Fast measurement of phosphates and ammonium in fermentation-like media: A feasibility study.

Real-time monitoring of bioprocesses plays a key-role in modern industries, providing new information on full-scale production, thus enabling control of the process and allowing it to run at optimal conditions while minimizing waste. Monitoring of phosphates and ammonium in fermentation processes has a twofold interest: they are important nutrients for living organisms while at the same time constituting environmental nutrient pollutants, for which unnecessary use and disposal must be avoided. In this report, the possibility of simultaneous analysis of phosphates and ammonium in fermentations was verified using spectroscopy-based methods combined with chemometrics to construct calibration models. To achieve this, the models were based on synthetic samples mimicking real fermentation media, providing a dataset where the analytes were completely uncorrelated. Different at-line techniques (mid- and near- infrared spectroscopy, MIR and NIR) were evaluated for their ability to monitor quickly both analytes, in a wide range of concentrations (10-100 mM), in three media of different complexities. Partial Least Squares (PLS) models on MIR spectroscopy gave very good results, with prediction errors lower than 5 % for both analytes in all datasets. In contrast, the results for PLS models on NIR spectroscopy were inferior (prediction errors between 3 and 26 %) for both analytes, as, in the case of phosphate, it could be demonstrated that the model was based on based on indirect predictions.

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures.

Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters. Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for.

CFD predicted pH gradients in lactic acid bacteria cultivations.

The formation of pH gradients in a 700 L batch fermentation of Streptococcus thermophilus was studied using multi-position pH measurements and computational fluid dynamics (CFD) modeling. To this end, a dynamic, kinetic model of S. thermophilus and a pH correlation were integrated into a validated one-phase CFD model, and a dynamic CFD simulation was performed. First, the fluid dynamics of the CFD model were validated with NaOH tracer pulse mixing experiments. Mixing experiments and simulations were performed whereas multiple pH sensors, which were placed vertically at different locations in the bioreactor, captured the response. A mixing time of about 46 s to reach 95% homogeneity was measured and predicted at an impeller speed of 242 rpm. The CFD simulation of the S. thermophilus fermentation captured the experimentally observed pH gradients between a pH of 5.9 and 6.3, which occurred during the exponential growth phase. A pH higher than 7 was predicted in the vicinity of the base solution inlet. Biomass growth, lactic acid production, and substrate consumption matched the experimental observations. Moreover, the biokinetic results obtained from the CFD simulation were similar to a single-compartment simulation, for which a homogeneous distribution of the pH was assumed. This indicates no influence of pH gradients on growth in the studied bioreactor. This study verified that the pH gradients during a fermentation in the pilot-scale bioreactor could be accurately predicted using a coupled simulation of a biokinetic and a CFD model. To support the understanding and optimization of industrial-scale processes, future biokinetic CFD studies need to assess multiple types of environmental gradients, like pH, substrate, and dissolved oxygen, especially at industrial scale.

Profiling of Indigenous Microbial Community Dynamics and Metabolic Activity During Enrichment in Molasses-Supplemented Crude Oil-Brine Mixtures for Improved Understanding of Microbial Enhanced Oil Recovery.

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil-brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil-brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil-brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.

A high-throughput method for quantifying metabolically active yeast cells.

By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of the method in fermenters and high-throughput systems proved equivalent, displaying reduction curves that interrelated directly with CFU counts. For growth rate estimation, the methylene blue reduction test (MBRT) proved superior, since the discriminatory nature of the method allowed for the quantification of metabolically active cells only, excluding dead cells. The drop in metabolic activity associated with the diauxic shift in yeast proved more pronounced for the MBRT-derived curve compared with OD curves, consistent with a dramatic shift in the ratio between live and dead cells at this metabolic event. This method provides a tool with numerous applications, e.g. characterizing the death phase of stationary phase cultures, or in drug screens with pathogenic yeasts.

Synthetic promoter library for modulation of actinorhodin production in Streptomyces coelicolor A3(2).

