Aligning Microbial Biodiversity for Valorization of Biowastes: Conception to Perception

Abstract

Generation of biowastes is increasing rapidly and its uncontrolled, slow and persistent fermentation leads to the release of Green-house gases (GHGs) into the environment. Exploration and exploitation of microbial diversity for degrading biowastes can result in producing diverse range of bioactive molecules, which can act as a source of bioenergy, biopolymers, nutraceuticals and antimicrobials. The whole process is envisaged to manage biowastes, and reduce their pollution causing capacity, and lead to a sustainable society. A strategy has been proposed for: (1) producing bioactive molecules, and (2) achieving a zero-pollution emission by recycling of the GHGs through biological routes.

Introduction

Exploration of microbial diversity is among the top of the curiosity driven research and development (R&D) agendas around the scientific world [1]. As per Ribosomal Database Project Release 11—3,356,809 aligned and annotated 16S rRNA sequences representing bacterial diversity and 125,525 fungal 28S rRNA sequences have been deposited (http://rdp.cme.msu.edu/misc/rel10info.jsp). It was estimated based on DNA reassociation kinetics and metagenomic studies that more than 1 million prokaryotic species exist and less than 0.1% have been identified so far [2–5]. Microbes are known to possess a diverse range of metabolic pathways. These unique features enable microbes to metabolize a diverse variety of organic matter rich materials of plant and animal origin. Because of these metabolic activities they produce and secrete bioactive molecules, which are necessary for plant, animal and human growth, development and health, especially nutraceuticals and antimicrobial drugs [6–8]. Plant and animal products are the major sources of food for human beings. Commercial scale processing of fruits, vegetables, crop plants, animals for producing food results in large quantities of waste. It has been estimated that world-wide 1.6 billion tons of waste are generated annually [9–11]. Biowaste disposal at landfills results in the release of green-house gases (GHGs), breeding of insects and pests, and spread infectious diseases. In addition, scarcity of land and heavy transport charges make it uneconomical. One of the best biological process, which degrades around 95% of the total organic matter content present in a biowaste is Anaerobic Digestion [12, 13]. Despite such high efficiency the process is economically very low. Incidentally, Anaerobic digestion is a multistep process, which provides opportunities to focus on each one of them and value-added products can be extracted at each stage [14–17]. A unique feature of the process is that effluent from each stage can be subjected to different set of bacteria in the next stage. During the last stage, effluent from all previous stages can be treated independently or in a pooled manner to generate methane rich biogas. The diverse value-added products which can be extracted include bioactive molecules, including volatile fatty acids (acetic acid), biofuels (hydrogen, H₂, and methane, CH₄), biopolymers (polyhydroxyalkanoates, PHAs), and antimicrobials (acylhomoserine lactonases, AHLases) [18–38].

In this article, microbial diversity has been explored using culturable, and comparative genomic approaches. The basic objectives have been to identify highly efficient microbes, which can result in complete degradation of biowastes under unsterile conditions. Since biowastes as feed carry inherent bacteria (as ‘contaminants’), there is a high risk of failure of biological processes, which are based on a few specific bacteria. Hence, the need is to identify industrially robust organisms (antibacterial/antipathogens), which can withstand contaminants and possess properties such as: (1) ability to hydrolyse biowastes, (2) produce (a) biofuels (H₂, CH₄) and (b) biopolymers (PHA) in the subsequent stages. Another worry, which still plagues this highly efficient process is the release of GHGs—carbon dioxide (CO₂) into the atmosphere. A brief strategy has been presented on the prospects of recycling of CO₂ and processing of CH₄ into a non-polluting energy rich fuel–methanol.

Culturable Microbial Diversity for

Hydrolysis of Biowastes

For utilizing bio-wastes as feed, the initial step is hydrolysis of complex organic chemicals such as carbohydrates, proteins and fats. Screening of around 3000 bacteria isolated from diverse environmental niches was carried out to identify bacteria with high hydrolytic activities for enzymes such as amylases, proteases and lipases. A set of bacterial strains belonging to Bacillus sphaericus, B. subtilis, B. thuringiensis, Bacillus sp., and Proteus mirabilis were used for the preparing different consortia.

