Joseph J. Vallino

Network rigidity and metabolic engineering in metabolite overproduction

In order to enhance the yield and productivity of metabolite production, researchers have focused almost exclusively on enzyme amplification or other modifications of the product pathway. However, overproduction of many metabolites requires significant redirection of flux distributions in the primary metabolism, which may not readily occur following product deregulation because metabolic pathways have evolved to exhibit control architectures that resist flux alterations at branch points. This problem can be addressed through the use of some general concepts of metabolic rigidity, which include a means for identifying and removing rigid branch points within an experimental framework.

Terrestrial inputs of organic matter to coastal ecosystems: An intercomparison of chemical characteristics and bioavailability

Dissolved and particulate organic matter (DOM and POM) collected from rivers or groundwater feeding five estuaries along the east and west coasts of the USA were characterized with a variety of biogeochemical techniques and related to bioavailability to estuarine microbes. Surface water was sampled from the Columbia, Satilla, Susquehanna and Parker Rivers and groundwater was sampled from the Childs River. Several geochemical descriptors (percent organic matter of suspended particulate matter, C/N, lignin phenol content, ratio of vanillic acid to vanillin) suggested an ordering of the systems with respect to POM lability: Satilla < Parker < Columbia < Susquehanna.DOC concentrations in these systems ranged from <100 μM for the Columbia River to >2000 μM for the Satilla River. Elemental analysis of DOM concentrates (>1000 D) was used to predict organic matter composition and to calculate degree of substrate reduction using two different modeling approaches. Models predicted aliphatic carbon ranging between 43 and 60% and aromatic carbon between 26 and 36%, with aliphatic content lowest in the Satilla and highest in the Columbia River. The degree of substrate reduction of the organic matter concentrates followed a pattern similar to that for aliphatic C, being lowest in the Satilla (3.5) and highest in the Columbia (4.0). Extracellular enzyme activity varied broadly across the systems, but again ordered sites in the same way as did aliphatic content and degree of substrate reduction. Bacterial growth rates ranged from 1.3 ug mg-1 d-1 DOC in the Satilla to 1.7 ug mg-1 d-1 DOC in the Parker River. Bioassays confirmed patterns of dissolved organic matter lability predicted by the chemical models. Between 67% to 75% of the variation in bacterial growth could be explained by differences in organic matter composition.

Efficient export of carbon to the deep ocean through dissolved organic matter

Oceanic dissolved organic carbon (DOC) constitutes one of the largest pools of reduced carbon in the biosphere. Estimated DOC export from the surface ocean represents 20% of total organic carbon flux to the deep ocean, which constitutes a primary control on atmospheric carbon dioxide levels. DOC is the carbon component of dissolved organic matter (DOM) and an accurate quantification of DOM pools, fluxes and their controls is therefore critical to understanding oceanic carbon cycling. DOC export is directly coupled with dissolved organic nitrogen and phosphorus export. However, the C:N:P stoichiometry (by atoms) of DOM dynamics is poorly understood. Here we study the stoichiometry of the DOM pool and of DOM decomposition in continental shelf, continental slope and central ocean gyre environments. We find that DOM is remineralized and produced with a C:N:P stoichiometry of 199:20:1 that is substantially lower than for bulk pools (typically >775:54:1), but greater than for particulate organic matter (106:16:1—the Redfield ratio). Thus for a given mass of new N and P introduced into surface water, more DOC can be exported than would occur at the Redfield ratio. This may contribute to the excess respiration estimated to occur in the interior ocean. Our results place an explicit constraint on global carbon export and elemental balance via advective pathways.

