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FAIR-CT96-3116
Improvement of a process technology to scale-up liquid cultures of biocontrol nematodes |
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Proposal No: | FAIR-CT96-3116 |
| Date Prepared: | July 2001, November 1999, September 1999 | |
| Source: | Final Report
Abstract and Executive Summary Progress Report September 1999 Progress Report August 1998 |
Final Report Abstract
Source: Final Report, February 2001
Consortium: The project was co-ordinated by the Institute for Phytopathology, Christian-Albrechts-University, Raisdorf (Germany) in partnership with Bionema AB, Umea (Sweden), e-nema GmbH Raisdorf (Germany), The Biology Department, St Patrick's College, Maynooth (Ireland) and the Department of Nematology, Volcani Center, Bet-Dagan (Israel).
Abstract
Biocontrol nematodes are safe antagonists of soil insect pests in ornamentals, vegetables, fruit and turf. The main aim of the PRONEMA project was to provide highly effective nematodes for outdoor control of insect pests at economically reasonable product prices. The primary objective was to develop standard operation protocols for low-cost liquid culture and downstream processing that had the potential to be scaled-up to the industrial scale. It was further planned to enhance the level of productivity of the nematodes by genetic improvement to produce strains better adapted to liquid culture conditions. Another aim was the development of quality control procedures.
During the course of the project (September 1997 to August 2000) most of the major objectives were met. A reduction of production costs was achieved through the scale-up of the technology to 3000 litre volume bioreactors, a reduction in the process time and and increase in product yields. Fundamental results concerning the biology of the nematodes and their symbiotic bacteria enabled a better understand the mechanisms of the symbiotic interaction and reproduction biology of the nematodes. This enabled the development of models to predict the yields of liquid culture processes. Based on these results standard operation protocols were improved and the risk of process failures was significantly reduced. In addition, improvements on the downstream technology have helped to reduce labour and to minimize losses during product recovery. Tests have been developed to monitor nematode quality.
Heterorhabditis spp. are symbiotically associated with bacteria of the species Photorhabdus luminescens, which are the essential food source for the nematodes. The nematode dauer juvenile (DJ) is the infective stage and carries cells of its symbiont in the intestine. It is adapted to long term survival and is therefore used for product formulation and storage until applied in the field by means of conventional spraying equipment.
After penetration into the insect, the DJs exit recover from this arrested stage, release the bacteria and develop to self-fertilizing hermaphrodites. Their offspring can either develop to amphimictic females and males or form pre-dauer juveniles (J2d). This option is determined during the first juvenile stage. High nematode population density and low concentrations of food induce the development to the J2d, whereas low nematode density and high concentration of food induces the development to amphimictic adults.
The nematode culture process in liquid media can be divided into four phases, all requiring specific and distinct process conditions to yield a maximum of dauer juveniles within a short time.
Research has enabled optimal process conditions to be defined for all process phases. It was found that the main reason for unstable results was the unpredictable, unsynchronized and low recovery of the inoculated dauers, varying between 18 and 90% over a period of several days. Low DJ recovery results in low yields and makes it necessary to carry out additional scale-up steps. Unpredictable recovery prevents a population management which is necessary in order to
Consequently, the key for to improvement of process technology of Heterorhabditis spp. is a reproducible and high dauer recovery.
The symbiotic bacteria are cultured and used to inoculate the DJs The metabolism of P. luminescens was investigated and conditions defined which can enhance bacterial density. Based on results obtained from continuous cultures with P. luminescens, models were developed to best describe the bacterial growth. A model, that can be used to monitor bacterial density in batch and fed-batch cultures, was developed based on data obtained from exhaust gas analysis. Culture conditions were defined which are able to provide a maximum signal activity of the bacterium to induce dauer recovery. Morphological and behavioural changes during DJ recovery were described. Bioassays were developed to test recovery of DJs.