The objective of this study was the application of the synthetic promoter library (SPL) technology for modulation of actinorhodin production in Streptomyces coelicolor A3(2). The SPL technology was used to optimize the expression of a pathway specific positive transcriptional regulator ActII orf4, which activates the transcription of the S. coelicolor actinorhodin biosynthetic gene cluster. The native actII orf4 promoter was replaced with synthetic promoters, generating a S. coelicolor library with a broad range of expression levels of actII orf4. The resulting library was screened based on the yield of actinorhodin. Selected strains were further physiologically characterized. One of the strains from the library, ScoSPL20, showed considerably higher yield of actinorhodin and final actinorhodin titer, compared to S. coelicolor wild type and S. coelicolor with actII orf4 expressed from a strong constitutive promoter. ScoSPL20 demonstrated exceptional productivity despite having a comparatively weak expression from the promoter. Interestingly, the ScoSPL20 promoter was activated at a much earlier stage of growth compared to the wild type, demonstrating the advantage of fine-tuning and temporal tuning of gene expression in metabolic engineering. Transcriptome studies were performed in exponential and actinorhodin-producing phase of growth to compare gene expression between ScoSPL20 and the wild type. To our knowledge, this is the first successful application of the SPL technology for secondary metabolite production in filamentous bacteria.

Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C6 glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

Cell mass and cell cycle dynamics of an asynchronous budding yeast population: experimental observations, flow cytometry data analysis, and multi-scale modeling.

Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model is a suitable tool for describing the microbial population dynamics in a bioreactor. This study therefore contributes towards the understanding of the development of heterogeneous populations during microbial cultivations. More generally, it consists of a step towards a paradigm change in the study and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation.

Identification of a transcription factor controlling pH-dependent organic acid response in Aspergillus niger.

Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis.

Physiological heterogeneities in microbial populations and implications for physical stress tolerance.

BACKGROUND: Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions--i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes. RESULTS: In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished. CONCLUSIONS: The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.

Comparison of laser diffraction and image analysis for measurement of Streptomyces coelicolor cell clumps and pellets.

Morphology is important in industrial processes involving filamentous organisms because it affects the mixing and mass transfer and can be linked to productivity. Image analysis provides detailed information about the morphology but, in practice, it is often laborious including both collection of high quality images and image processing. Laser diffraction is rapid and fully automatic and provides a volume-weighted distribution of the particle sizes. However, it is based on a number of assumptions that do not always apply to samples. We have evaluated laser diffraction to measure cell clumps and pellets of Streptomyces coelicolor compare to image analysis. Samples, taken five times during fed-batch cultivation, were analyzed by image analysis and laser diffraction. The volume-weighted size distribution was calculated for each sample. Laser diffraction and image analysis yielded similar size distributions, i.e. unimodal or bimodal distributions. Both techniques produced similar estimations of the population means, whereas the estimates of the standard deviations were generally higher using laser diffraction compared to image analysis. Therefore, laser diffraction measurements are high quality and the technique may be useful when rapid measurements of filamentous cell clumps and pellets are required.

Robust, small-scale cultivation platform for Streptomyces coelicolor.

BACKGROUND: For fermentation process and strain improvement, where one wants to screen a large number of conditions and strains, robust and scalable high-throughput cultivation systems are crucial. Often, the time lag between bench-scale cultivations to production largely depends on approximate estimation of scalable physiological traits. Microtiter plate (MTP) based screening platforms have lately become an attractive alternative to shake flasks mainly because of the ease of automation. However, there are very few reports on applications for filamentous organisms; as well as efforts towards systematic validation of physiological behavior compared to larger scale are sparse. Moreover, available small-scale screening approaches are typically constrained by evaluating only an end point snapshot of phenotypes. RESULTS: To address these issues, we devised a robust, small-scale cultivation platform in the form of MTPs (24-square deepwell) for the filamentous bacterium Streptomyces coelicolor and compared its performance to that of shake flasks and bench-scale reactors. We observed that re-designing of medium and inoculum preparation recipes resulted in improved reproducibility. Process turnaround time was significantly reduced due to the reduction in number of unit operations from inoculum to cultivation. The incorporation of glass beads (ø 3 mm) in MTPs not only improved the process performance in terms of improved oxygen transfer improving secondary metabolite production, but also helped to transform morphology from pellet to disperse, resulting in enhanced reproducibility. Addition of MOPS into the medium resulted in pH maintenance above 6.50, a crucial parameter towards reproducibility. Moreover, the entire trajectory of the process was analyzed for compatibility with bench-scale reactors. The MTP cultivations were found to behave similar to bench-scale in terms of growth rate, productivity and substrate uptake rate and so was the onset of antibiotic synthesis. Shake flask cultivations however, showed discrepancy with respect to morphology and had considerably reduced volumetric production rates of antibiotics. CONCLUSION: We observed good agreement of the physiological data obtained in the developed MTP platform with bench-scale. Hence, the described MTP-based screening platform has a high potential for investigation of secondary metabolite biosynthesis in Streptomycetes and other filamentous bacteria and the use may significantly reduce the workload and costs.

