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Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled cost competitive reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production, while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. The effects of ascorbic acid, L- cysteine, and dithiothreitol (DTT) on ABE fed-batch production using Clostridium acetobutylicum was investigated in current study. NADH, ATP, extracellular amino acid secretion, and NADH-dependent butanol dehydrogenase (BDH) assays were performed to study the metabolic modifications triggered by reducing agents. Incidentally, L- cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. Elevated NADH, BDH, and ATP levels in fermentation broth reflected in enhanced ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress tolerance in this study. The results demonstrate that addition of reducing agents in fed-batch ABE fermentation operation enables efficient utilization of glucose with significant improvement in solvent production.

Cultivation of aerobic microorganisms typically requires rapid, but gentle and simultaneously energy efficient mixing in bioreactors to prevent high gradients between local concentrations. The mixing efficiency in a pilot scale airlift bioreactor with helical flow promoters was investigated with varying air inputs. Helical flow promoters were helical tubes attached to riser or downcomer, or both and besides acting as flow promoter they were used as additional gas spargers. Mixing time, circulation time, dimensionless mixing time and Bodenstein number were used as assessment parameters to analyze the mixing efficiency of different airlift setups with helical flow promoters. Introducing air through the helical flow promoter in the downcomer zone prolonged the circulation time, but shortened the mixing time and decreased the Bodenstein number. Low Bodenstein number indicated an improved axial dispersion. Mixing was more related to dispersion in the airlift reactor with helical flow promoter in the downcomer zonewhereas in a regular airlift reactor mixing was related to liquid circulation speed.

Maize ( Zea mays L.) is one of the main crops, which is easily susceptable to Aspergillus flavus infection resulting in huge losses worldwide. This study was carried out to investigate the effect of combining heat and irradiation treatments in controlling the fungal growth in maize grains. Surface disinfected maize grains were artificially contaminated with spores of Aspergillus flavus Link NRRL 5906, and then exposed to gamma radiation with doses of 3.0, 4.0 and 5.0 kGy. The samples were additionally heat treated at 60 °C for 30 min. The heat and irradiation treatments showed a synergistic effect on controlling Aspergillus flavus growth. The heat treatment reduced the required radiation dose of about 0.5–1.0 kGy when 4.0 kGy or 5.0 kGy irradiation was used. The combined heat and irradiation treatment of moisture reduced the average CFU by 8 log cycles when 4 kGy or 5 kGy irradiation was used and by 7 log cycles when 3 kGy irradiation was used. The heat treatment of moisture alone reduced the average CFU by only by 0.8 log cycles. Combining irradiation with heat treatment to reduce the required radiation dose is very useful especially when there is a concern over biological side effects of irradiation.

A novel pilot scale helix airlift reactor (helix-ALR) consisting of a regular draft tube airlift reactor (Ad/Ar = 1.17) and helical tubes attached to riser or downcomer, or both, was studied in different configurations with varying air inputs. The helical tubes acted as helical flow promoters (HFP) and additional gas spargers. The aim was to investigate the performance of these HFP-gas spargers and study their effect on oxygen transfer in ALR. HFP-gas spargers improved the volumetric oxygen transfer coefficients of the ALR radically even without additional energy, reaching up to 3 times higher kLa-values (over 0.1 l/s) when compared to regular ALR. Small bubbles from the HFP-gas spargers played an important role in the oxygen transfer, and downcomer helix had greater effect in general on kLa and hydrodynamics than riser helix. Empirical correlations regarding mass transfer and gas holdups were formed. When compared to stirred tank reactors (STR) the required specific power input of the helix-ALR for a level of kLa 0.1 l/s was around 0.34 kW/m3 while for STR 2.0 kW/m3, suggesting that the helix-ALR is very energy efficient. HFP-gas spargers can significantly improve the performance of ALRs regarding oxygen transfer, lower their operating costs and improve the total energy economy.

The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from 13 C Metabolic Flux Analysis ( 13 C-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40 n-% rhamnose, 34 n-% glucose, 13 n-% mannose, 10 n-% galactose, and 2 n-% arabinose. The EPS was studied to have butanol adsorbing property, 70 (butanol) mg (EPS) g −1 at 37 °C.