The construction of a recovery-reporter strain (daf7:GFP) had to be cancelled due to problems with the transformation of male or hermaphrodite nematodes. All attempts to transform H. bacteriophora failed. DJ recovery was therefore detected using morphological characters. The influence of process parameters on DJ recovery was investigated. The carbon dioxide concentration had a major influence on DJ recovery, other parameters were of minor importance. The pretreatment of inoculum DJs had minor effects on the DJ recovery.
Mutant strains were produced which have an early recovery genotype. Results from crossbreeding indicate that the phenotype is a result of dominant gene mutation. Synchronisation of the recovery can significantly increase process stability. The available mutants can therefore be of commercial value.
Differential display has been used to identify genes expressed during recovery. A cDNA library was prepared and will be used to isolate genes with an effect on dauer recovery. Although significant progress has been made to overcome problems related to the recovery of DJs in liquid culture, observations made during scale-up suggest that this parameter continues to be the major reason for process instability.
Process parameters were defined which can increase the DJ yields of liquid cultures. The influence of process parameters on the alternative development to either amphimictic (male and female) or automictic (DJs and hermaphrodites) populations and on the endotokle matricide (intrauterine development of DJs) was identified. The different phases of the life cycle were studied in detail in order to identify critical periods with influence on the success of the liquid culture process.
Based on population data from several cultures performed under variable culture conditions a decision support system was developed which can be used to predict the DJ yield at an early state of the culture. In order to avoid the occurrence of non-reproductive amphimictic adults, it was planned to produce hermaphrodite-constitutive mutants. Due to problems encountered during the development of the necessary bioassay to select for the mutants, attempts to produce hermaphrodite-constitutive mutants had to be cancelled.
Attempts to identify genes involved in sex determination of H. bacteriophora also failed.
Experiments using various types of bioreactor, revealed that internal loop technology is superior to conventionally stirred bioreactors and air-lift columns. The scale-up of the liquid culture to the industrial scale succeeded and yields and process stability could be increased. A downstream process was developed making use of a separator and a centrifugal sifter for product recovery at losses of less than 10%.
Quality control methods were developed, which use the migration of DJs on a thermal gradient. Data correlated well with the results of infectivity tests. The analysis of the DJ fat content, fatty acid, glycogen and trehalose composition revealed that these parameters cannot indicate DJ performance.
Four differrent batches from liquid culture were field tested against grubs in turf. After 2 weeks 30% control was recorded, reaching 95% after 8 weeks. Recycling of the nematode in the pest insect seems to contribute to the increase in insect control.
Although all batches passed the laboratory quality control, two batches were inferior in the field.
The project significantly contributed to the basic understanding of the biology of the nematode-bacterium complex and its reproduction in liquid culture. Together with progress on the development of quality control methods, this information supported the successful scale-up of the culture technology, which resulted in a significant reduction of the production costs. As a consequence of the results of the project a major reduction of nematode product prices was possible leading to an augmentation of nematode use against soil insect pests.
For example, almost all tree nurseries in Germany are using nematodes against larvae of the Black Vine Weevil (BVW) and nematodes have been introduced into outdoor markets (against BVW in strawberries in France and against grubs in turf grass in Central Europe). Providing the development of innovative formulation technology is successful, nematodes will also be used on the plant surface in the near future.
Objectives
The aim of this project is to develop process technology for the industrial scale production of highly effective, low-cost nematode products for the biological control of insect pests. It is hoped to decrease the process duration and operation costs and increase process stability, nematode yields and nematode quality. Successful completion of the project is dependent on achieving six main project objectives;
Activities
Entomopathogenic nematodes (Heterorhabditis spp.) are symbiotically associated with the bacterium Photorhabdus luminescens, which is the essential food supply for the nematode. For liquid culture production the bacteria are pre-cultured prior to inoculation of nematode dauer juveniles (DJs) which recover, develop to hermaphrodites and produce offspring. Experiments to increase the bacterial densities by addition of increasing glucose concentrations revealed a maximum concentration tolerated by P. luminescens. With a glucose fed-batch technique the bacterial density could be nearly doubled in liquid culture. The amino acid preferences of P. luminescens have been defined and the data has been used for medium optimisation as well as for establishing stochiometric growth models.
