Bioaugmentation in anaerobic digesters: A systematic review

1 Bioaugmentation in anaerobic digesters: A systematic review 1 Mozhdeh Alipoursarbani1, Jeroen Tideman2, Mitzy López1,3, Christian Abendroth1* 2 3 4 1 Brandenburg University of Technology Cottbus-Senftenberg, Chair of Circular Economy, 5 Cottbus, Germany. 6 2 Bioclear earth B.V., Groningen, the Netherlands 7 3 Universidad Nacional Autónoma de México, Mexico City, Mexico 8 9 *Corresponding author ([email protected]) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 2 Abstract: 25 Bioaugmentation, the intentional introduction of specific microorganisms into anaerobic 26 digestion (AD) systems, has shown promise in enhancing methane production and in 27 mitigating stressful conditions, particularly in systems operating below optimal performance. 28 This review presents a systematic literature review (SLR) and meta-analysis to evaluate the 29 efficacy of bioaugmentation strategies in AD. This review identified and analyzed studies 30 meeting predefined eligibility criteria through a structured methodology involving research 31 protocol, search, appraisal, synthesis, analysis, and reporting. A notable innovation of this 32 review is its comprehensive critical comparison of different controls used in bioaugmentation 33 studies, which has been inadequately addressed in previous literature. To facilitate the 34 functional understanding, strains for bioaugmentation were grouped into the four phases of 35 anaerobic digestion (hydrolysis, acidogenesis, acetogenesis and methanogenesis). A highly 36 diverse set of microbes has been described for bioaugmentation, especially from the families 37 Clostridiaceae, Pseudomonadaceae and Syntrophomonadaceae. Most works are related to 38 hydrolysis. The few works that address acidogenesis are mostly related to dark 39 fermentation. Several studies used methanogenic archaea as well as syntrophic acetate 40 oxidising bacteria, despite the difficulties in culturing them. On the other hand, studies 41 applying strains for acetogenesis were largely underrepresented. Especially works on 42 syntrophic propionate and butyrate oxidation (SPO and SBO) were missing. 43 44 Key words: Bioaugmentation, anaerobic digestion, biogas plants, microorganisms, 45 microbiomes, defined cultures. 46 47 48 49 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 3 1. Introduction 50 Fossil fuels continue to play a central role in supplying energy for both industrial and 51 domestic purposes. However, this fuel presents two significant issues: its finite nature 52 leading to eventual depletion and its environmental impact through greenhouse gas 53 emissions, contributing to global warming. Thus, the development of renewable and 54 environmentally friendly alternative energies is crucial to address these limitations. 55 In recent years, there has been an increasing trend towards the utilisation of anaerobic 56 digestion (AD) for the production of biogas and renewable energy. AD offers a sustainable 57 and environmentally friendly solution for the treatment of organic waste and the generation 58 of valuable resources (Tian et al., 2019). The utilisation of AD to convert organic waste into 59 biogas is a highly effective approach to waste management (Liu et al., 2023b). AD is widely 60 acknowledged as a technology for extracting energy (CH4) from organic waste through the 61 action of various microbial communities within anaerobic conditions (Im et al., 2020). This 62 process encompasses several stages including hydrolysis, acidogenesis, acetogenesis, and 63 methanogenesis, with each phase being facilitated by specific microbial consortia (Li et al., 64 2021). The efficiency of AD depends on the metabolic activities and interactions of the 65 microorganisms (Xu et al., 2023). 66 The alteration in microbial balance within bioreactors, often caused by the inhibition of 67 certain groups of microorganism or the proliferation of others, is predominantly triggered by 68 various inhibitory factors. These factors encompass elevated levels of inorganic toxicants 69 like ammonium, phosphate, sulfate, and metal ions. Additionally, fluctuations in parameters 70 such as temperature, pH, organic loading rate (OLR), and the resistance of feedstock to 71 biodegradation contribute to decreased efficiency in AD. Among the proposed mitigation 72 strategies are feedstock pretreatment (including ensilage) or dilution, implementation of 73 multi-phase bioreactors, and precise control of temperature and pH, among others. While 74 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 4 these strategies show promise, they may also extend the duration and consequently 75 increase the cost of the AD process (Jain et al., 2015). 