The cell immobilization potential of a novel xylan based disulfide-crosslinked hydrogel matrix reinforced with cellulose nanocrystals was studied with continuous cultivation of Propionibacterium acidipropionici using various dilution rates. The cells were immobilized to hydrogel beads suspended freely in the fermentation broth or else packed into a column connected to a stirred tank reactor. The maximum propionic acid productivity for the combined stirred tank and column was 0.88 g L-1 h-1 and the maximum productivity for the column was determined to be 1.39 g L-1 h-1. The maximum propionic acid titer for the combined system was 13.9 g L-1 with a dilution rate of 0.06 h-1. Dry cell density of 99.7 g L-1 was obtained within the column packed with hydrogel beads and productivity of 1.02 g L-1 h-1 was maintained in the column even with the high circulation rate of 3.37 h-1.

Cultivations of Streptomyces peucetius in two types of medium were monitored on-line using a Fourier transform infrared (FTIR) spectrometer combined with an attenuated total reflection probe. The quantitative measurements of the glucose, starch and acetate concentrations were implemented using partial least squares calibration models. These were regressed on spectral and concentration information obtained by adding together single constituent spectra of the main constituents in the medium according to a full factorial design. The accuracy achieved was considered to be satisfactory, with an average root mean square error of prediction of 1.5 g/l for glucose and 0.25 g/l for acetate. The methodology used is considered to be a rapid technique for generation of calibration data, and a step towards the use of library type data for calibration purposes in quantitative FTIR spectroscopy applications in bioprocesses.

Inhomogeneous mixing in industrial-sized fermentation processes causes oscillations in process parameters such as temperature or pH value in the cultivation medium, which causes stress to the bacteria being cultivated. In this work, the impact of extracellular pH oscillations on the production of Lactobacillus rhamnosus, a well-studied probiotic bacteria, were investigated by means of a scale-down batch process, simulating inhomogeneous pH values by controlling the pH value of the medium on sinusoidal trajectories. Effects of pH stimulation on the bacteria were assessed by testing storage and freeze-drying stability of harvested cells, two factors relevant for the industrial process. Furthermore, gene expressions of six selected genes, i.e. atpA, fat, cfa, groEL, hrcA, and pstS, known to be related to stress response were monitored. Although storage stability is only slightly negatively affected by pH stimulation of the bacteria, gene expression of four of the studied genes, i.e. fat, hrcA, groEL, and pstS show to correlate with amplitude and frequency of the oscillation.

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.

This study presents an analysis of a Bifidobacterium longum ATCC 15707 optimization study. Kinetic growth models were fitted to cultivations from a central composite circumscribed (CCC) experiment design for three variables (temperature as well as glucose and yeast extract concentration). The parameters of these kinetic models, μ max , K S , Y XS and m S , were used as responses of the experiment design. This novel concept of combining optimization and modeling presented slightly different optimal conditions for B. longum growth from the original optimization study. However, the optimum of this study could be based on more scientific arguments. The parameters of the kinetic model represent the physiological effects that the cultivation parameters impose on the organism. A difference was observed in the optimum of the initial glucose concentration, which was originally thought to benefit process efficiency. This re-analysis showed that it is better for all aspects of cell physiology (substrate efficiency and growth rate) to use a lower initial glucose concentration.

The aim of the work was to generate a flexible measurement, control and simulation environment for laboratory and pilot scale experiments. Various measurement devices were connected and synchronized to an integrated system with many bioreactors. Integrated devices were for example biomass monitoring, mass spectrometer and glucose control. Microcontroller-based interface modules and device drivers capable of transferring data to Multi Fermentor Control System (MFCS) via software interface were developed for each integrated device. A simple network TCP/IP interface enabled to distribute measurement devices in the laboratory intranet. The final result was an enhanced and flexible bioreactor data acquisition system.

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants mu (max) over 0.10 h(-1), Y (XS) 0.536 g g(-1), and m(S) 0.54 mg g(-1) h(-1). The product of secondary metabolism, epsilon-rhodomycinone, was produced with characteristics Y (PX) 12.99 mg g(-1) and m (P) 1.20 mg g(-1) h(-1). Significant correlations were found for phosphate and glucose consumption with biomass and epsilon-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated epsilon-rhodomycinone flux.

