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FAIR-CT95-0191
A New Approach to Bacterial Cellulolysis to Improve Biogas Production From Cellulosic Materials in Agricultural and Municipal Solid Wastes |
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Contract No: | FAIR-CT95-0191 |
| Date Prepared: | July 2001, May 1999 | |
| Source: | Final Report
Abstract and
Executive Summary
Second Annual Progress Report |
Final Report Abstract
Source: Final report of May 1999
Consortium: This project was co-ordinated by the Institute of Biological Sciences, University of Wales (UK), in partnership with Laboratoire de Chimie Biologique, Univ. Henri Poincaré, Nancy (France) and Steinmuller-Valorga SARL (France).
It is generally considered that the rate of digestion of lignocellulosic wastes is limited by the initial hydrolysis of cellulose, since intermediate compounds such as volatile fatty acids (mainly acetate) or H2 cannot be detected. The principal objective of the project was to bring recent advances in our understanding of bacterial cellulolysis to bear upon this problem in order to speed up biogas production from municipal solid waste (MSW). Moreover, MSW is subject to substantial seasonal variation. Garden waste is a major component during the summer & autumn, whereas at other times it contains a higher proportion of paper and cardboard. These compositional variations can impair bioreactor performance and biogas production becomes sluggish once the proportion of paper and cardboard exceeds ca. 25 percent.
The initial aim was therefore to optimise the process of cellulolysis, so that digesters can operate efficiently during periods when MSW contains higher proportions of paper and cardboard. European waste management policy has evolved during the 1990s. In several countries, bulk waste collection has been superseded by a system of separation at source. As a result, anaerobic digesters now use a more uniform feedstock of putrescible biowaste (vegetable and garden waste together with fruit - VGF) and cellulose overload resulting from paper and cardboard waste is no longer encountered.
The scientific advance that underpinned the project was the demonstration, by the Nancy laboratory, that cellulose-degrading clostridia (one of the major groups of bacteria in the bioreactor) undergo a cyclic process of adhesion to and colonisation of cellulose fibrils, followed by cellulose hydrolysis and release of the bacteria. During hydrolysis the bacteria break down the semi-crystalline substrate and use the cellobiose produced to support their growth and energy production. Cellulolytic bacteria are therefore subjected to periodic cycles of substrate sufficiency (when adherent) and substrate starvation (when released). They are also capable of (a) endospore formation, as a result of which they no longer contribute actively to cellulose hydrolysis, and (b) storage polymer production, whereby sugars are converted into glycogen for future use.
The overall scientific goal was to understand the inter- relationships between these different physiological processes and to manipulate them so as to maximise the efficiency of bacterial cellulolysis, in line with the fluctuating composition of MSW. A combined physiological, biochemical and genetical approach was taken to achieve these goals, using Clostridium cellulolyticum. This organism is very closely related to the population of cellulolytic clostridia that inhabit MSW digesters. It was originally isolated and partially characterised by the Nancy laboratory, while the group of J-P. Bélaich in Marseilles had identified many of the components of the C. cellulolyticum cellulosome (the enzymatic machinery responsible for cellulose hydrolysis).
In spite of the previous work, it was realised how little was fundamentally understood about the carbon metabolism of C. cellulolyticum. Attempts to improve rates of carbon source utilisation met with failure and we were forced to the conclusion that cellular regulation in C. cellulolyticum has become strictly adapted life, at low carbon availability. As the flux of carbon increases (using cellobiose, the immediate product of cellulose hydrolysis, as substrate) the organism can no longer dispose of the reducing power it generates. C. cellulolyticum, and its close relatives in the bioreactor, are essentially oligotrophs that are adapted to low carbon source availability. C. cellulolyticum has also adapted to a consortial lifestyle. In its natural environment, it lives "cheek by jowl" with methanogenic bacteria.
These latter organisms are ultimately responsible for the production of biogas, since they use "waste products" of the cellulose fermentation (acetate and hydrogen) to generate methane. In conformity with this, C. cellulolyticum grows to a higher cell density and uses higher substrate concentrations when cultivated within a dialysis sac bathed in continuously renewed culture medium. Many clostridia undergo a metabolic switch towards the end of exponential growth in batch culture, leading to the accumulation of more reduced end products. Laboratory conditions were identified permitting a metabolic switch in C cellulolyticum. As a result, growth became bi-phasic and lactate (a more highly reduced end product) was produced during the second phase instead of acetate. It is unlikely that this switch occurs in the bioreactor environment.