Industrial glucoamylase fed-batch benefits from oxygen limitation and high osmolarity.

The market for glucoamylase is large and very competitive and the production process has been optimized through several decades. So far a thorough characterization of the process has not been published, but previous academic reports suggest that the process suffers from severe byproduct formation. In this study we have carried out a thorough characterization of a process as close as possible to the industrial reality. The results show that the oxygen-limited phases of the process have the highest glucoamylase yields on carbon and that the byproducts are efficiently reused in late phases of the process. An alternative process with low glucose concentration show that high osmolarity is beneficial for the process, and we conclude that oxygen limitation, high osmolarity, and the associated byproduct metabolism are important for the efficiency of the process.

Multi-objective optimization of glycopeptide antibiotic production in batch and fed batch processes.

Fermentation optimization involves potentially conflicting multiple objectives such as product concentration and production media cost. Simultaneous optimization of these objectives would result in a multiobjective optimization problem, which is characterized by a set of multiple solutions, knows as pareto optimal solutions. These solutions gives flexibility in evaluating the trade-offs and selecting the most suitable operating policy. Here, ε-constraint approach was used to generate the pareto solutions for two objectives: product concentration and product per unit cost of media, for batch and fed batch operations using process model for Amycolatopsis balhimycina, a glycopeptide antibiotic producer. This resulted in a set of several pareto optimal solutions with the two objectives ranging from (0.75 g l(-1), 3.97 g $(-1)) to (0.44 g l(-1), 5.19 g $(-1)) for batch and from (1.5 g l(-1), 5.46 g $(-1)) to (1.1 g l(-1), 6.34 g $(-1)) for fed batch operations. One pareto solution each for batch and for fed batch mode was experimentally validated.

Topology optimized microbioreactors.

This article presents the fusion of two hitherto unrelated fields--microbioreactors and topology optimization. The basis for this study is a rectangular microbioreactor with homogeneously distributed immobilized brewers yeast cells (Saccharomyces cerevisiae) that produce a recombinant protein. Topology optimization is then used to change the spatial distribution of cells in the reactor in order to optimize for maximal product flow out of the reactor. This distribution accounts for potentially negative effects of, for example, by-product inhibition. We show that the theoretical improvement in productivity is at least fivefold compared with the homogeneous reactor. The improvements obtained by applying topology optimization are largest where either nutrition is scarce or inhibition effects are pronounced.

Application of mechanistic models to fermentation and biocatalysis for next-generation processes.

Mechanistic models are based on deterministic principles, and recently, interest in them has grown substantially. Herein we present an overview of mechanistic models and their applications in biotechnology, including future perspectives. Model utility is highlighted with respect to selection of variables required for measurement, control and process design. In the near future, mechanistic models with a higher degree of detail will play key roles in the development of efficient next-generation fermentation and biocatalytic processes. Moreover, mechanistic models will be used increasingly in the frame of multi-objective decision-making under uncertainty and to promote increased selectivity of products.

Sensor combination and chemometric variable selection for online monitoring of Streptomyces coelicolor fed-batch cultivations.