Bifidobacteria are used as probiotics mainly in the dairy industry as cell suspensions or as freeze-dried additives. So far there have been no reports on a thorough investigation on factors influencing the production process or a statistical approach to the optimization thereof. A 2(8-4) fractional factorial design was used in determining the critical parameters influencing bioreactor cultivations of Bifidobacterium longum ATCC 15707. Glucose, yeast extract and l-cysteine concentrations were found critical for the cultivation of this strain. Glucose and yeast extract concentrations were further studied together with temperature in a three factor central composite design. The optimized cultivation conditions were temperature 40 degrees C, yeast extract concentration 35 gl(-1) and glucose concentration 20 gl(-1). Freeze-drying of frozen cell suspensions of B. longum was studied first in controlled temperatures and thereafter with temperature programming experiments. The results were statistically evaluated. A temperature program with a 2 h temperature gradient from -10 to 0 degrees C, a 10 h temperature gradient from 0 to +10 degrees C and a 12 h temperature hold at +10 degrees C was found best for the freeze-drying process. Temperature programming reduced drying times by over 50% and improved the product activity by over 160%.

Cross-linked crystals of glucose isomerase (CLGI) were characterized as a liquid chromatographic separation material. The experiments were done with crystals having an average diameter of 83 µm. Porosity (epsilon(p)) and pore size distribution of the CLGI crystals were measured with size exclusion chromatography using D(2)O and polyethylene glycols as probes. CLGI material was capable of separating1000 g/mol polyethylene glycols. Fifty two percent (epsilon(p) = 0.47) of the total crystal volume was in pores. Pore size measurement showed that CLGI crystals were microporous material, having an average apparent pore diameter of 29 +/- 0.08 Å. CLGI material separated n-alcohols C(1) to C(8) based on the hydrophobic interaction between the protein material and the carbon chain of the alcohols. Height equivalent to a theoretical plate (HETP, in millimeters) ranged from 1.6 to 0.89 for the C(1) to C(7) n-alcohol series. Despite the large crystal size, CLGI as a chirally active phase effectively separated D- and L-arabitol (R(s) = 0.58) and showed potential for chiral separation of amino acids.

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

For the purpose of producing thermoplastic starch, barley starch was extruded in mixtures of water and glycerol in a twin-screw extruder equipped with a specially constructed slit die. The process parameters — water feed (15–25 g/min), glycerol feed (15–25 g/min), barrel temperature (150–200 °C) and screw speed (272–400 rpm) — were varied according to a statistical experimental design consisting of 28 trials. The values of the process parameters were selected to lie within ranges technically feasible with the equipment. Starch amylopectin was clearly depolymerized, and starch retained on average molar mass at the level of (13–37) × 106g/mol. Intrinsic viscosities decreased from 230 mL/g to about 150 mL/g. The tensile strength depended only on glycerol content in the experimental area, varying from 4.5 to 16 MPa. The specific mechanical energy consumption was 85–240 W.h/kg. In all trials, water had only a slightly greater effect than did glycerol on the dependent variables. All the models of dependent variables were statistically very significant. In the experimental range used it was possible to produce thermoplastic starch with relatively high molar mass and mechanical strength.

In this study a step-wise optimization procedure was developed to predict solvent production using continuous ABE fermentation with immobilized cells. The modeling approach presented here utilizes previously published constraint-based metabolic model for Clostridium acetobutylicum without direct flux constraints. A recently developed flux ratio constraint method was adopted for the model. An experimental data set consisting of 25 experiments using different sugar mixtures as substrates and differing dilution rates was simulated successfully with the modeling approach. Converted to end product concentrations the mean absolute error for acetone was 0.31 g/l, for butanol 0.49 g/l, and for ethanol 0.17 g/l. The modeling approach was validated with another data set from similar experimental setup. The model errors for the validation data set was 0.24 g/l, 0.60 g/l, and 0.17 g/l for acetone, butanol, and ethanol, respectively.

Intelligent computer programs capable of state estimation and prediction into the future would be helpful as ‘software sensors’ when dealing with bioprocesses characterized by uncertainties and complexity. Fuzzy logic has been shown to be a valuable tool in dealing with vague and incomplete information, and in incorporating human expert knowledge into process models. Neural network programs capable of learning from past experience are useful when no exact mathematical information on the process under investigation is available. This paper discusses the state of the art of such novel tools of artificial intelligence, with examples demonstrating their potential in food-related applications.

In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.