H₂ Production

Screening of around 500 bacteria isolated from diverse environmental niches enabled identification of high H₂ producing bacterial strains of Bacillus cereus, B. thuringiensis, B. pumilus, B. megaterium, Bacillus sp. and Bordetella avium. These bacteria were selected as these could produce H₂ from a wide range of pure sugars. Mixed microbial cultures based on these defined bacteria were used for producing H₂ from different biowaste either individually or well-defined mixtures [39–47].

PHA Production

Strains of Bacillus cereus, B. thuringiensis and Bacillus sp. were selected after screening around 200 different isolates. These cultures have been used for PHA production from different biowastes (1) directly as feed, (2) after pre-treatment with hydrolytic bacteria, and/or (3) from the effluent of H₂ production stage [39, 48–56].

Methanogenesis

For methanogenesis, isolation and maintenance of strictly anaerobic bacteria is required. It is a costly process and the use methanogens as pure culture is not efficient. Methanogens were thus enriched by incubating cattle dung slurry (3% w/v) for 3 weeks [57–62].

Biofilm Production

Around 1000 different bacterial isolates from cattle dung have been characterized and segregated based on their high hydrolytic activities: protease, lipase and amylases. Two hundred fifty bacteria were grown on 30 different media to identify the conditions under which they can form biofilms. A specific medium helped to identify 4 bacteria with very strong biofilm forming abilities. Bacterial DNA from around 100 have been subjected to PCR amplification and sequencing [63].

Bioactive Molecules from Biowastes

Food and Fruit processing plants, Municipal markets, restaurants etc. dispose of biowastes in large quantities at a single location. Some of the biowastes used in these studies for producing H₂, PHA and CH₄ were apple pomace, pea-shells, damaged wheat grains, mixed vegetables (Radish, cabbage, cauliflower), Kitchen waste, and Biodiesel industry waste (glycerol). The preliminary works were done on pure sugars for identifying the potential of microbes to produce these bioactive molecules.

H₂ and CH₄ Generation

Completely damaged wheat grains unfit for human consumption or any other applications were used for producing H₂ [64]. Continuous culture methanogenesis of damaged wheat grains carried out over a period of 60 days generated 545 L biogas/kg volatile solid (VS) containing 69.1% CH₄. Recycling of the effluent, with the objective of saving waster used for preparing slurry was effective over a period of 82 days, which helped to enhance biogas and CH₄ yields to 693 and 480 L/kg VS, respectively. Methanogenesis was effective even on recycling as the CH₄ content was almost similar at 69.3% [57]. Continuous culture digestion of apple pomace resulted in generation of 275 L biogas/kg total solids (TS) having 54% CH₄ content. Switching the biowaste to vegetable waste and rotten cabbage was also quite effective in generating 210 L and 320 L biogas/kg TS, respectively [58]. Pea-shells being highly fibrous in nature could be digested under batch culture conditions using mixed culture leading to the production of biogas having 33% H₂, i.e.,119 L H₂/kg VS reduced. On the other hand, methanogenesis of pea-shells under strictly anaerobic conditions produced biogas rich in CH₄ (43%) i.e. 218 L CH₄/kg TS [59]. Under batch culture mesophilic conditions banana stem waste could be converted to produce biogas which had 196 L/kg TS as CH₄, equivalent to 72%. Whereas, methanogenesis under thermophilic conditions lead to the production of 171 L CH₄/kg TS i.e. 79% of the total biogas. Anaerobic digestion of banana stem waste was 2.4 times faster under thermophilic conditions than those observed at mesophilic temperature range [60]. Ecobiotechnological approach was employed to digest vegetable waste and kitchen food waste for producing H₂ and CH₄ under unsterile conditions. Biowastes hydrolyzed with bacterial cultures were able to produce 17 and 85 L H₂/kg TS and 61.7 and 63.3 L CH₄/kg TS of vegetable waste and food waste, respectively, under continuous culture. Bacterial hydrolysis helped in enhancing H₂ yield up to 2.8-fold and CH₄ yield up to threefold [62].