Two model strategies, a formal kinetic approach and a model based on gas analysis, were used to mathematically describe the growth of P. luminescens. Modelling the consumption of glucose showed that the data was in good accordance with Monod's law. In the last reports the first steps of the growth modelling of P. luminescens culture under constant conditions of pH, temperature and oxygen supply, with glucose as limiting substrate were described. Now models are available that include the responses of the bacteria to changes in pH, dissolved oxygen and rate of aeration. The determination of the oxygen uptake profiles during the course of growth in various media led to an improved model linking bacterial growth and glucose uptake.
The exit from the developmentally arrested DJ stage (recovery) is a response to a yet unknown food signal. This food signal is produced by P. luminescens and excreted into the culture medium. In order to test various stimuli for their potential to trigger signal transduction which subsequently induces recovery, it was planned to construct a transgenic nematode carrying the green fluorescent protein (GFP) reporter gene under the control of the daf7 promoter (daf = dauer formation gene). Since the transformation protocols described for the rhabditide nematodes Caenorhabditis elegans and C. briggsae could not be easily adapted to H. bacteriophora, this activity was stopped. Other, methods that enabled an early detection of DJ recovery, using fluorescent dyes (SYTO-12, DiO, Molecular Probes Inc. and FITC, Sigma) were developed. These dyes stain specific neurons or the amphids and phasmids when the recovery process has been induced.
Following a detailed description of the behavioural and morphological changes during recover it was possible to define measures that enhanced the predisposition of the DJ to recover. These results are useful in improving the mass production process. Stress factors prior to inoculation, such as storage, temperature shifts or short term pre-treatments of the DJs with solutions of high osmotic potential promote the predisposition of the DJs to recover. In contrast, high osmolarity in the medium and a pH < 7.0 can suppress the induction of recovery. The bacterial food signal and CO2 have a synergistic effect on the recovery. The proportion of recovered DJs could be nearly doubled in bacterial cultures kept under 4 % CO2, atmosphere. However, an apparent negative influence of C02 on the subsequently development of the recovered DJs to adult hermaphrodites has also been assumed.
Attempts have been made to improve the offspring production target with the reduction of amphimictic adults in order to increase the DJ population. The nematode density in relation to the bacterial biomass defines one or other of the developmental pathways. Without the symbiotic bacteria the nematode offspring are unable to surpass the J 1 stage, even if they develop in vivo inside an insect larva. Most of the DJs that can be harvested from liquid cultures develop inside the parent hermaphrodites. In contrast, juveniles which hatch in the liquid medium tend to develop to a second adult generation. Hence, the DJ yield after inoculation can be correlated with the hermaphrodite density. This correlation was used to create a preliminary decision support model, which can predict the maximal possible yield at a defined hermaphrodite density 3 days after inoculation.
Strain improvement has two targets, an increase in recovery and a hermaphrodite constitutive strain or mutant. A bioassay was developed to detect recovery in a mutagenised population (maturity assay). Mutagenised populations were screened for early maturity in G. mellonella cadavers. So far, six putative mutant lines were found which recover faster then the wild type dauers. Initial data suggest that the early recovery phenotype is the result of a dominant gene mutation.
In order to identify genes regulating sex determination in H. bacteriophora, the homologue of the transformer-2 (tre-2) gene from C. elegans was searched for in the H. bacteriophora genome. Several pairs of PCR primers derived from conserved regions in C. elegans were designed. One of the PCR products of 290 bp was cloned into the pGEM vector, It is similar in size to the product obtained from the control (a plasmid carrying the tre-2 cDNA of C. elegans) and the sequence was found to be 99% homologous to the tre-2 gene cloned from C. briggsae. In order to clone the entire putative tre-2 gene from H. bacteriophora, a cDNA library from this nematode was constructed, which was screened with the PCR-amplified fragment as a probe. Homologues of the sex- determination genes of C. elegans could not be found in a cDNA library from adult H. bacteriophora. Recently it was shown that sex determination occurs in the Jl -stage of Heterorhabditis spp. Hence, in the next period it is planned to generate a cDNA library from this stage.
