The inoculation of microorganisms in composting processes: need or commercial strategy?

M Acevedo, L Acevedo, N Restrepo-Sánchez  and C Peláez

Grupo Interdisciplinario de Estudios Moleculares (GIEM),
Instituto de Química-Facultad de Ciencias Exactas y Naturales,
Universidad de Antioquia. A. A. 1226, Medellín, Colombia.
giem@matematicas.udea.edu.co

Abstract

In the first study blends of poultry manure and sawdust (200 kg quantities) were treated with a commercial inoculum or were not treated.  In a second study, microorganism populations were measured in seven piles of mushroom production waste (2000 kg/pile) that had been previously inoculated with five commercial products, with a natural inoculum (manure) and an absolute control (corresponding to residue without inoculation). In the third study. wastes resulting from export quality bananas were used as raw material to which commercial inocula were also applied, with piles of about 1000 kg in weight. The effect of inoculation was evaluated in terms of time required to obtain a stable product and product quality based on physicochemical variables of water retention capacity, organic carbon, cation exchange capacity and microorganism population kinetics.

There were no differences in the efficiency of the composting process nor on compost quality between inoculated and control treatments.

Keywords: Banana wastes, composting, inoculation, manure, mushroom wastes, physicochemical variables

Introduction

In 1864 Louis Pasteur declared in front of the French Academy of Science his famous speech "About the spontaneous generation" where he presented evidences of microbial occurrence in the environment, giving end to the old controversy with the vitalists (Latour 1998; Pasteur 2000).  Simultaneously the British surgeon Joseph Lister proved that the microbes present in the hospitals´ environment were responsible for recurrent infections in the procedures applied to patients and it was with the implementation of his disinfection programs that initiated the beginning of aseptic techniques which are nowadays the basis of surgical and food safety (Madigan et al 1999).  It can be said then, that it was XIX century science what established the paradigms about the cellular theory and the omnipresence of microorganisms in the environment (Lodish et al 2001).

On the other hand, the consolidation of biological sciences was constituted in a new tool of analysis for the theoretical comprehension of the emerging properties of the ecosystems, so articulating the ecology theoretical speech up to understanding why the microorganisms are present there "not just because" and establishing that their occurrence in a specific habitat is a lot more than the consequence of their introduction in a media, giving that their geographical consolidation is highly determined through the interaction with the abiotic media and other alive systems present (McNaughton and Wolf 1984).

When the thermodynamics approached the study of the alive world, it found an endless source of new challenges among which can be emphasized; the conceptualizations about cellular organization, aging, death and reproduction, to cite just some. The evolutive implication of earth being a closed system, became the focal point of all this new problematic (Juo and Llebot 1989; Schrödinger 1983; Pregonine 1996), and it was such a condition that pushed the organisms to become specialized in three big independent groups; producers, consumers and decomposers, being the last ones responsible for, regardless of being a closed system, the earth being able to count on the possibility of generating new biomass.

Moreover, since about 3500 millions years the "natural engineering" has been specializing all this complex system up to the articulation of the macrosystem that we know today as biosphere. The Neolithic revolution pointed out the new path of humane culture, with technological challenges that as agriculture finished up consolidating a new lifestyle; the sendatarism. However, this new life condition resulted to be just a new beginning with new problems such as climate change and production of antropogenic contaminants that have compelled us to rethink the production systems (Rynk 2003; Comisión Europea 2000)^.

The agriculture consolidation in the Neolithic era not only represented challenges of domestication, since in a parallel way it was necessary the creation of technological processes that such fertilization went beyond the vegetal material. In this sense it can be considered that agricultural practices divided the cultures between those that recognized the importance of fertilization, incorporating them as an agronomic tool and those that did not include the amendment as part of their "technological package" and in consequence required the design of a semi-nomad agriculture tied to long resting periods of the agro systems (Evans 1995).