A novel approach to produce H₂ from glycerol, present in the effluent generated by biodiesel industry involved the use of H₂ producers with biofilm forming ability. This biofilm acted as support for immobilizing Bacillus strain and to produce H₂ from glycerol under continuous culture conditions [46]. Subsequently, co-digestion glycerol waste and domestic wastewater was shown to be effective in H₂ production and recycling of the effluent was also demonstrated [47]. Direct biomethanation of glycerol waste and the effluent generated from the H₂ production stage has also been demonstrated.

PHA Production

Bacteria generally produce homopolymers of PHA, such as polyhydroxybutyrates (PHBs). PHBs are industrially undesirable because of their poor physical strength and brittle nature. PHA co-polymers have better physical properties. PHA production from pure chemical substrates is costly, with feed cost accounting for 45% of the total production cost [65, 66]. Hence, to reduce cost of producing PHA co-polymers, biowastes as feed are recommended [48, 49]. PHA producing abilities of different feeds such as pea-shells, onion peels, potato peels, apple pomace was done using pure bacterial culture largely Bacillus strains. Bacillus strains were identified with unique abilities to produce homo- and co-polymers of PHA from single biowastes or their combinations in different ratios as feed. Copolymers of PHA having high valerate content up to 17% could be produced under optimum conditions [50–56].

Unique Features of the Biowaste to Bioactive Molecule Production Bioprocesses

• First demonstration of using defined mixed cultures for different steps involved in degradation of biowaste: hydrolysis, production of biofuel and biopolymer.

• First report among dark fermentative bacteria with ability to produce H₂ and PHA.

• H₂ yields were observed up to 2.6 mol/mol of hexose.

• Hydrogen yields of 50–200 L/kg TS of biowaste fed from peas-shells, damaged wheat grains, mixed vegetables, kitchen waste and biodiesel industry waste (glycerol).

• H₂ constituting 35–65% of the total biogas.

• Reproducible H₂ production up to 20 L reactor scale.

• PHA yields of up to 2 g/kg TS of biowaste fed from Peas-shells and Biodiesel industry waste (Glycerol).

• Homo-polymers and co-polymers (up to 17% hydroxyvalerate (HV) content) could be produced by manipulating feed composition: Combinations of pea-shells, onion peel, apple pomace and glycerol.

• Continuous culture CH₄ production (up to 250 L CH₄/kg TS fed) from apple pomace, municipal market wastes, pea-shells, damaged wheat grains, biodiesel industry waste and kitchen waste.

Immobilization of bacteria

• Lignocellulosic biowastes as support material for immobilizing bacteria in the reactor for continuous culture production of bioactive molecules.

• Novel technique of using biofilm producing bacteria as support material for immobilizing bacteria.

• Using bacteria with biofilm forming and high H₂ yielding abilities.

• Developed a method to identify biofilm producing bacteria.

Antimicrobial Molecules

Bacteria can communicate among themselves through the phenomenon of Quorum sensing. It operates through diffusible chemical signals. This mechanism allows bacteria to protect from predators by producing antibacterial agents especially quorum sensing inhibitors (QSIs). The objective was to evaluate H₂ and PHA producers for their potential to produce QSIs such as acylhomoserine lactone (AHL) lactonase, which will enable them to compete with contaminants and allow the bioprocess to continue even under unsterile conditions [67–79]. Forty-two Bacillus strains were found to possess gene aiiA encoding for AHL-lactonase [80]. Based on this information, a few Bacillus strains were found to inhibit Aeromonas hydrophila-induced infections in goldfish (Carrasius auratus).