76 Given that the factors mentioned above induce shifts in microbial community dynamics, 77 bioaugmentation emerges as a potential alternative strategy to address these limitations. In 78 regard to anaerobic digestion, bioaugmentation involves the introduction of specific stress79 resistant or efficient microorganisms into the underlying microbial community with the aim 80 of bolstering its capacity to produce biomethane. This approach has demonstrated success 81 in aerobic biodegradation scenarios, particularly in soil and wastewater, targeting 82 contaminants typically resistant to degradation (Nzila et al., 2016; Semrany et al., 2012; 83 Tyagi et al., 2011). 84 However, despite the advancements in AD technology, there are still significant challenges 85 to overcome, particularly in the area of biodegradation enhancement or bioaugmentation. 86 Bioaugmentation involves the addition of specific microbial consortia or enzymes to improve 87 the breakdown of complex organic substrates and enhance the biogas production process. 88 The successful implementation of bioaugmentation techniques holds great potential for 89 optimising biogas yields, improving process stability / robustness, and facilitating the 90 digestion of challenging feedstocks (Zhang et al., 2018). This literature review aims to 91 explore the current state of AD for biogas production, with a specific focus on the challenges 92 associated with bioaugmentation. 93 94 2. Material and methods 95 2.1 Data collection 96 This review follows a systematic literature review (SLR) approach inspired by, Mengist et al. 97 (2020) aiming to ensure thoroughness, transparency, and reproducibility in identifying and 98 analyzing relevant literature. The systematic process utilised for conducting the search for 99 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 5 relevant literature is illustrated in Fig. 1. The authors conducted a comprehensive 100 examination of Clarivate's Web of Science (WoS) core collection to identify all publications 101 related to the subject of canaerobic OR biogas AND bio$augmentation). The search was 102 performed on December 4, 2022, encompassing a period of 25 years from January 1, 1999, 103 to March 31, 2024. A total of 1058 documents were retrieved from the search. The parsing 104 and analysis of the WoS corpus were carried out using the bibliometrix package in R. 105 Subsequently, 130 review articles were excluded from the analysis, resulting in 928 articles 106 for further examination. Among these articles, 293 specifically focused on bioaugmentation 107 and included defined taxonomic affiliations. Within this subset of bioaugmentation papers, 108 89 articles specifically addressed bioaugmentation in the context of anaerobic digestion, 109 with taxonomic affiliations also being defined. Of these articles, some could not be accessed, 110 so only the abstracts were evaluated. In total, 635 articles were excluded due to the lack of 111 taxonomic affiliations, with a subset of these focusing on anaerobic digestion. 112 113 Figure 1. Flow chart depicting the methodology for conducting a systematic review by using Web of 114 Science with the search terms “anaerobic OR biogas AND bio$augmentation”. 115 116 117 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 6 3. Results and discussion 118 3.1 Exclusion criteria 119 The distribution of various types of articles are displayed in Fig. 2 (a). The predominant type 120 of article identified was research papers. This finding suggests that researchers have 121 dedicated significant efforts to investigating and contributing new knowledge in the subject 122 area. Although bioaugmentation in the context of anaerobic digestion is a young field of 123 research (beginning in 1999), there is already a considerable amount of review articles 124 (130). Web of Science groups several articles into “Other” and “Meetings”. Considering that 125 after applying the exclusion criteria, relatively few articles remained, it was decided to also 126 take into account “Other” articles and as well articles from “Meetings”. Recently, it has been 127 highlighted as a problem that many review articles are currently reviewing other review 128 articles (Kirchherr, 2023). In agreement with this finding, all review articles were excluded 129 from the reviewed set of articles. Nevertheless, the article should be differentiated from 130 previous review articles in order to better emphasise the importance of the article. This has 131 been detailed in in section 3.2. 