In the Roman culture and a bit before our era, it had been paid attention to the importance of applying "compost" - organic matter stabilized through biotransformations - to the agricultural soils to keep their fertility. This thesis was held and understood for most farmers, being a tradition that in many cases remains until this day and that for many centuries meant the only alternative to application of  fertilizers.

The consolidation of the cities meant new challenges that were translated in the use of new applications for processes that like composting had exclusive agricultural uses. It is so as in 1992 the utility of composting as a degradation process, was complemented by being seen as a bioremediation alternative by Geovani Beccari, who patented a process for the elimination of solid organic residues with an initial anaerobic phase and a final aerobic one.

In 1925 Howard in Indore (India) pushed the first program of systematic composting, named Indore that is based on the formation of ditches with a depth of up to a meter with alternate layers of organic material: vegetal wastes, manure or municipal solid wastes, that are mixed once or twice in a six months period. This process was later on improved while more frequent aerations were done and in 1931, J. Borada modified the original model of Beccari eliminating the anaerobic phase.

In Europe and specifically in Holland appeared a process named van Maanem which was an adaptation of  the Indian process and dates from year 1932. In the third and fourth decades of the  XX century, a good number of composting plants were launched both in Europe and in America, but the lack of knowledge about biochemical processes involved, brought serious troubles and many of the plants had to be closed (Keener et al 1993; Orozco 1980). Nevertheless, during this period it was clear that the aerobic process was more applicable (at least in terms of time and costs) in order to get a suitable management of solid wastes.

In 1951 the University of California started a series of research projects about the aerobic treatment of municipal solid wastes which, linked to those efforts done by the  University of Michigan at a laboratory scale, are considered as the beginning of the elucidation of the laws that rule the process and the establishment of temperature, pH, moisture and aeration as macroscopic variables determining of the efficiency. The practical component of the process, as being considered an alternative to a growing environmental problem, has allowed its popularization and has even driven the research in this field towards an eminently practical component (Camps 1987; Bond and Straub 1973; Kononova 1966)^.

Fertilization as a quantitative problem had its origin in the research done by Liebig who in 1849 through a series of experiments established that some bio-elements contributed, by means of the use of manure and other mineral products, were responsible for the increase in the agricultural production (McNaughton and Wolf 1984; UNIDO 1998). This basic observation was generalized in 1905 by Blackman through the formulation of the Law of Limiting Factors (McNaughton and Wolf 1984).  However, the development of chemical fertilizers and very specifically the design of composed fertilizers, pointed out the beginning of the "industrialization of agriculture" and simultaneously meant the disuse of organic materials in fertilization programs (McNaughton and Wolf 1984; Chen and Inbar 1993). In the last three decades, studies about the behavior of the soils have shown the importance of organic matter presence in the agroecosystems. They have defined what are the mechanisms of genesis-degradation and finally they have stimulated the development of technologies based on bio-oxidative processes of organic matter as an alternative to the use or disposal of organic residues in an indiscriminate manner, in such a way that the impact of antropogenic activities over the biogeochemical cycles can be minimized (Kononova 1966; Schlesinger 1991; Gómez 2000a; Gómez 2000b).

These new research efforts have centered on understanding how the molecular changes work, which determine the appearance of stable organic substances that were originally considered exclusive to soil, as well as in rescuing the importance of organic matter addition in agricultural soils as an integral part of fertilization programs for appropriate and sustainable production, even in the so called intensive agriculture (Chen and Inbar 1993; Blanco and Almendros 1997; Roig et al 1988; Brewer 2003; Cooperband et al 2003). If it is taken into account that agricultural production in the last fifty years has turned into an extractive system, the soil degradation became the most important limiting factor to productivity of the agroecosystems. . This degradation process that is associated with the decrease in the capacity of soil  to accomplish functions such as a growth medium for vegetative regulation of hydric regimens and as an environmental filter, can happen either for natural reasons or induced by humans, being the evident symptomatology the damage in physical, chemical and biological properties that drive to the strengthening of negative effects in vegetal productivity and in environmental quality (Fassbender 1980).