Anti-cancer, Anti-diabetes, Anti-inflammation and Anti-infectivity molecules

Around 1500 different bacterial cultures out of a total of 25,000 bacterial cultures (CSIR-Institute of Genomics and Integrative Biology, IGIB) isolated under the Department of Biotechnology (DBT) funded project had the potential to produce bioactive molecules against the metabolic disorders. The bioactive molecules were characterized at Industrial level by Piramal Life Sciences, Mumbai, India. Cultures are being maintained at DBT-Microbial Culture Collections (MCC) and International Depositary Authority (IDA), National Center for Cell Science (NCCS), Pune, India. The bacterial cultures are available for Industry and Researchers in Biotechnological and Pharmaceutical areas.

Comparative Genomics

For Novel H₂ Producers

Conventional methods of searching efficient and high H₂ producing bacteria have been quite successful. However, the R&D efforts during the last few decades have revealed only a few new genera and species of H₂ producers. Another major constraint in this area is the inability to enhance H₂ yields. Invariably, the yields by either dark or photo-fermentative H₂ production routes have been reported to reach up to 3.3 mol H_2/mole of hexose sugars. The recent approaches to find new H₂ producers have been reported through metagenomic techniques. On the other hand, advantage has been taken of the availability of sequenced microbial genomes. Comparative genomics have proved helpful in revealing some novel H₂ producers which are well adapted to extreme stress conditions and are also able to utilize biowastes as feed material [81]. To evaluate the evolution of H₂ production among diverse bacteria, it is necessary to comprehend the underlying mechanisms. It is expected to reduce environmental emissions, ensure sustainability and energy security. Analysis of factors responsible for gene transmission and evolution revealed events of horizontal transfer of genes in taxonomically diverse H₂ producers. Based on this analysis, certain bacteria were identified, which can be transformed from their present status as ‘non’-H₂ producers into producers. It is a biotechnological advancement for mimicking natural genetic transmission processes to produce novel H₂-producers [82].

For Novel Bioplastic Producers

Compared to conventional methods for isolating and identifying PHA producers, comparative genomics of sequenced microbial genomes offer an opportunity to achieve the same target without culturing them. In silico analysis of 123 sequenced genomes for three genes responsible for PHA biosynthesis revealed certain bacteria which had not been shown previously as PHA producers. The most promising PHA producers were: Novosphingobium aromaticivorans, Burkholderia fungorum, Microbulbifer degradans, and Rhodopseudomonas palustris, which could grow well in industrial wastewater, and degrade environmental pollutants. Thus, waste degradation and PHA production can be achieved simultaneously. In addition, this study revealed certain dark fermentative bacterial strains which could produce both H₂ and PHA. This property was previously reported exclusively among photosynthetic microbes [83]. The phylogenetic analysis of three PHA synthesis genes—phaA, phaB and phaC representing 253 sequenced microbial genomes reveled certain phylogenetics discrepancies. Horizontal gene transfer was observed in 24 organisms, with some being more prone to genetic transmission provide an opportunity to genetically manipulate them and transform non-PHA producers to PHA producers, e.g. Burkholderia sp., Staphylococcus epidermidis, Brucella suis, Streptomyces coelicolor and Leptospira interrogans [84, 85].

Reconstitution of Novel Routes for PHA Production

PHA biosynthesis relies on acetyl-CoA, an intermediate of tricarboxylic acid cycle and fatty acid metabolism. Acetyl-CoA is very critical for many metabolic pathways under normal conditions. Attempts to find out an acetyl-CoA independent pathway for PHA synthesis was done by in silico analysis of metabolic pathway database (KEGG). Organisms such as Brucella, Xanthomonas, Thermoanaerobacter, and Deinococcus, were found to have the pathways necessary for this alternative PHA synthesis routes. This study also revealed the presence of these novel routes in Clostridium, Ralstonia, Pseudomonas, and Mesorhizobium, which are also known for their PHA synthetic potential. Thus, the range of PHA producers can be widened to utilize new feed materials [86].