132 Beginning with a few items in 1999 and the early 2000s, the number of items increased and 133 now indicates exponential growth (Fig. 2b). This trend indicates a significant increase in the 134 attention and interest of the scientific community towards this topic. 135 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 7 Figure 2. Article types and number of publications: Article types of raw data (a); number of publications 136 per year before and after the application of exclusion criteria (b). 137 Important keywords, their frequency, important journals as well as geographical distribution 138 is shown in Fig. 3. Analysing the occurrence of important terms, it appears that the focus of 139 interest was shifting over the years (Fig. 3a). The terms are ranked by frequency on the right 140 side, reflecting their prominence in the literature. The connections between terms show the 141 evolving research focus over time, revealing how topics have shifted as the field has 142 developed. The early phase of research, represented by the leftmost terms, shows a strong 143 focus on chemical processes involving chlorinated compounds. Terms such as 144 "dehalogenation," "tetrachloroethene," "chlorinated ethenes," "reductive dechlorination," 145 and "vinyl chloride" appear frequently and are closely connected. This suggests that early 146 studies were primarily concerned with understanding and mitigating the environmental and 147 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 8 health impacts of specific chlorinated organic pollutants. As research progressed, there was 148 a noticeable shift towards microbial processes and biodegradation mechanisms. Terms 149 such as "culture," "biodegradation," and "reduction" begin to appear, indicating that 150 researchers started focusing on biological approaches for breaking down these pollutants. 151 This shift reflects an increased interest in utilizing natural microbial communities and 152 bioaugmentation to enhance pollutant degradation, which is evident from the term 153 bioaugmentation, one of the most frequently occurring topics in the graph. In more recent 154 years, the focus has expanded to encompass broader environmental applications and 155 sustainable waste management practices. Terms like "methane production," "co-digestion," 156 "food waste," and "stability" suggest a growing emphasis on integrating waste management 157 practices with environmental sustainability goals. This trend indicates a movement towards 158 addressing not just pollutant removal, but also harnessing by-products (such as methane) 159 in the process, thereby contributing to a circular economy approach. 160 The frequency data highlights some core topics that have sustained attention over time. 161 "Bioaugmentation" (249 occurrences) and "degradation" (144 occurrences) are particularly 162 prominent, emphasizing the consistent research interest in enhancing microbial 163 communities to break down pollutants effectively. The significant frequency of 164 "biodegradation" (102 occurrences) and "methane production" (125 occurrences) further 165 underscores a dual focus on both pollutant degradation and energy/resource recovery. 166 Figure 3b provides a comprehensive overview of research trends in environmental 167 biotechnology over nearly two decades. It highlights the diverse journals through which 168 knowledge has been disseminated and illustrates the geographical distribution of research 169 efforts. The figure effectively portrays the global landscape of contributions, emphasizing 170 the interconnectedness of leading countries, key research topics, and significant scientific 171 publications in the field. Using a Sankey diagram, it highlights how major research areas 172 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 9 such as "bioaugmentation," "anaerobic digestion," "bioremediation," and "microbial 173 community" studies are distributed