The degradative processes of soils are generally initiated with decrease in organic matter levels and biological activity, that is translated into unfavorable effects on soil physical structure and on water retention capacity, that are in turn manifested by the superficial sealing, compacting, limited radicular development, poor drainage, frequent droughts, excessive leaching and accelerated erosion (Fassbender 1980; Sánchez 1981).  Brams describes the changes in typical profiles of organic fractions in tropical zones when soil function has stopped being a support for a natural ecosystem to become an agroecosystem and in all of them a significant lessening of organic matter is established (Sánchez 1981).  It is so evident that organic matter contribution is constituted a necessary agronomic practice for preservation of soils with appropriate characteristics for agricultural activities given the impact that this kind of material has over different soil properties such as structure, by strengthening bigger aggregates formation and improving porosity; and aeration and drainage that considerably augment the organic matter content. The water retention capacity also increases directly with the increment in organic matter and as a consequence it allows soil humidity regulation. Soil color change is another consequence of organic matter fraction restitution and in most cases it implies a favorable environment for germination and root development. It turns out to be equally important the increase in cationic exchange capacity (CEC) given that it avoids nutrient losses through lixiviation. Finally it has to be mentioned that organic matter implies an increase in pH buffering capacity (Planas and Peláez 2001).

Studies performed in the last three decades of the XX century  about fertility of agro-ecosystems, clearly showed the importance of incorporating organic matter in fertilization programs (Hoitink and Keener 1993) and big steps were simultaneously given to standardize the processes that lead to industrial production of safe organic materials (Camps 1987; Stoffella and Kahn 2001).

The industrialization of organic matter stabilization processes such as composting or anaerobic digestion supposed in a first instance a significant increase of the organic matter to be biotransformed, which meant a radical change in natural conditions of matter recycling and therefore it required a quantitative estimative of the dependent and independent variables that define the biotransformation, such as residence time, stability and maturity parameters that allows the safe use of the material, and the biotic successions that drive the process (Hoitink and Keener 1993).  One of the first aspects considered in the industrialization process was the establishment of optimal process time, a fact that was rapidly associated to the biomass degradation rate by the saprophytic microorganisms; being then to one of the first working hypotheses (from the stoicheometric perspective) that microorganisms present are constituted in the "limiting reagent" of the process. This fact was quickly taken by some companies as a new business opportunity (Anon 2005a; Anon 2005b; Anon 2005c).

Inoculation, that is the action and effect of inoculate is derived from Latin inoculare, which means to introduce an organism or substance in an artificial manner (Real Academia de la Lengua Española 2004).  Though it was originally defined in terms of diseases, the microbial inoculation is currently understood as the process through which some population of microorganisms, selected for specific goals, is added to a medium looking for a different response from that which is obtained in natural conditions (Madigan et al 1999; Rynk et al 1992; Goldstein 1994). As a strategy, inoculation has been associated to three processes. In the first one, it is used as a substrate (liquid or solid) with a significant microorganism population concentration (or enzymes) previously isolated and selected. The second inoculation method involves the "reuse" of a fraction of a material in an intermediate phase of a biotransformation process, as an inoculum's for the material that is just starting the process. The third inoculation method implies the use of organic residues with high concentration of saprophytic microorganisms, such is the case of manures.

The commercial relevance of the methods expounded is centered on the application of inocula produced through isolation and massive reproduction programs. In this last case, one starts from the premise that the selection implies the choosing of special microorganisms whose pure strains have been cultivated in the laboratory basically because of two facts: They present a high, organic matter decomposing or nitrogen fixation capacity. Though in principle this method is commendable, significant difficulties for its implementation have been found in the practice, provided that in the first place microbial populations  are rarely constituted as limiting factors in the degradation process. It has equally been established that under regular environmental conditions of operation in biotransformation processes, the native microorganisms usually represent the best adapted populations for the specific raw materials. It means then that raw materials act as "natural selectors" of microorganisms. A suggested biochemical alternative involves the use of enzymes; however the experimental assays have shown that extra-costs so generated are unnecessary given that microorganisms present in the process are equally efficient to produce the required enzymes.