Determination of metabolites from an anaerobic digester with an acid base titration is considered as superior method for many reasons. This paper describes a practical at line compatible multipoint titration method. The titration procedure was improved by speed and data quality. A simple and novel control algorithm for estimating a variable titrant dose was derived for this purpose. This non-linear PI-controller like algorithm does not require any preliminary information from sample. Performance of this controller is superior compared to traditional linear PI-controllers. In addition, simplification for presenting polyprotic acids as a sum of multiple monoprotic acids is introduced along with a mathematical error examination. A method for inclusion of the ionic strength effect with stepwise iteration is shown. The titration model is presented with matrix notations enabling simple computation of all concentration estimates. All methods and algorithms are illustrated in the experimental part. A linear correlation better than 0.999 was obtained for both acetate and phosphate used as model compounds with slopes of 0.98 and 1.00 and average standard deviations of 0.6% and 0.8%, respectively. Furthermore, insensitivity of the presented method for overlapping buffer capacity curves was shown.

Biomass measurement is one of the most critical measurements in biotechnological processes. The technologies developed for the measurement of biomass in situ have developed over the years. Because it has been over 10 years since the last review concentrating on practical issues concerning biomass measurements, it is time to evaluate recent developments in the field. This review concentrates on the applications of dielectric spectroscopy, optical density, infrared spectroscopy, and fluorescence for in situ measurement of biomass. The advantages offered by these methods and an economic way of estimating biomass concentration, the software sensors, are considered.

Two on-line probes for biomass measurement in bioreactor cultivations were evaluated. One probe is based on near infrared (NIR) light absorption and the other on dielectric spectroscopy. The probes were used to monitor biomass production in cultivations of several different microorganisms. Differences in NIR probe response compared to off-line measurement methods revealed that the most significant factor affecting the response was cell shape. The NIR light absorption method is more developed and reliable for on-line in situ biomass estimation than dielectric spectroscopy. The NIR light absorption method is, however, of no significant use, when the cultivation medium is not clear, and especially in processes using adsorbents or solid matrix for the microorganism to grow on. The possibilities offered by dielectric spectroscopy are impressive, but the on-line probe technology needs to be improved.

The bacterial species Enterobacter amnigenus was isolated from sugar beets harvested in Finland. It produced an exopolysaccharide rich in l-fucose, which gave viscous water solutions. Its primary structure was determined mainly by NMR spectroscopy and ESIMS of oligosaccharides and a polysaccharide with decreased molecular weight, obtained by Smith degradation of the O-deacetylated native polymer [carbohydrate structure: see text]

Streptomyces peucetius var. caesius is an aerobic bacterium that produces doxorubicin as a secondary metabolite. A mixture design was applied for the screening of suitable complex medium components in the cultivation of S. peucetius var. caesius N47, which is an epsilon-rhodomycinone-accumulating mutant strain. epsilon-Rhodomycinone is a non-glycosylated precursor of doxorubicin. Best growth results were obtained with soy peptone and beef extract. A central composite face-centered (CCF) experimental design was constructed for the investigation of pH, temperature and dissolved oxygen (DO) effects on the cultivation growth phase. Another CCF was applied to the production phase to investigate the effects of aeration, pH, temperature and stirring rate on epsilon-rhodomycinone production. An increase in cultivation temperature increased both cell growth and glucose consumption rate. Best epsilon-rhodomycinone productivities were obtained in temperatures around 30 degrees C. DO control increased all growth phase responses, but aeration in the production phase coupled with pH decrease resulted in rapid epsilon-rhodomycinone decay in the medium. In non-aerated production phases a pH change resulted in better productivity than in experiments without pH change. A pH increase with a temperature decrease seemed most beneficial for productivity. This implies that dynamic control strategies in batch production of epsilon-rhodomycinone could increase the overall process productivity.

Neural networks are computer programs initially developed to mimic the function of brain. They are characterized by their ability to learn from past experiences. The superwised backpropagation learning procedure presented in the late 1980s opened up the way to practical engineering applications involving dynamic, highly non-linear systems. Neural networks have been demonstrated to be a powerful tool in control, estimation, fault detection, time-series analysis, etc. The paper discusses briefly the principles of multilayer, backpropagation networks and describes a program package written in Microsoft® VisualC++™ for Windows™. Determining the end point of β-galactosidase fermentation by multistep ahead prediction, estimating of substrate and product in lysine fermentation, and dynamic control of an extrusion cooker are given as examles of neuroengineering applications.