across various countries and published in specific 174 journals. China and the USA are identified as the most significant contributors, with 175 substantial research outputs across multiple topics, followed by other active countries like 176 India, Korea, and Italy. The diagram reveals that Bioresource Technology, Environmental 177 Science & Technology, and Water Research are among the top journals publishing these 178 studies, with Bioresource Technology standing out as a primary publication venue across 179 diverse topics. The interconnected flows in the diagram emphasize how different countries 180 focus on similar research areas and target common journals, creating a cohesive 181 international research network. This visualization effectively captures the interdisciplinary 182 and collaborative nature of environmental biotechnology research, underscoring the global 183 commitment to advancing sustainable biotechnological solutions. 184 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 16 Wang et al. (2008) 1.48 m2 reactor (batch) No microbial additive apart from the inoculum 2 Yes (H2) 0.20* Yan et al. (2022) 2.3 L reactor (batch) Pure culture 4 Yes (CH4) 0.10* Atasoy & Cetecioglu, (2020) 2000 mL reactor (batch) Mixed culture and pure culture 4 No Granular seed sludge: 1.42* Clostridium butyricum: 0.10* Morales-Martínez et al. (2020) 120 mL glass bottles (batch) Pure culture 4 Yes (H2) 0.004* Tartakovsky et al. (2001) UASB reactor Pure culture 4 No 1.00* Puyol et al. (2011) 2.5 L batch Mixed culture 5 No - Yan et al. (2020) CSTR reactors with a total and working volume of 4.5 Pure culture 4 Yes (CH4) 0.60* .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 17 Sharma & Melkania, (2018) 500 mL bottles (batch) Pure culture 4 Yes (H2) 1.04* Cirne et al. (2006) 100 cm3 serum bottles (batch) Pure culture 3, 4 Yes (CH4) 1.35* Cayetano et al. (2021) 150 mL serum bottles (batch) Pure culture 4 Yes (CH4) 8.07* Shanmugam et al. (2019) 100 mL serum bottle (batch) No microbial additive apart from the inoculum 2 Yes (CH4) 5.56* Sinha et al. (2021) 2 l heavy-duty vacuum bottle (batch) Pure culture 4 Yes (CH4) - Kumar et al. (2015) 225 mL bath fermentation (batch) Pure culture 4 Yes (H2) 0.06* Wang et al. (2018) 280 mL serum bottles (batch) Pure culture 4 Yes (H2) 0.10* Kavitha et al. (2017) 250 mL conical flask (batch) Pure culture 4 Yes (CH4) 7.00* .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 18 Mulat et al. (2018) 120 mL bottles (batch) Pure culture 4 Yes (CH4) 0.06* Fotidis et al. (2017) 118 mL batch reactor (batch) Mixed culture 4 Yes (CH4) 0.04* Sarkar et al. (2017) 100 mL serum vials (batch) Pure culture 4 No (hydrocarbondegrading) 1.00* Ozbayram et al. (2018) 100 mL bottle (batch) No microbial additive apart from the inoculum - No 0.02* Zagrodnik et al. (2020) 120 mL serum bottles (batch) Pure culture 4 No 0.10* Larsen et al. (2009) 300 mL glass bottles (batch) Pure culture 4 No 1.36* Dams et al. (2016) 250 mL serum bottle flasks (batch) Pure and mixed culture 4 Yes (H2) Goat rumen liquid: 0.05* Flocculent sludge: 0.03* Granular sludge: 0.06* .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 19 Poszytek et al. (2018) 1 L glass bottles Pure culture 4 Yes (CH4) 1.00* Poszytek et al. (2019) 1 L bottles (batch) Pure culture 4 Yes (CH4) 1.00* Jain et al. (2021) 10 m3 reactor (batch) Pure culture 4,6 Yes (CH4) - Peng et al. (2014) Not clear Pure culture 3,4 Yes (CH4) 2 Kovács et al. (2013) 5 L CSTR Pure culture 4 Yes (CH4) 0.26* Lü et al. (2014) Sequenced batch reactor Pure culture 4 Yes (CH4) 0.01* Vidmar et al. (2017) Not clear Pure culture Not clear Yes (CH4) Not clear Town & Dumonceaux, (2016) 100 mL syringe (batch) Pure culture 2, 6 Yes (CH4) 0.0008* Yang et al. (2020) 1200 mL bottle (batch) Pure culture 4, 6 Yes (CH4) 0.01* .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 20 Senko et al. (2019) 120 mL reactor (batch) Pure culture 4 Yes (CH4) 1.00* Zheng et al. (2022) 500 mL batch bioreactor Pure culture 4 Yes (CH4) BD10: 0.08* BD30: 0.24* BD50: 0.40* BD70: 0.56* Tale et al. (2011) UASB reactor Not clear 2 Yes (CH4) 0.40* Loureiro et al. (2020) 125 mL glass bottles Pure culture 2 No 3.33* Barua et al. (2018) 1 L reactor bottles (batch) Pure culture 4, 6 Yes (CH4) 0.01* Chen et al. (2018) 6 L digester Pure culture 5, 6 Yes (CH4) 1.00 Yang et al. (2019) 500 mL reactor (batch) Pure culture 4, 5 Yes (CH4) 2.00 Lin et al. (2018) 1.5 L MBBRs reactor Pure culture 2, 6 No 0.09* Xiao et al. (2019) 2 L bottle (batch) Pure culture 4, 6 Yes (CH4) 4.00* .