tightly regulated in response to gas sparging. We discuss the potential impacts of dissolved H2 on electron carrier (NADH, NADPH, ferredoxin) oxidation and how these electron carriers can redirect carbon and electron flux and regulate adhE transcription.

Insights into electron flux through manipulation of fermentation conditions and assessment of protein expression profiles in Clostridium thermocellum.

While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H2 versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H2 production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO2 was only 20 % of that of the decrease in H2, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR.

Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

Removal of nutrients from aquaculture wastewater using cattail (Typha spp.) constructed wetlands.

The aquaculture industry is among the fastest growing food production sectors in the world. Land-based aquaculture systems continue to increase in popularity as they offer the benefits of controlling diseases, managing water quality, and minimizing threats to wild populations of fish. However, these systems discharge wastewater high in N and P. The ability of cattail (Typha spp.) constructed wetlands (CWs) to remove N and P from aquaculture wastewater (AWW) was examined here. Cattail CWs were established in mesocosms and had a gradient of AWW applied weekly for a total of 5 weeks. Total N and P loadings ranged from 13.7 to 209.2 mg m-2 and 3.01 to 45.97 mg m-2 over 28 days, respectively. Additions of AWW did not cause elevations in total dissolved N, total ammonia N, or nitrite N in CW water; however, concentrations of nitrate N and P in CW water were related to nutrient loading conditions. Elevations in P persisted for 3-4 weeks among high nutrient loading treatments, providing an opportunity for eutrophic conditions to develop in CW systems. However, after 33 days of treatment, >95% total P concentration reduction was achieved in all mesocosms with final concentrations <0.05 mg L-1, equivalent to reference conditions. High-loading treatments achieved greater P load reduction (856.8-955.0 mg m-2 year-1) than low-loading and reference treatments (591.7-792.7 mg m-2 year-1). This study demonstrates the effectiveness of cattail CWs to remove nutrients during AWW treatment and highlights the potential for end-of-season use in northern climates, providing insights regarding the operational timeline of such systems.

Characterization of enriched aerotolerant cellulose-degrading communities for biofuels production using differing selection pressures and inoculum sources.

Ethanol production from direct cellulose fermentation has mainly been described as a strictly anaerobic process. The use of air-tolerant organisms or consortia for this process would reduce the need for prereduction of the medium and also permit continuous feed of aerobic feedstock. To this end, moderately thermophilic (60 °C) consortia of fermentative, cellulolytic bacteria were enriched from 3 distinct environments (manure, marsh, and rotten wood) from a farm in southeast Saskatchewan, Canada. Community phenotypic and metabolic profiles were characterized. Selection methods included direct plating under an aerobic atmosphere and repeated passaging; the methods were designed to select for robust, stable aerotolerant cellulose-degrading communities. Several of the isolated communities exhibited an increase in total cellulose degradation and total ethanol yield when compared with a monoculture of Clostridium thermocellum DSMZ 1237. Owing to stringent selection conditions, low diversity enrichments were found, and many appeared to be binary cultures via density gradient gel electrophoresis analysis. On the basis of 16S rRNA gene sequencing, aerobic conditions selected for a mix of organisms highly related to C. thermocellum and Geobacillus species, while anaerobic conditions led to the development of consortia containing strains related to C. thermocellum with strains from either the genus Geobacillus or the genus Thermoanaerobacter. The presence of a Geobacillus-like species appeared to be a prerequisite for aerotolerance of the cellulolytic enrichments, a highly desired phenotype in lignocellulosic consolidated bioprocessing.

Investigation of chemical-free nutrient removal and recovery from CO2-rich wastewater.

The feasibility of a bench-scale system for removal and recovery of phosphorus (P) as struvite from CO2-rich wastewater was tested. A continuous 12 L reactor system combining a fluidized seedbed and aeration for pH increase was developed and tested using synthetic feed. For a 100 mL min(-1) influent rate, an aeration and recycle rate combination of 7 L min(-1) and 700 mL min(-1) was sufficient for increasing and maintaining the reactor pH from 6.7 to between 7.6 and 8.0. Significant P removal was achieved in 6 h runs without a seedbed (91-92%), while neither the struvite nor sand seedbeds improved P removal (91-96%). Struvite was recovered in all runs, with additional calcium (Ca) precipitation in the seedbed runs. Reactor operation was possible for an extended period of time, up to 46 h without any major adjustment during long-term run. The average P removal was 88%, and precipitate collected after 24 h was found to be mainly struvite, while the final precipitate had a Ca: total phosphorus molar ratio of 0.56 and also contained calcite. This study has demonstrated the technical feasibility of an aerated crystallization reactor system for chemical-free struvite removal and recovery from CO2-rich wastewater such as stored livestock manure.

Critical review of phytoremediation for the removal of antibiotics and antibiotic resistance genes in wastewater.

Antibiotics in wastewater are a growing environmental concern. Increased prescription and consumption rates have resulted in higher antibiotic wastewater concentration. Conventional wastewater treatment methods are often ineffective at antibiotic removal. Given the environmental risk of antibiotics and associated antibiotic resistant genes (ARGs), finding methods of improving antibiotic removal from wastewater is of great importance. Phytoremediation of antibiotics in wastewater, facilitated through constructed wetlands, has been explored in a growing number of studies. To assess the removal efficiency and treatment mechanisms of plants and microorganisms within constructed wetlands for specific antibiotics of major antibiotic classes, the present review paper considered and evaluated data from the most recent published research on the topics of bench scale hydroponic, lab and pilot scale constructed wetland, and full scale constructed wetland antibiotic remediation. Additionally, microbial and enzymatic antibiotic degradation, antibiotic-ARG correlation, and plant effect on ARGs were considered. It is concluded from the present review that plants readily uptake sulfonamide, macrolide, tetracycline, and fluoroquinolone antibiotics and that constructed wetlands are an effective applied phytoremediation strategy for the removal of antibiotics from wastewater through the mechanisms of microbial biodegradation, root sorption, plant uptake, translocation, and metabolization. More research is needed to better understand the effect of plants on microbial community and ARGs. This paper serves as a synthesis of information that will help guide future research and applied use of constructed wetlands in the field antibiotic phytoremediation and wastewater treatment.

Natural wetlands contribution on phosphorus removal in small northern communities in Canada.

This study focuses on understanding the role of passive wastewater treatment (wastewater lagoon plus wetland) in reducing the phosphorus discharge levels in a northern small community in Manitoba, Canada. The facultative lagoon system of that small community treats domestic wastewater and seasonally discharges effluent into a wetland that connects to Lake Manitoba. This research assesses phosphorus removal efficiency through the natural wetland during the vegetation growing season. The average total phosphorus (TP) concentration reduction for the observed treatment area of 1.3 ha was more than 70%, achieving the desired TP discharge concentration below 1 mg/L. Data analysis showed that the main accumulation of TP occurred at the 21-40 cm soil depth, which indicates the potential of natural wetland treatment applications under cold continental climate conditions as an effluent polishing step to satisfy regulatory requirements for phosphorus reduction.

Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria.

BACKGROUND: Fermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism's potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae. RESULTS: Bioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do. CONCLUSIONS: Here we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.

Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46.

Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P. putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P. putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12 h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48 h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C(6)-C(14) saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20 mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8 mol%) in P. putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88 mol%).