Multifunctional properties of phosphate-solubilizing microorganisms grown on agro-industrial wastes in fermentation and soil conditions

One of the most studied approaches in solubilization of insoluble phosphates is the biological treatment of rock phosphates. In recent years, various techniques for rock phosphate solubilization have been proposed, with increasing emphasis on application of P-solubilizing microorganisms. The P-solubilizing activity is determined by the microbial biochemical ability to produce and release metabolites with metal-chelating functions. In a number of studies, we have shown that agro-industrial wastes can be efficiently used as substrates in solubilization of phosphate rocks. These processes were carried out employing various technologies including solid-state and submerged fermentations including immobilized cells. The review paper deals critically with several novel trends in exploring various properties of the above microbial/agro-wastes/rock phosphate systems. The major idea is to describe how a single P-solubilizing microorganism manifests wide range of metabolic abilities in different environments. In fermentation conditions, P-solubilizing microorganisms were found to produce various enzymes, siderophores, and plant hormones. Further introduction of the resulting biotechnological products into soil-plant systems resulted in significantly higher plant growth, enhanced soil properties, and biological (including biocontrol) activity. Application of these bio-products in bioremediation of disturbed (heavy metal contaminated and desertified) soils is based on another important part of their multifunctional properties.     

NMR studies on heavy metal immobilization in biosorbents and mineral matrices

Magnetic resonance imaging and otherNMR methods have a potential for thenon-destructive observation of environmentallyrelevant processes with both spatial andtemporal resolution. Among other applications,such methods can be used to study transport andimmobilization of paramagnetic heavy metal ionsin biosorbents and other matrices. Thisoverview covers various NMR approaches to studysuch processes and illustrates them withexamples of imaging on alginate-basedbiosorbents and on heavy-metal doped gypsumpastes. Experimental challenges in studies ofother matrices are shortly addressed as well.     

Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives

Solid-state fermentation (SSF) is accompanied inevitably by development of concentration and temperature gradients within the substrate particles and microbial biofilms. These gradients are needed for driving the transport of substrates and products. In addition, concentration gradients have been suggested to be crucial for obtaining the characteristics that define the products of SSF; nevertheless, gradients are also known to result in reduced productivity and unwanted side reactions. Solid-state fermentations are generally batch processes and this further complicates their understanding as conditions change with time. Mathematical models are therefore needed for improving the understanding of SSF processes and allowing their manipulation to achieve the desired outcomes. Existing models of SSF processes describe coupled substrate conversion and diffusion and the consequent microbial growth. Existing models disregard many of the significant phenomena that are known to influence SSF. As a result, available models cannot explain the generation of the numerous products that form during any SSF process and the outcome of the process in terms of the characteristics of the final product. This review critically evaluates the proposed models and their experimental validation. In addition, important issues that need to be resolved for improved modeling of SSF are discussed.     

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.     

Controlled gas environments in industrial fermentations

The gas environment is of major importance in controlling aerobic fermentation processes for the manufacture of microbial products. Oxygen and carbon dioxide levels in gas-liquid equilibria affect productivity and energy consumption in such processes and appear to be implicated in the regulation of microbial metabolism. Gas-liquid transfer has been intensively studied by many investigators for Newtonian and non-Newtonian fluids, primarily in terms of oxygen-limitation in biomass and product formation. More recentreports show that microbial growth and product formation are affected by levels of oxygen and carbon dioxide in the gas environment, suggesting that microbial metabolism may be directed towards specific products by the control of such environments. High product concentrations may also be obtained by solid substrate fermentations with mycelial organisms cultured on semi-solid agricultural products at low moisture contents. Such methods are commonly used in the Orient for the manufacture of enzymes and traditional fermented foods and could probably be extended to other microbial products. This review covers fundamental aspects of engineering research in microbial processes that suggest applications for controlled gas environments in submerged culture and solid substrate fermentations of potential industrial interest.     