In detailed studies about the behavior of microorganisms in the composting process it could be established that their presence obey essentially to succession events with emerging characteristics dependent upon biota-medium complex interactions. In this sense, the University of California indicates that treatments with inocula have not represented an acceleration of the process, neither an improvement in product quality. Even natural inocula have presented better results than isolated microorganism formulations (Golueke 1991; Gouin 1992). An important fact to be taken into account is the different approach to the inoculation term according to the discipline. While for microbiology the inoculum corresponds to a small amount of microorganisms that when added modified substantially the behavior of a system, for engineering an inoculum corresponds to an important mass of microorganisms (Torsvik et al 1990).  A different case is developed  when the problem is environmental and the subject of inoculation is essentially the bioremediation since for this circumstance clear examples of convenience had been established in the use of inocula. Finally, it is important to establish unambiguous differences between the use of inocula in the composting process and the use of compost as an inoculum (Ogram and Feng 1996).

A large series of difficulties have appeared in the implementation of inoculation as an industrially suitable process, which have their origin in a principle up to now impassable: It is estimated that  less than 1% of microorganisms present in biotransformation processes are susceptible to being isolated (Torsvik et al 1990; Ogram and Feng 1996).  A second factor that has impeded the inoculation implementation has to do with the genetic expression and related evolutive aspects. A concrete case is the so called enzymatic induction phenomenon through which the organisms modify specific enzyme synthesis rates in response to environmental changes.

Since the beginning of XX century, it had been recognized that bacteria adapt themselves to their environment producing enzymes that metabolize products of specific surroundings. So, E. coli can synthesize approximately 3000 different types of polypeptides, but the amount of copies generated varies considerably. While for ribosomal proteins it can be obtained up to 1 x 10⁴ copies per cell, for some regulating proteins it does not get even to 10 copies per cell. This fact allowed classification of enzymes produced in an approximately constant way, as constitutive and those synthesized at variable rates as inducible, and in consequence the first ones are considered as of media independent production and the second group as media dependent. This characteristic of producing a series of proteins only when the substances to be metabolized are present, has been interpreted as an environmental adaptation capacity since it avoids the weakening on not permanently synthesizing big amounts of unnecessary substances (Voet and Voet 1992).

In the fifties, De Cohn gave quantitative evidences of enzymatic induction processes on growing E. coli strains in a medium without lactose. On evaluating b-galactosidase contents, it was established that a few copies per cell of this enzyme were obtained. When adding lactose to the medium, the bacteria incremented the synthesis rate of these proteins in a factor of about 1000, maintaining it until lactose was not available any more. In this context, an inducer was defined as a substance that is responsible of the metabolic response that leads to the massive production of a specific enzyme (Anon 2003). At the beginning of the 60^s, Jacob and Monod, based on the concept of enzymatic induction, proposed a model called operon that explains the regulation mechanisms of the genes that control the production of the enzymes involved in lactose metabolism. It is clear then, that the different chemical environments and the molecular mechanisms for their recognition, are transcendental aspects in the understanding of microbial and biochemical behavior of composting (Jacob and Monod 1961).

Giving then that composting is basically an industrial adaptation of soil oxidative bio-transformations whose final result implies the obtaining of a partially mineralized organic material (Peláez 2000), the transition from the natural to the industrial process supposes the recognizing and management of the variables that define it, this is; 1) Raw materials and their composition, 2) Process variables, 3) Stability-maturity parameters and 4) Residence time. The appropriate combination of these variables allows not only obtaining a good quality product but also a profitable process from the economic point of view and that can simultaneously be environmental and sanitary safe.