Fed-batch cultivations of Streptomyces coelicolor, producing the antibiotic actinorhodin, were monitored online by multiwavelength fluorescence spectroscopy and off-gas analysis. Partial least squares (PLS), locally weighted regression, and multilinear PLS (N-PLS) models were built for prediction of biomass and substrate (casamino acids) concentrations, respectively. The effect of combination of fluorescence and gas analyzer data as well as of different variable selection methods was investigated. Improved prediction models were obtained by combination of data from the two sensors and by variable selection using a genetic algorithm, interval PLS, and the principal variables method, respectively. A stepwise variable elimination method was applied to the three-way fluorescence data, resulting in simpler and more accurate N-PLS models. The prediction models were validated using leave-one-batch-out cross-validation, and the best models had root mean square error of cross-validation values of 1.02 g l(-1) biomass and 0.8 g l(-1) total amino acids, respectively. The fluorescence data were also explored by parallel factor analysis. The analysis revealed four spectral profiles present in the fluorescence data, three of which were identified as pyridoxine, NAD(P)H, and flavin nucleotides, respectively.

Reducing the variability of antibiotic production in Streptomyces by cultivation in 24-square deepwell plates.

Highly reproducible production values of the aminocoumarin antibiotic novobiocin were achieved by cultivation of a heterologous Streptomyces producer strain in commercially available square deepwell plates consisting of 24 wells of 3 ml culture volume each. Between parallel cultivation batches in the deepwell plates, novobiocin accumulation showed standard deviations of 4-9%, compared to 39% in baffled Erlenmeyer flasks. Mycelia used as inoculum could be frozen in the presence of 20% peptone and stored at -70 degrees C, allowing repeated cultivations from the same batch of inoculum over extended periods of time. Originally, novobiocin titers in the deepwell plate (5-12 mg l(-1)) were lower than in Erlenmeyer flasks (24 mg l(-1)). Optimization of the inoculation procedure as well as addition of a siloxylated ethylene oxide/propylene oxide copolymer, acting as oxygen carrier, to the production medium increased novobiocin production to 54 mg l(-1). The additional overexpression of the pathway-specific positive regulator gene novG increased novobiocin production to 163 mg l(-1). Harvesting the precultures in a defined section of growth phase greatly reduced variability between different batches of inoculum. The use of deepwell plates may considerably reduce the workload and cost of investigations of antibiotic biosynthesis in streptomycetes and other microorganisms due to the high reproducibility and the low requirement for shaker space and culture medium.

Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina.

A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially and constitutively expressed protein isoforms, which were associated with 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2-DE reference maps covering 3-10, 4-7 and 4.5-5.5 pH gradients available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism intermediates, were upregulated during antibiotic production. qRT-PCR analysis revealed that 8 out of 14 upregulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two nonproducing mutants, restricted to a sub-set of differentially expressed proteins, showed that most proteins required for the biosynthesis of balhimycin precursors are downregulated in both mutants. These findings suggest that primary metabolic pathways support anabolic routes leading to balhimycin biosynthesis and the differentially expressed genes are interesting targets for the construction of high-yielding producer strains by rational genetic engineering.

In situ near infrared spectroscopy for analyte-specific monitoring of glucose and ammonium in streptomyces coelicolor fermentations.

There are many challenges associated with in situ collection of near infrared (NIR) spectra in a fermentation broth, particularly for highly aerated and agitated fermentations with filamentous organisms. In this study, antibiotic fermentation by the filamentous bacterium Streptomyces coelicolor was used as a model process. Partial least squares (PLS) regression models were calibrated for glucose and ammonium based on NIR spectra collected in situ. To ensure that the models were calibrated based on analyte-specific information, semisynthetic samples were used for model calibration in addition to data from standard batches. Thereby, part of the inherent correlation between the analytes could be eliminated. The set of semisynthetic samples were generated from fermentation broth from five separate fermentations to which different amounts of glucose, ammonium, and biomass were added. This method has previously been used off line but never before in situ. The use of semisynthetic samples along with validation on an independent batch provided a critical and realistic evaluation of analyte-specific models based on in situ NIR spectroscopy. The prediction of glucose was highly satisfactory resulting in a RMSEP of 1.1 g/L. The prediction of ammonium based on NIR spectra collected in situ was not satisfactory. A comparison with models calibrated based on NIR spectra collected off line suggested that this is caused by signal attenuation in the optical fibers in the region above 2,000 nm; a region which contains important absorption bands for ammonium. For improved predictions of ammonium in situ, it is suggested to focus efforts on enhancing the signal in that particular region.