For Antibacterial Producers

Using different genomic and metabolic databases bacteria with genetic machinery to cephalosporin and penicillin could be identified. A few bacteria presently known as ‘non’-producers of these antibacterials can be supplemented the gene(s) which they lack to transform them into producers. [87, 88]. Most bacteria possess QSI genes for producing either AHL-lactonases and -acylases. Mining of sequenced bacterial genomes revealed certain that (1) Photorhabdus luminescens subsp. laumondii TTO1, (2) Hyphomonas neptunium ATCC15444, and (3) Deinococcus radiodurans R1, had abilities to produce both the enzymes. These organisms can be used for inhibiting bacterial infections, prevent food from getting spoiled and bioremediation [68, 89].

Biomarkers for Bacterial Identification

Bacteria are generally identified ion the basis of their rrs gene sequence. The approach is quite effective but become limited in case the organisms are quite closely related or possess multiple copies of rrs gene in its genome. In general, the issue is resolved by analyzing up to 8 other highly conserved genes (housekeeping genes). A new approach based on restriction endonuclease pattern and the presence of 15–50 nucleotide long signature sequences have been reported to allow identification of organisms easily even up to the species level. The strategy can be extended even in medical field for identifying disease causing organisms [90–101].

Prospects

Nanotechnological Approach

In past few years, a significant advance in the area of nanotechnology has resulted in the development of unique materials for biotransformation reactions, biofuel production, environmental and anti-microbial purposes [102–123]. Microbial H₂ production involves metal dependent enzymes such as [NiFe]-, [FeFe]- and [Fe]-hydrogenases. Using the Dark-fermentative H₂ production route, 33% of the theoretical H₂ yield can be achieved i.e. around 4 mol/mol of hexose [124]. This process is limited due the production of intermediates such as acetate, butyrate, ethanol, lactate and propionate [125]. Different strategies including screening of H₂ producers, optimization of process parameters, metabolic engineering and use of defined mixed cultures, have been adopted to enhance H₂ yield [126–128]. Nanoparticles (NPs) have been shown to significantly altered H₂ production primarily by acting as antimicrobial agents, and as support materials [113, 129]. It has been due to the surface and quantum size effect of NPs, which affect electron transfer rate and catalytic activity of the enzyme hydrogenase [113]. The need is to replace chemically synthesized NPs with biologically synthesized NPs to overcome the issue of biocompatibility [130].

Utilization of GHGs

The increasing emission of GHGs, such as CH₄ and CO₂ is a major concern among Environmentalists due their harmful environmental effects [131]. CH₄ has around 25-fold higher pollution causing potential than CO₂, over a 100-year of time scale. Utilization of GHGs for methanotrophs to produce biofuels is going to prove beneficial in the long term [131–134]. Also, CH₄ can be used a substrate to generate electricity through microbial fuel cell [135].

Zero-Emission Circular Bioeconomy

The long-term goals of these works are to completely utilize biowastes and produce many value-added products. And the final end products either gases (especially CO₂) or liquids (wastewater as effluent) should be recycled to make it a circular bioeconomy with zero-level of pollution emissions. The different Bacillus strains isolated in these studies can be combined to develop consortia which can be used for producing hydrolytic enzymes, H₂-production, PHA production, antibacterial (AHL-lactonase) production, antibiotic production [39, 77, 80]. The intermediates of the dark fermentative H₂-prodction stage can be integrated with photosynthetic H₂-production, PHA-production, methanogenesis, methanol production, bioelectrogenesis, and producing anti-bacterials [136, 137]. These Bacillus stains can be evaluated for their potential as probiotics and used in human gut for fighting pathogenic bacteria and improving human health [138]. The use of glycerol rich biodiesel industry as feed and recycling of the wastewater emanating from the processes of H₂ and CH₄ production, takes us from the conception to perception [15, 16, 22, 26, 32, 46, 47, 51, 52, 56].