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 21 Wang et al. (2023) MFC Pure and mixed culture 4, 6 No - Akila & Chandra, (2010) Not clear Pure culture 4, 6 Yes (CH4) - Yan et al. (2021) 1.5 L batch reactor Not clear 2, 6 Yes (CH4) 0.02* Fotidis et al. (2014b) 118 mL batch reactor No microbial additive apart from the inoculum 2, 6 Yes (CH4) 2.00 Costa et al. (2012) 1 L bottle (batch) Pure culture 4, 6 Yes (CH4) 0.37* Mazzurco Miritana et al. (2023) 120 mL serum bottles (batch) Pure culture 4 Yes (CH4) 0.19* Gállego-Bravo et al. (2023) 125 mL serum bottles (batch) Pure culture 2 Yes (CH4) MC10: 0.11* MC25: 0.33* MC50: 1.00* MC75: 3.00* Xiao et al. (2024) CSTRs with 5 L volume Pure culture 4, 6 Yes (CH4) 2.00 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 22 Zhang et al. (2023) 250 mL bottles (batch) Pure culture 4, 5, 6 Yes (CH4) 1.00 Wang et al. (2024) 400 mL bottles (batch) Pure culture 4, 6 Yes (CH4) 2.00 Liu et al. (2023) 200 mL bottles (batch) Pure culture 4 Yes (CH4) 1.00 * Calculated after reviewing the respective references 246 Comparing the “types of experiment” in table 1, it becomes clear that the Biochemical 247 Methane Potential (BMP) assay is common choice. This assay is a valuable method for 248 determining the ultimate biodegradability and methane conversion yield of organic 249 substrates (Angelidaki et al., 2009). A critical parameter in the BMP assay is the inoculum250 to-substrate ratio (ISR), which significantly influences the efficiency of anaerobic 251 degradation, the relevance of the degradation test to full-scale digesters and the accuracy 252 of the assay. Research has shown that a higher ISR can improve the ultimate practical 253 methane yield. For example, a batch digestion test on microalgae found that an ISR of 2, 254 compared to 1 and 0.33, resulted in the highest methane productivity, ranging from 188 to 255 395 mL CH₄/g VS added across different microalgae types (Alzate et al., 2012). The 256 digestion of sunflower oil cake (SuOC) at an ISR of 3, compared to lower ratios, produced 257 the highest methane yield (Raposo et al., 2009). However, at lower ISRs, while the maximum 258 specific methane production rate was higher, the overall methane yield was lower, as 259 observed in BMP tests of maize at various ISRs (Raposo et al., 2009). This lower yield at 260 low ISRs was linked to the accumulation of longer-chain acids within the system, which could 261 inhibit methanogens, particularly due to the acetate produced during digestion at high 262 substrate concentrations (Maya-Altamira et al., 2008). Increasing the ISR, which involves 263 diluting the substrate, can help enhance practical methane yield. Most studies have focused 264 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 23 on the impact of ISR on methane yield for single substrates, with limited documentation on 265 its effects in co-digestion scenarios. Additionally, the source of inoculum is crucial, especially 266 when dealing with complex substrate mixtures, due to the diverse microbial consortia 267 involved. Calculating the ISR is important because it directly impacts the methane 268 production efficiency and overall yield, which is illustrated in table 1. Different substrates 269 produce varying methane outputs, which can be effectively assessed by considering the ISR 270 in the BMP assay. It is hypothesized by the authors a low ISR, or usage of an inoculum 271 source that is unsuitable for the substrate of choice can lead to false-positives on the effect 272 of bioaugmentation. 273 The ISR values presented in Table 1 show significant variability across different studies, 274 reflecting diverse experimental setups and microbial additives. In this review only 34% of 275 the studies meet the minimum required ISR of >1, and only 15% of the studies meet the 276 minimum desired ISR of >2. This threshold is based on Holliger et al. (2016). Among the 277 entries, the highest ISR is reported by Zhang et al. (2018), with a value of 9.4, achieved 278 using a pure culture in 100 mL serum bottles aimed at biogas production. This indicates that 279 the selected microbial strains and experimental conditions can greatly influence ISR 280 outcomes. In contrast, studies such as Arkatkar et