The Relationships Among Man's Activities in Watersheds and Estuaries: A Model of Runoff Effects on Patterns of Estuarine Community Metabolism

Activities of man in rivers and their watersheds have altered enormously the timing, magnitude, and nature of inputs of materials to estuaries. Despite an awareness of large-scale, long-term changes in river-estuarine watersheds, we do not fully understand the consequences to estuarine ecosystems of these activities. Deforestation, urbanization, and agriculturalization have changed the timing and nature of material inputs to estuaries. Conversion of land from forest to almost any other land use promotes overland flow of storm runoff; increases the timing, rate and magnitude of runoff; and increases sediment, organic matter, and inorganic nutrient export. It has been estimated that total organic carbon levels in rivers have increased by a factor of 3–5 over natural levels. Man’s activities have also changed the magnitude of particulate organic carbon relative to dissolved organic carbon export and the lability of the organic matter. Historically, rivers and streams had different features than they do today. Two of man’s activities that have had pronounced effects on the timing and quality of river water are channelization and damming. Agricultural drainage systems, channelized and deepened streams, and leveeing and prevention of overbank flooding have had the combined effect of increasing the amplitude and rate of storm runoff, increasing sediment load, increasing nutrient delivery downstream, and decreasing riparian wetland productivity. Dams on the other hand have altered natural discharge patterns and altered the downstream transfer of sediments, organic matter, and nutrients. Patterns of estuarine community metabolism are sensitive to variations, in the timing, magnitude, and quality of material inputs from watersheds. The autotrophic-heterotrophic nature of an estuary is determined by three primary factors: the ratio of inorganic to organic matter inputs, water residence time, and the overall lability of allochthonous organic matter inputs. A simulation model is used to explore the effects of man’s activities in watersheds on the spatial patterns of production and respiration in a generalized estuarine system. Examined are the effects of variations in the ratios of inorganic and organic nitrogen loading, the residence time of water in the estuary, the degradability of allochthonous organic matter, and the ratio of dissolved to particulate organic matter inputs. Simulations suggest that the autotrophic-heterotrophic balance in estuaries is more sensitive to variations in organic matter loading than inorganic nutrient loading. Water residence time and flocculation-sedimentation of organic matter are two physical factors that most effect simulated spatial patterns of metabolism in estuaries.

Decomposition of dissolved organic matter from the continental margin

Decomposition of dissolved organic carbon, nitrogen and phosphorus (DOC, DON, DOP) was measured for surface and bottom waters of the middle Atlantic bight (MAB) and deep slope water adjacent to the MAB on two occasions in March and August 1996. We used standard bottle incubation techniques to measure the decrease in dissolved organic matter (DOM) concentrations over a 180-day interval. Generally DOM concentrations in the MAB were elevated (125mM DOC, 10.2mM DON and 0.30mM DOP) relative to the surface ocean and deep slope water (46.7mM DOC, 2.76mM DON, 0.03mM DOP). On average the C:N:P ratio of shelf DOM (431:36:1) was substantially higher than the Redfield ratio, but not nearly as high for that of deep slope water (2700:215:1). Decomposition time course data were fit to a three-pool (very labile, labile, and recalcitrant pools) multi-G model using a Marquardt fitting routine. The threepool model was superior to a simple exponential decay model assuming a single pool of DOM. We observed no significant changes in concentration of DOM in deep-water samples, attesting to the old age of this material, its recalcitrant nature, and the cleanliness of our technique for measuring decomposition. There were major differences in the relative amount of very labile, labile and recalcitrant fractions of shelf-water DOC, DON and DOP as a result of preferential remineralization of P over N and N over C. Averaged over stations, the decomposable portion of the bulk DOC, DON and DOP pools increased from 30% to 40% to 81% for C, N and P. There was a wide range in decay coefficients for the very labile and labile DOM pools: average decay coefficient for the very labile pool was 0.219 d � 1 , and 0.018 d � 1 for the labile pool. Average half-lives calculated from the decay coefficients were 4, 12 and 8 days for the very labile DOC, DON and DOP pools, and 54, 113 and 90 days for the labile DOC, DON and DOP pools. On the basis of pool turnover times relative to shelf-water residence time (B100 days) we conclude that autochthonous algal production is the source of the very labile DOM pools. Its rate of production is sufficient to sustain estimated rates of bacteria C demand in continental margins. Our results for the MAB indicate that while substantial amounts of DOM are remineralized in the same time frame as shelf-water residence time, there is substantial DOM remaining that is depleted in N and P relative to C. Strong concentration gradients in DOM occur between shelf and ocean waters and between surface and deeper waters. Coupled with appropriate vertical and horizontal advective and eddy diffusive transports, DOM export from the MAB and other shelf systems may be a significant component of ocean C dynamics. r 2002 Elsevier Science Ltd. All rights reserved.