A reason that is often wielded to explain the success of composting as a bio-oxidative process is that it allows the management of big volumes, which results in a significant reduction in production cost. At the end, the residence time is the factor to determine if the process is being optimized. The optimal time can be understood as the minimum necessary to reach the stability and maturity for its safe application as an organic substrate. This minimum time implies the adjusting in such a way that the acceleration of the process can be achieved and therefore the manipulation of variables such as: appropriate mixture of raw materials, adapting of particle size, humidity, aeration and microbial populations (Anon 2003).

Methodology

For WRC determination, a qualitative filter paper was moistened up to saturation in a glass funnel. The weight was recorded and the filter placed over an erlenmeyer flask. To the weighed sample, water was added in excess, until completely filtered and the funnel weighed again with the wet sample and the difference between initial and final weight recorded. The water capacity retention, expressed as ml/g of sample, is calculated according to the following equation:

WRC = (Vr / W)

Where:

W: Sample initial weight in g
Vr: Water volume retained by sample, which is calculated as the difference between sample final and initial weight.
Vr = W_final - W_initial

For ash determination, 2 g of sample are weighted in a porcelain crucible previously tared. The crucible with the sample is placed in a muffle preheated at 250°C, heated up to 550ºC and allowed to incinerate during four hours. The crucible is taken out of the oven, allowed to cool in a desiccator and weighed. Ash content expressed as percentage is calculated according to the following equation:

Ash % = (B - C)/(A - C) x 100

Where:

A: Crucible + sample weight in g
B: Crucible + ashes weight in g
C: Empty crucible weight in g

To determine the organic carbon 1.0 g (0.5 g or less, if sample comes from high content organic matter) of sample is placed in a previously dried erlenmeyer flask and passed through a mesh of 350 mm. Then, 10.0 ml of potassium dichromate 1 N are added followed immediately by 20 ml of concentrated sulphuric acid with vigorous shaking during one minute. The mixture is left to stand during 30 minutes, after which 200 ml of water and 10 ml of phosphoric acid are added. Next, 0.5 ml of barium diphenylaminosulphate are added and the mixture is titrated with FeSO₄.7H₂O until a light "phosphorescent green" is obtained.

The CEC is determined by weighing 5 g of dry sample (size < 2mm) which is transferred to a 250 ml erlenmeyer and 35 ml of NH₄OAc 1N (pH = 7) solution are added. The mixture is shaken for several minutes and left to stand for 12 h, after which it is centrifuged at 3000 rpm during 10 min and the supernatant is kept for extractable cation analysis. The pellet is transferred to a Kjeldhal flask and 400 ml of water are added followed by 10 g of NaCl, 5 g of antifoaming mixture, 1-2 zinc granules and 40 ml of 1N NaOH. About 200 ml of the mixture are distilled off over 50 ml of 2% boric acid solution which includes 3 drops of mixed indicator and 2 drops of green bromocresol.

In the determination of total microorganisms 10 g of sample are weighted, diluted with 90 ml of peptone water and the dilution is mixed during 2 minutes. The mixture is left to stand at room temperature for 15 min and from this dilution (10^-1) a 1 ml aliquot is re-diluted (dilution 10^-2) with 9 ml of peptone water and the mixture homogenized. The process is repeated up to 10^-6 final dilution. From each dilution (10^-1, 10^-2, 10^-3, 10^-4, 10^-5 y 10^-6), 1 ml aliquot is taken and seeded in Petri dishes by duplicate. Cultures are prepared in plates and those corresponding to a same dilution (from duplicates) which show between 30 - 300 colonies are selected and number of microorganisms per g is calculated.

Statgraphics version 5.0 was used for the study of the behavior of the different variables.

Results and Discussion

Starting from the premise that inoculation is applied to reduce the duration of the process, to increase product quality or in best of cases to obtain both benefits, the inoculation effect evaluation will be centered on these two issues.