al. (2020) report much lower ISR values, 281 such as 0.01, due to the use of a pure culture in a microbial fuel cell (MFC) setup. The 282 discrepancies in ISR values across authors can be attributed to differences in the type of 283 cultures used (mixed vs. pure), the experimental design (batch vs. continuous systems), and 284 the specific aims of the research, such as methane or hydrogen production. Therefore, while 285 some ISRs suggest strong potential for biogas formation, others indicate challenges that 286 may require further optimization or different microbial approaches. Overall, identifying the 287 most effective ISR for biogas production depends on the specific research context and 288 microbial strains utilized. 289 290 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 24 3.4 Manipulation of hydrolysis 291 The present work tries to distinguish applied microbes according to the different phases of 292 anaerobic digestion (hydrolysis, acidogenesis, acetogenesis and methanogenesis). This 293 separation is not always feasible, as there are overlaps. For example, some hydrolytic 294 bacteria yield organic acids, which results in an overlap between hydrolysis and 295 acidogenesis. This simultaneous involvement blurs the boundaries between the hydrolysis 296 and acidogenesis phases. Similar challenges arise in other phases, such as acetogenesis, 297 where certain microorganisms may contribute to both acidogenesis and acetate production, 298 creating further complexities in differentiation. The difficulty of dividing found articles into the 299 various phases of anaerobic digestion is also made clear in a work by Zhang et al. (2019). 300 Zhang et al. (2019) used the hydrolytic Thermoanaerobacterium thermosaccharolyticum 301 W16 mixed with undefined methanogenic granular sludge. It did not only improve hydrolysis, 302 but also syntrophic relations, which are rather related to the later phases of anaerobic 303 digestion. Nevertheless, the authors of the present study tried to distinguish the phases as 304 clearly as possible, starting with hydrolysis. Hydrolysis contemplates the first stage of 305 anaerobic digestion. During hydrolysis, complex organic compounds (e.g., carbohydrates, 306 proteins, and lipids) will be transformed into simpler molecules, like sugars, long chains of 307 fatty acids and amino acids due to the enzymatic attack made by different types of anaerobic 308 microorganisms. During this stage, various obstacles may arise that limit the AD process, 309 as well as the performance and adequate production of biogas. 310 The use of bioaugmentation within hydrolysis has been studied for various purposes with 311 the overall goal of improving the efficiency and overall stability of the AD process. The 312 systematic literature search performed in the present work resulted in 33 articles, which were 313 predominantly focused on the inoculation of hydrolytic microorganisms for bioaugmentation 314 within the first stage of AD. Many of them are focussed on the improved degradation of fibre315 rich material. For example, two of them highlighted the use of bioaugmentation to increase 316 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 25 the methane yield from cattle manure and brewery spent grain. Hydrolytic organisms are 317 promising here, as the mentioned substrates contain lignocellulosic biomass, which usually 318 degrades very slowly and, therefore, the hydrolysis takes longer to complete (Nielsen et al., 319 2007; Čater et al., 2015). In a similar approach, improved lignocellulose degradation has 320 been shown with Citrobacter werkmanii VKVVG4, Bordetella muralis VKVVG5 and 321 Paenibacillus sp. VKVVG1, but for water hyacinth (Barua et al., 2018). Another promising 322 approach was presented by Peng et al. (2014), who was able to enhance wheat straw 323 hydrolysis and to improve the biochemical methane potential (BMP) from wheat straw due 324 to the application of the cellulolytic anaerobic bacterium Clostridium cellulolyticum. The 325 outcomes showed BMPs of 342.5 ml g-1 VS and 326.3 ml g-1 VS, representing a 13.0% and 326 7.6% increase, respectively, compared to the BMP without bioaugmentation, which was 327 303.3 ml g-1 VS. Similar to Peng et al., Ecem Öner et al. (2018) worked on the degradation 328 on wheat straw too. They used Clostridium thermocellum to enhance methane yield from 329 lignocellulosic