SUSCEPTIBILITY OF SALT MARSHES TO NUTRIENT ENRICHMENT AND PREDATOR REMOVAL

Salt marsh ecosystems have been considered not susceptible to nitrogen overloading because early studies suggested that salt marshes adsorbed excess nutrients in plant growth. However, the possible effect of nutrient loading on species composition, and the combined effects of nutrients and altered species composition on structure and function, was largely ignored. Failure to understand interactions between nutrient loading and species composition may lead to severe underestimates of the impacts of stresses. We altered whole salt marsh ecosystems (;60 000 m 2 /treatment) by addition of nutrients in flooding waters and by reduction of a key predatory fish, the mummichog. We added nutrients (N and P; 15-fold increase over ambient conditions) directly to the flooding tide to mimic the way anthropogenic nutrients are delivered to marsh ecosystems. Despite the high concentrations (70 mmol N/L) achieved in the water column, our annual N loadings (15-60 g Nm � 2 � yr � 1 ) were an order of magnitude less than most plot-level fertilization experiments, yet we detected responses at several trophic levels. Preliminary calculations suggest that 30-40% of the added N was removed by the marsh during each tidal cycle. Creek bank Spartina alterniflora and high marsh S. patens production increased, but not stunted high marsh S. alterniflora. Microbial production increased in the fertilized creek bank S. alterniflora habitat where benthic microalgae also increased. We found top-down control of benthic microalgae by killifish, but only under nutrient addition and in the opposite direction (increase) than that predicted by a fish-invertebrate-microalgae trophic cascade. Surprisingly, infauna declined in abundance during the first season of fertilization and with fish removal. Our results demonstrate ecological effects of both nutrient addition and mummichog reduction at the whole-system level, including evidence for synergistic interactions.

Improving marine ecosystem models: Use of data assimilation and mesocosm experiments

Our inability to accurately model marine food webs severely limits the prognostic capabilities of current generation marine biogeochemistry models. To address this problem we examine the use of data assimilation and mesocosm experiments to facilitate the development of food web models. The components of the data assimilation demonstrated include the construction of measurement models, the adjoint technique to obtain gradient information on the objective function, the use of parameter constraints, incorporation of discrete measurements and assessing parameter observability. We also examine the effectiveness of classic and contemporary optimization routines used in data assimilation. A standard compartment-type food web model is employed with an emphasis on organic matter production and consumption. Mesocosm experiments designed to examine the interaction of inorganic nitrogen with organic matter provide the data used to constrain the model.Although we are able to obtain reasonable e ts between the mesocosm data and food web model, the model lacks the robustness to be applicable across trophic gradients, such as those occurring in coastal environments. The robustness problem is due to inherent structural problems that render the model extremely sensitive to parameter values. Furthermore, parameters governing actual ecosystems are not constants, but rather vary as a function of environmental conditions and species abundance, which increases the sensitivity problem. We conclude by briee y discussing possible improvements in food web models and the need for rigorous comparisons between models and data (a modeling workbench) so that performance of competing models can be assessed. Such a workbench should facilitate systematic improvements in prognostic marine food web models.