Nematode cultures were scaled-up successfully to a full-scale volume of 3000 1. Conventional stirred bioreactors were compared with internal loop and airlift reactors. The internal loop system was found most promising. In the 3000 1 vessel yields of up to 0.2 billion DJ/1 were reached.
So far, a separator has been found to be the most reliable instrument for downstream processing. Losses of DJs during separation usually were in the range of 10 to 15 %. Viscosity of the nematode suspension had a significant effect on separation efficiency. An additional cleaning procedure to remove particulate organic matter from the DJs is still in development.
Quality control measures covering nematode activity and infectivity have been investigated. Microscopic examination of DJs was used to determine the active dispersal of DJs at different temperatures. The movement was video-taped. It could be demonstrated that migration behaviour of the DJs can be used as an indicator for their pathogenicity. Methods for the measurement of quality indicating biochemical parameters, such as total fat, trehalose, cholesterol and free fatty acids, were established. Results showing the correlation between the amounts of these substances and nematode quality will be available by the end of the project.
Progress
In most tasks the progress is in accordance with the expected timetable. As far as strain improvement is concerned, delays have been caused due to the labourious nature of assays limiting the number of mutants that can be screened. A transgenic nematode with a green fluorescent reporter gene, aimed at indicating the recovery induction at an early state, could not be constructed. The adaptation of the gene transfer protocols used for the nematode Caenorhabditis elegans and other organisms failed. Such a transgenic nematode is not essential for the success of the project, as alternative methods to detect early recovery were developed. Hermaphrodite constitutive mutants were not found.
Achievements
The scale-up to a 3000 1 plant was successful.. Bacterial culture condition' were improved in order to produce high density cell populations and optimise food signal production. These improvements are accompanied by first results in modelling of bacteria] growth. Methods are available to enhance the DJ predisposition to recover from the dauer stage and to optimise the liquid culture process with regard to the recovery. Mutant strains could be achieved which recover earlier in liquid culture than the wild type. Quality control methods are available to monitor DJ activity In the laboratory.
Future activities
The project will continue within the lines of the outlined research plan. At this time no changes of the objectives and research tasks are necessary.
Introduction Entomopathogenic nematodes (Heterorhabditis spp.) are symbiotically associated with the bacterium Photorhabdus luminescens, which is the essential food supply for the nematode. For liquid culture production the bacteria are pre-cultured prior to inoculation of nematode dauer juveniles (DJs), which recover, develop to hermaphrodites and produce offspring.
Objectives The objective is the development of a process technology for the industrial scale production of highly effective, low-cost nematode products for the biological control of insect pests. The project is aiming at a decrease of the process duration and operation costs and an increase of process stability, nematode yields and nematode quality. Successful completion of the project is dependent on achieving six main objectives:
Activities Experiments to increase the bacterial densities by addition of increasing glucose concentrations revealed a maximum concentration tolerated by P. luminescens. The biomass production was positively correlated with the initial glucose concentrations. However, the increase per unit glucose decreased with increasing glucose concentration. The exit from the developmentally arrested DJ stage (recovery) is a response to an as yet undescribed food signal. A food signal is produced by P. luminescans and excreted into the culture medium. The maximum food signal production was recorded during the late exponential growth phase. In order to increase recovery, DJs should be inocculated at the late logarithmic growth phase of P. luminescans.
For testing of different stimuli on their potential to trigger signal transduction which subsequently induces recovery, a transgenic nematode carrying the green flourescent protein (GFP) reporter gene under the control of the daf7 promoter (daf = dauer formation gene) will be constructed. A method to inject DNA into pre-adult hermaphrodites was developed.