Pulsed feeding during fed-batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression

The goal in this study was to determine if pulsed addition of substrate could be used to alter filamentous fungal morphology during fermentation, to result in reduced broth viscosity. In all experiments, an industrially relevant strain of Aspergillus oryzae was grown in 20-liter fermentors. As a control, cultures were fed limiting substrate (glucose) continuously. Tests were performed by altering the feeding strategy so that the same total amount of glucose was fed in repeated 300-s cycles, with the feed pump on for either 30 or 150 s during each cycle. Variables indicative of cellular metabolic activity (biomass concentration, oxygen uptake rate, base consumed for pH control) showed no significant difference between continuous and pulse-fed fermentations. In addition, there was no significant difference between total extracellular protein expression or the apparent distribution of these proteins. In contrast, fungal mycelia during the second half of pulse-fed fermentations were approximately half the size (average projected area) of fungi during fermentations with continuous addition of glucose. As a result, broth viscosity during the second half of pulse-fed fermentations was approximately half that during the second half of continuous fermentations. If these results prove to be applicable for other fungal strains and processes, then this method will represent a simple and inexpensive means to reduce viscosity during filamentous fungal fermentation.     

Equipment for the automatic aseptic inoculation and sampling of microbial cultures

Equipment has been designed and constructed for the automatic aseptic inoculation and sampling of microbial cultures. It enables fermentations to be started and samples to be taken round-the-clock, according to a predetermined program. The equipment is suitable for microbial processes on both a laboratory and a factory scale.     

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.     

Scale-up of monoclonal antibody purification processes

Mammalian cell culture technology has improved so rapidly over the last few years that it is now commonplace to produce multi-kilogram quantities of therapeutic monoclonal antibodies in a single batch. Purification processes need to be scaled-up to match the improved upstream productivity. In this chapter key practical issues and approaches to the scale-up of monoclonal antibody purification processes are discussed. Specific purification operations are addressed including buffer preparation, chromatography column sizing, aggregate removal, filtration and volume handling with examples given.     

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.     

Koji – where East meets West in fermentation

Almost all biotechnological processes originate from traditional food fermentations, i.e. the many indigenous processes that can be found already in the written history of thousands of years ago. We still consume many of these fermented foods and beverages on a daily basis today. The evolution of these traditional processes, in particular since the 19th century, stimulated and influenced the development of modern biotechnological processes. In return, the development of modern biotechnology and related advanced techniques will no doubt improve the process, the product quality and the safety of our favourite fermented foods and beverages. In this article, we describe the relationship between these traditional food fermentations and modern biotechnology. Using Koji and its derived product soy sauce as examples, we address the mutual influences that will provide us with a better future concerning the quality, safety and nutritional effect of many fermented food products.     

Management of the recurrent, benign tumor of the parotid gland.

On occasion, the salivary gland surgeon is faced with clinical management questions related to the treatment of parotid gland tumors. Some of these are intraoperative and include margin of resection, management of the facial nerve when tumor abuts its branches, judicious use of lymphadenectomy, and the appropriateness of nerve grafting. Other questions concern postoperative care-such as the use of radiation therapy, reoperation when a malignancy is reported after surgery, timing of follow-up, and management of a recurrent, benign tumor. Although all of these issues cannot be addressed in one review article, the author has attempted to offer a rational approach to one of the most perplexing-the recurrent, benign tumor. The recommendations offered are based on the literature and a clinical experience of two decades of salivary gland surgery. Within this review a number of other management issues noted above are also addressed.     