Storage polysaccharide production in C. cellulolyticum is also regulated in line with its adaptation to a low carbon flux. Most organisms accumulate glycogen towards the end of exponential growth in batch culture (when the carbon source is present in excess). When grown under these conditions, C. cellulolyticum makes glycogen during early exponential growth. This is in line with the difficulty these bacteria experience in disposing of their excess reducing power. By making glycogen in early exponential phase, they effectively 'siphon off' excess carbon skeletons and sequester them in an insoluble, osmotically inactive form for subsequent use.
A significant achievement of the project was the development of several alternative methods for undertaking genetic exchange and transposon mutagenesis in C. cellulolyticum. This advance is likely to influence future research strategies, on bacterial cellulolysis, since the separate contributions of the various cellulosome components can now be analysed in vivo. Conjugative transfer of several plasmids and of Tn1545 was obtained by co-cultivation of the oligotrophic recipient with copiotrophic donors. Electrotransformation procedures were also developed, using in vivo methylation (M.BsuFI) to protect the incoming DNA from the host encoded restriction system (CceI). However, gene transfer frequencies were never high enough to permit the construction of defined mutants in the particular genes in which we were interested and the full potential of the methods we have developed has still to be realised. Moreover, we also found it difficult to isolate mutants in the absence of direct selection for the mutant phenotype, since even after extensive optimisation, the organism showed a very poor plating efficiency (ca. 1%). As a result, the great majority of the mutants in the bacterial population were lost when organisms were plated on their agar-solidified growth medium.
Genes encoding Spo0A, the master regulator of the post-exponential phase gene expression, and GlgC, an enzyme concerned with glycogen production, were isolated and characterised. A third gene, encoding AbrB, a subsidiary transition state regulator, was also detected. The products of spo0A and glgc were very similar to their counterparts in other Gram- positive bacteria, but in the absence of defined mutants, the effects of an inability to sporulate (spo0A), or to make glycogen (glgC) on bacterial cellulolysis and survival in the bioreactor environment could not be assessed.
The responses of C. cellulolyticum to several different stresses were characterised and several candidate stress-induced -proteins were identified. In response to carbon starvation, the bacteria showed enhanced tolerance of heat or osmotic stress. In natural substrates, cellulose fibrils are embedded within an amorphous hemicellulose matrix, containing xylans, mannans and arabinans. C. cellulolyticum elaborates a cellulosome- associated xylanase with which it attacks the hemicellulose to expose cellulose fibrils for colonisation.
These experiments have shown more clearly than ever before, that the protein composition of the clostridial cellulosome shows considerable variation in relation to the precise nature of the substrate on which the organism is growing. The question of how a free- living organism senses and/or encounters its insoluble substrate was briefly considered, but not resolved.
Microcosms resembling the bioreactor were established and operated using VGF as substrate. When seeded with an additional inoculum of C. cellulolyticum, there was no enhancement of cellulolysis or biogas production. However, there was enhancement if shredded newspaper was also added to the VGF feedstock in the reactor. The current practise of separation of biowaste at source produces a recalcitrant substrate that is only incompletely digested even after residing for 21 days in the bioreactor. The work indicated that increased biogas production is attainable on an industrial scale by adding some paper wastes to VGF.
Objectives
The objective of the project is to improve biogas
production by anaerobic digestion of cellulosic materials in agricultural
and municipal solid wastes. To achieve this we need to understand, and
deal with the periodic carbon starvation experienced by anaerobic,
cellulose-degrading bacteria, such as Clostridium cellulolyticum, during
their natural growth cycle.
Specific objectives for the current reporting period were (1) characterisation of regulators that control gene expression as the cellulose-degrading bacteria cease active growth and prepare for stationary phase, (2) development Or methods for genetically manipulating these organisms, (3) characterisation of bacterial growth when different nutrients are limiting, (4) evaluation of the responses of the bacteria to stress (particularly nutrient limitation), (5) isolation and characterisation of mutant strains and (6) optimisation of bioreactor conditions and evaluation of potential beneficial effects of adding C. cellulolyticum to the bioreactor.