Studies of kinetic evolution of an organic biodegradable matrix suffering a composting process, have indicated that a series of variables can be used as parameter to measure product quality and to establish process final point (bioland 2005; microtack 2005) These quality variables can be classified as: physicochemical and biological.  Among the physicochemical variables; apparent density, water retention capacity, cationic exchange capacity, ash and organic carbon content, account in a quantitative and statically significant manner, of the kinetics of the bio-oxidative process that rules in composting. This conclusion derives from the fact that the nature of the process, indicates that organic carbon content is the independent variable while others are considered as dependent.

According to the statements above, to quantitatively evaluate the impact of commercial inocula on the composting process, Study 1 was dedicated to the comparison of physicochemical variables in the final products. Samples were considered as final product when the temperature curve showed asymptotic behavior, which can be taken as indicative of stability if the term is understood as slow rate of change of the organic matter.

According to current Colombian regulations (ICONTEC 2003) the water retention capacity (WRC) for a material suitable for agricultural use has to be equal toits own weight. This means that one gram of organic material should retain at least one gram of water. Table 1 shows the average WRC values for six repetitions of inoculated and control (without inoculation) cases in Study 1. From the comparisons it is established that for both cases the averages are very high with respect to the minimum required by the Colombian regulation and no significant differences were obtained for the inoculated cases with respect to controls. 

Considering that composting is a bio-oxidative process, the organic carbon content has to decrease as the process goes on, up to an asymptotic behavior. In Table 1, it can be seen the OC comparison showed no statistically significant difference between inoculated and control samples, concluding that there was similar catalytic activity.

The third regulated variable according to the Norma Técnica Colombia (NTC) (ICONTEC 2003) to give account of quality of organic materials for agricultural use is the cation exchange capacity (CEC). There were no differences between inoculated and control treatments for this variable (Table 1).

In the bio-oxidative process, the ash content increase as the composting process goes on; presenting a kinetic inflexion peak that is interpreted as the initial point of the stabilization phase. The results obtained for this variable (Table 1 – entry 4) showed no significant differences and therefore, in quality terms, once again there is  no evidence to support the need of using inoculation.

Catabolic processes are associated with exothermic reactions and the temperature of a pile in a composting process is estimated to be a function of the heat generated in the bio-oxidation and the system dissipative capacity. Provided that the superficial area remains constant, system temperature varies with direct proportionality to the metabolic activity and temperature kinetics is an indication of system bio-oxidative behavior. As observed in this study (Figure 1), the behavior of the process temperature was very alike, essentially for the final cooling or stabilization phase, indicating that there were no significant differences between the inoculated and not-inoculated treatments.

A second aspect that has to be considered is the effect of the the inocula on the microbial population (Table 2 and Figure 2). The microbial populations were of the same magnitude for the control as for the commercial inocula with an indication of higher values when manure was used. The microbial population observed at day 30 was superior to that on the first day, with the result obtained for manure being higher than that for the other treatments.

An important fact that has to be emphasized is that given the cost of the commercial products, the equivalent amount of manure added corresponded to a 8% of the mass of the initial weight of the pile

The concentration of micro-organisms (Table 2) was highest for the compost inoculated with manure (94 times higher than in treatment with inoculum 3 that had the lowest value)..

It is usually argued that the microorganisms supplied in the commercial inocula cannot be quantitatively evaluated, as they are specialized microorganisms that promote more efficient specific  transformations regardless of their concentrations. However, it is evident that the experimental data in the present study do not support such a hypothesis.

Conclusions

It is concluded that:

• There were no significant differences among a range of microbial inocula and an un-inoculated control when these were evaluated in terms of capacity to reduce residence time or to improve desirable characteristics of the organic substrates used in the present study.
  
  
• The addition of inocula to the organic substrates traditionally used in composting processes (municipal solid waste and farm residues) has not been translated into statistically significant differences that justify their application.
  
  
• The difficulty in isolating the different microbial species present in decomposing organic matter is the main barrier to the selection of the more efficient species in the bio-oxidative transformation processes.
  
  
• Mechanisms such as substrate enzymatic induction and natural selection establish that the raw materials quickly lead to the selection of endemic specialized populations.

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