Effects of controlled gas environments in solid-substrate fermentations of rice

The gas environment is solid-substrate fermentations of rice significantly affected levels of biomass and enzyme formation by a fungal species screened for high amylase production. Constant oxygen and carbon dioxide partial pressures were maintained at various levels in fermentations by Aspergillus oryzae. Control of the gas phase was maintained by a “static” aeration system admitting oxygen on demand and stripping excess carbon dioxide during fermentation. Constant water vapor pressures were also maintained by means of saturated salt solutions. High Oxygen pressures stimulated amylase productivity significantly. On the other hand, amylase production was severely inhibited at high carbon dioxide pressures. While relatively insensitive to oxygen pressure, maximum biomass productivities were obtained at an intermediate carbon dioxide pressure. High oxygen transfer rates were obtained at elevated oxygen pressures, suggesting, in view of the stimulatory effect of oxygen on amylase production, a stringent oxygen requirement for enzyme synthesis. Solid-substrate fermentations were highly advantageous as compared with submerged cultures in similar gas environments. Not only were amylase productivities significantly higher, but the enzyme was highly concentration in the aqueous phase of the semisolid substrate particles and could be extracted in a small volume of liquid. Results of this work suggest that biomass and product formation in microbial processes may be amenable to control by the gas environment. This is believed to offer an interesting potential for optimizing selected industrial fermentation processes with respect to productivity and energy consumption.     

Estimation of yield, maintenance, and product formation kinetic parameters in anaerobic fermentations

In many anaerobic fermentation processes, high energy bonds in adenosine triphosphate (ATP) are produced when available electrons are converted from organic substrate into extracellular organic products such as ethanol. The true growth yield and maintenance parameters are directly related to the product formation kinetic parameters for these anaerobic processes. Methods are presented which allow all of the experimental measurements to be used simultaneously to estimate these parameters. Results are presented for several different anaerobic fermentations.     

Continuous Fermentation in high flow rate fermenter systems

In commercial batch processes the productivity of product formation is low. But a significant increase of productivity can be achieved in continuous fermentations. By using high flow rate fermenter systems characterized by a relatively long retention time of biomass in comparison with the retention time of the liquid we can realize a high-performance fermentation. The problem of holding back the biomass within the reactor could be solved by means of membranes being impenetrable to the cells, but permeable to the hydraulic phase. Such a process technology was successfully tested for its applicability in alcoholic and lactic acid fermentations. The maximum productivities obtained on this way were ? = 120 g/l. · h for ethanol production and ? = 51 g/l. h for lactic acid fermentation, respectively.     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

Pure Culture Fermentation of Brined Cucumbers

The relative abilities of Pediococcus cerevisiae, Lactobacillus plantarum, L. brevis, and several other species of lactic acid bacteria to grow and produce acid in brined cucumbers were evaluated in pure culture fermentations. Such fermentations were made possibly by the use of two techniques, gamma radiation (0.83 to 1.00 Mrad) and hot-water blanching (66 to 80 C for 5 min), designed first to rid the cucumbers of naturally occurring, interfering, and competitive microbial groups prior to brining, followed by inoculation with the desired lactic acid bacteria. Of the nine species tested, strains of the three common to cucumber fermentations, P. cerevisiae, L. plantarum, and L. brevis, grew to the highest populations, and produced the highest levels of brine acidity and the lowest pH values in fermentations at 5.4 to 5.6% NaCl by weight; also, their sequence of active development in fermentations, with the use of a three-species mixture for inoculation, was in the species order just named. This sequence of occurrence was similar to that estimated by others for natural fermentations. The rates of growth and acid production in fermentations with a mixture of P. cerevisiae, L. plantarum, and L. brevis increased as the incubation temperature was increased from 21 to 27 to 32 C; however, the maximal populations and acidities attained were essentially the same for fermentations at each temperature. Further, these same three species were found to be the most salt tolerant of those tested; their upper limit for appreciable growth and measurable acid production was about 8% salt, whereas thermophilic species such as L. thermophilus, L. lactis, L. helveticus, L. fermenti, and L. delbrueckii exhibited a much lower salt tolerance, ranging from about 2.5 to 4.0%. However, certain strains of L. delbrueckii grew very rapidly in cucumbers brined at 2.5 to 3.0% salt, and produced sufficient acid in about 30 hr at 48 C to reduce the brine pH from above 7.0 to below 4.0. An inexpensive, pure culture fermentor which was suitable for gamma radiation, resistant to salt and acid, and which permitted repeated aseptic sampling of the fermenting brine, is illustrated and the specifications are given.