Description of work
In the first year Or
the project, we showed that C. cellulolyticum grows best under
conditions of limited carbon flux. With this improved understanding of
bacterial physiology (i.e. oligotrophy) we have been able to demonstrate
gene transfer by conjugation from E. coli donor strains to C.
cellulolyticum Wide use has been made of standard molecular techniques
for gene detection, isolation and sequencing. Chemostat culture has been
extensively employed for assessing cellular physiology and biochemistry
under different kinds of nutrient limitation, and the extent of
cross-protection afforded by pre-exposure to different kinds Or stress has
been evaluated. Physiological characterisation of mutant strains and an
assessment of the effects of adding C. cellulolyticum to samples
of cellulosic materials suspended in the bioreactor environment have also
been undertaken.
State of progress
The gene isolation work is
well advanced. Moreover, very significant progress has been made with gene
transfer technology. However, our initial objective, which was to make
defined mutations (gene replacement / knockout) in selected genes in the
bacterial chromosome has not yet been realised. This will require further
optimisation of the gene transfer methods we have developed. Stress
responses are currently being studied and physiological characterisation
of the organism with respect to growth under nutrient limitation is
complete. Our experiments have not yet revealed any obvious ways whereby
the rate cellulolysis in the bioreactor environment can be enhanced.
Inoculation of cellulosic samples, suspended in the bioreactor, with C.
cellulolyticum had no noticeable effect on cellulolysis, suggesting
that under the current operating conditions at least, (18% of combustible
solids represented by cellulose), biogas production in the bioreactor is
not limited by the natural population of cellulolytic organisms.
Achievements
Physiology & metabolic
regulation Bacterial responses to carbon- and nitrogen-starvation have
been determined. It has been shown that NADH re-oxidation plays a cardinal
role in controlling bacterial growth. cellulolyis has been optimised and
conditions favouring glycogen accumulation and glycogen utilisation have
been determined.
Gene transfer and mutagenesis Plasmids have been transferred to C. cellulolyticum using conjugative mobilisation from Escherichia coli donors. Methods have been established for (a) isolating spontaneous mutants and (b) undertaking transposon mutagenesis with the conjugative transposon, Tn1545.
Gene isolation and characterisation The spo0A gene whose product is an ambiactive regulator that orchestrates the transition state in clostridia (and bacilli) has been isolated and characterised. The gene encoding another regulator, AbrB, which controls many aspects of cellular metabolism during the transition state has also been detected. A third gene, glgC, whose product is involved in the production of a storage polymer resembling glycogen, has been partially isolated and characterised.
Optimisation of biogas production in the bioreactor.We now have a more complete understanding of microbial dynamics in the bioreactor environment. In the laboratory environment that the most favourable conditions for growth of, and cellulose degradation by, C. cellulolyticum are those that (a) permit interspecies hydrogen transfer, (b) avoid end-product accumulation and (c) avoid accessibility of excess soluble substrate. These three conditions already exist in the bioreactor. Under conditions of cellulose overload, the addition of C. cellulolyticum could initially speed up the rate of cellulose degradation. However, this would occasion an imbalance in the natural bacterial consortium and the limiting factor would become interspecies hydrogen transfer and acetate consumption by methanogens, leading eventually to process failure.
Future actions
1. The glgC gene will be
characterised and we will attempt to disrupt it in the bacterial
chromosome. Concomitantly, spontaneous mutants unable to make glycogen
will be isolated using a simple screening procedure. These parallel
approaches will allow us to assess the role of glycogen accumulation in
the bacterial growth cyclic.
2. Attempts will be made further to
optimise gene transfer, since this will afford the possibility of
undertaking gene replacement mutagenesis, to create defined mutations in
the bacterial chromosome. If successful, this new technology will, for the
first time, open up the bacterial cellulosome to functional analysis in
vivo.
3. Further experiments with the bioreactor will concentrate on
(a) biogas production from the recalcitrant lignocellulosic material in
digested residue (b) biogas production in a reactor running continuously
(fed with household waste) and (c) biogas production from newspaper in
batch reactors. Additional experiments will be conducted using mutant
strains (see above) as and when they become available.
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Updated
by CPL Press:
03/07/2007
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