Attempts were made to improve the offspring production target to reduce the occurrence of amphimictic adults in order to increase the DJ development. The nematode population density in relation to the bacterial biomass defines one or other developmental pathway. Morphological changes during the development from J 1 to adults were described and an exact distribution of the different juvenile and adult stages in course of a liquid culture was documented.
Strain improvement has two targets, an increase in recovery and a hermaphrodite constitutive strain or mutant. A bioassay was developed to detect recovery in a mutagenised population (maturity assay). Mutagenised populations were screened for early maturity in G. mellonella cadavers. Three putative mutant lines appear to express an early maturing phenotype. Another bioassay was developed in which nematode development can be recorded. With this assay the, the influence of environmental factors on the rate of development of the different sexual stages (males/females/hermaphrodites) was determined. Within the range of 21-30°C no effect on sex ratio was found. Preliminary data from assays testing the influence of bacterium (primary vs. secondary or symbiotic bacterium from closely related nematode) and crowding effect indicate that most progeny shift to hermaphrodite upon exposure to adverse conditions. Once the ratio amphimicitic to automictic in the wildtype is known, the mutant screening will begin.
In order to identify genes regulating sex determination in H. bacteriophora, the homologue of the transformer-2 (tra-2) gone from C. elegans was searched for in the H. bacteriophora genom. Several pairs of PCR primers derived from conserved regions in C. elegans were designed. One of the PCR products of 290 bp was cloned into the PGEM vector. It is similar in size to the product obtained from the control (a plasmid carrying the tra-2 cDNA of C. elegans) and the sequence was found to be 99% homologous to the tra-2 gone cloned from C. briggsae. In order to clone the entire putative tra-2 gene from H. becteriophora, a cDNA library from this nematode was constructed., which will be screened with the PCR-amplified fragment as a probe.
Two model strategies - a formal kinetic approach and a model based gas analysis - were used to mathematically describe the growth of P. luminescens. Modeling the consumption of glucose following Monod's law showed this to be in good agrement with the experimental data. In contrast, the substrate-dependend biomass formation deviated from the Monod model. Part of the glucose seems to be utilized for the production of secondary metabolites. Using the oxygen uptake rate as determined on-line, the estimation of the biomass is possible. Incorporating the formal kinetic approach, the resulting model allows the estimation of glucose consumption and the production of the secondary metabolite.
Nematode cultures were scaled-up from 10 1 and 15 1 bioreactors to a 500 1 bioreactor and further to 4000 l. Reproduction of H. bacteriophora was seen in all bioreactors. The 500 1 reactor gave consistently lower yields than the 10 1 reactors, although both systems have a similar vessel design and stirring system. Lower yield in 500 1 was correlated with low DL recovery and, subsequently, low hermaphrodite density. One upstream line included an 80 1 and a 200 1 reactor to produce sufficient nematodes to inoculate a 4000 1 reactor. Due to low recovery the process in the 4000 1 vessel totally failed. For testing an air-lift system a 500 1 tank was installed. For scale-up experiments a 3000 1 bioreactor was installed. Both systems have passed sterility testing and will soon be tested for nematode production.
Downstream processing currently uses a separator. Experiments with a decanter were less promissing. Quality control was introduced to measure nematode activity and infectivity. Microscopic examination of DJs has been carried out to determine the active dispersal of DJs at different temperatures. The movement was video-taped. The behavior of fermented versus insect cultured nematodes was compared. Penetration capacity and pathogenicity of DJs from different fermentor batches have been determined. Methods for measurment of quality indicating biochemical parameters, such as total fat, trehalose, cholesterol and free fatty acids, were established.
Field application of liquid culture produced nematodes caused high mortality among grubs (Phyllopertha horticola) and vine weevils (Otiorhynchus sulcatus).
Discussion In most tasks the progress has been in accordance with the technical annex. In strain improvement delays have been cause due to laborious assays limiting the number of mutants that can be screened.
Otherwise the main achievements have been as follows.
Future activities The project will continue along the lines of the research plan At present no change in the objectives or research tasks are necessary.
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