A review of recent developments in estuarine scalar flux estimation

The purpose of this contribution is to review recent developments in calculation of estuarine scalar fluxes, to suggest avenues for future improvement, and to place the idea of flux calculation in a broader physical and biogeochemical context. A scalar flux through an estuarine cross section is the product of normal velocity and scalar concentration, sectionally integrated and tidally averaged. These may vary on interannual, reasonal, tidal monthly, and event time scales. Formulation of scalar fluxes in terms of an integral scalar conservation expression shows that they may be determined either through “direct” means (measurement of velocity and concentration) or by “indirect” inference (from changes in scalar, inventory and source/sink terms). Direct determination of net flux at a cross section has a long and generally discouraging history in estuarine oceanography. It has proven difficult to extract statistically significant net (tidally averaged) fluxes from much larger flood and ebb transports, and the best mathematical representation of flux mechanisms is unclear. Observations further suggest that both lateral and vertical variations in scalar transport through estuarine cross sections are large, while estuarine circulation theory has focused on two-dimensional analyses that treatment either vertical or lateral variations but not both. Indirect estimates of net fluxes by determination of the other relevant terms in an integral scalar conservation balance may be the best means of determining scalar import-export in systems with residence times long relative to periods of tidal monthly fluctuations. But this method offers, little insight into the interaction of circulation modes and scalar fluxes, little help in verifying predictive models, and may also be difficult to apply in some circumstances. Thus, the need to understand, measure, and predict anthropogenic influences on transport or carbon, nutrient, suspended matter, trace metals, and other substances across the land-margin brings a renewed urgency to the issue of how to best carry out estuarine scalar flux determination. An interdisciplinary experiment is suggested to test present understanding, available instrument, and numerical models.

Extended local similarity analysis (eLSA) of microbial community and other time series data with replicates

BackgroundThe increasing availability of time series microbial community data from metagenomics and other molecular biological studies has enabled the analysis of large-scale microbial co-occurrence and association networks. Among the many analytical techniques available, the Local Similarity Analysis (LSA) method is unique in that it captures local and potentially time-delayed co-occurrence and association patterns in time series data that cannot otherwise be identified by ordinary correlation analysis. However LSA, as originally developed, does not consider time series data with replicates, which hinders the full exploitation of available information. With replicates, it is possible to understand the variability of local similarity (LS) score and to obtain its confidence interval.ResultsWe extended our LSA technique to time series data with replicates and termed it extended LSA, or eLSA. Simulations showed the capability of eLSA to capture subinterval and time-delayed associations. We implemented the eLSA technique into an easy-to-use analytic software package. The software pipeline integrates data normalization, statistical correlation calculation, statistical significance evaluation, and association network construction steps. We applied the eLSA technique to microbial community and gene expression datasets, where unique time-dependent associations were identified.ConclusionsThe extended LSA analysis technique was demonstrated to reveal statistically significant local and potentially time-delayed association patterns in replicated time series data beyond that of ordinary correlation analysis. These statistically significant associations can provide insights to the real dynamics of biological systems. The newly designed eLSA software efficiently streamlines the analysis and is freely available from the eLSA homepage, which can be accessed at http://meta.usc.edu/softs/lsa.

Relationships between soil organic matter, nutrients, bacterial community structure, and the performance of microbial fuel cells.

Microbial fuel cells (MFCs) offer the potential for generating electricity, mitigating greenhouse gas emissions, and bioremediating pollutants through utilization of a plentiful renewable resource: soil organic carbon. We analyzed bacterial community structure, MFC performance, and soil characteristics in different microhabitats within MFCs constructed from agricultural or forest soils in order to determine how soil type and bacterial dynamics influence MFC performance. Our results indicated that MFCs constructed from agricultural soil had power output about 17 times that of forest soil-based MFCs and respiration rates about 10 times higher than forest soil MFCs. Agricultural soil MFCs had lower C:N ratios, polyphenol content, and acetate concentrations than forest soil MFCs. Bacterial community profile data indicate that the bacterial communities at the anode of the high power MFCs were less diverse than in low power MFCs and were dominated by Deltaproteobacteria, Geobacter, and to a lesser extent, Clostridia, while low-power MFC anode communities were dominated by Clostridia. These results suggest that the presence of organic carbon substrate (acetate) was not the major limiting factor in selecting for highly electrogenic bacterial communities, while the quality of available organic matter may have played a significant role in supporting high performing bacterial communities.