biomass by up to 39%. Ozbayram et al. (2018), also worked with wheat straw 330 as a substrate, enriching methanogenic communities from cow and goat rumen fluid and a 331 biogas reactor. The dominant strains in the enriched cultures were Bacteroidaceae spp. 332 (rumen) and Porphyromonadaceae spp. (reactor), with an increased abundance of 333 Ruminococcaceae spp. (Firmicutes). Similarly, Sinha et al. (2021) employed the cellulolytic 334 strains Microbacterium sp. DSB1 and Arthrobacter sp. DSB12 for lignocellulose degradation 335 of Lantana camara, achieving enhanced biogas production with methane yields of 57% and 336 60%, respectively. Based on the afore mentioned articles it stands out that especially 337 bacteria from the phylum Firmicutes are used abundantly to improve the degradation of 338 lignocellulose. In this regard, another study by Shanmugam et al. (2019) can be highlighted, 339 which focused on the strain Clostridium sp. WST. After isolating it from mangrove sediments, 340 this strain improved the degradation of lignocellulosic biomass degradation due to 341 bioaugmentation in anaerobic digestion experiments. 342 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 32 found that bioaugmentation with MJ2 significantly increased hydrogen production by 497 95.31%, and the addition of biochar further enhanced this by an impressive 158.10%. 498 In addition, Camargo et al. (2021) used a strain closely related to Enterococcus casseliflavus 499 for hydrogen production. The strain was isolated from citrus by-products and demonstrated 500 significant hydrogen production from xylose. Additionally, In a study by Kumar et al. (2015), 501 bioaugmentation with Escherichia coli XL1-Blue and Enterobacter cloacae DSM 16657 502 significantly improved hydrogen production from beverage industrial wastewater. The 503 addition of facultative anaerobic bacteria, combined with nutrients such as yeast extract and 504 tryptone, led to a remarkable increase in hydrogen production, especially when both the 505 bacteria and nutrients were used together. As mentioned already further above, it can be 506 promising to use more than one strain in a combined approach. This is also possible in 507 hydrogen production. In this regards, Laocharoen et al. (2015) investigated bioaugmentation 508 for hydrogen production by adding Rhodobacter sphaeroides KKU-PS5 and Lactobacillus 509 delbrueckii ssp. bulgaricus TISTR 895 into anaerobic digesters. While the co-cultivation 510 faced challenges due to differences in metabolic types, this study highlighted the potential 511 of combining these strains to improve hydrogen production through bioaugmentation. In a 512 similar approach, Sharma & Melkania, (2018) evaluated the effect of bioaugmentation with 513 three bacterial species (Escherichia coli, Bacillus subtilis, and Enterobacter aerogenes) on 514 hydrogen production from the organic fraction of municipal solid waste. It is also possible to 515 improve methanogenic communities due to the inoculation of hydrolytic strains. In this 516 regards, Jung (2012) investigated the impact of bioaugmentation with the mesophilic 517 cellulose-degrading strain Clostridium cellulolyticum H10 on anaerobic digestion of cattle 518 manure and wastewater sludge. This strain breaks down cellulose into hydrogen, acetate, 519 and ethanol, which enhance methanogenesis. One year later, a work from Kovács et al. 520 followed, where they specifically aimed for the inoculation of hydrogenic bacteria to improve 521 methanogenesis. Kovács et al. (2013) investigated the roles of pure hydrogen-producing 522 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 33 cultures of Caldicellulosiruptor saccharolyticus and Enterobacter cloacae in thermophilic 523 and mesophilic natural biogas-producing communities, respectively. Their findings indicated 524 that enhancing biogas production was associated with an increased abundance of hydrogen 525 producers, with the loading rate of total organic solids playing a crucial role in maintaining 526 an altered population balance. Promising results with Enterobacter cloacae have again been 527 demonstrated by Ács et al. in 2015. 528 In another study by Morales-Martínez et al. (2020), the production of hydrogen gas from 529 pretreated agave biomass. Cellulose-degrading microorganisms obtained from bovine 530 ruminal fluid were used to enhance H2 production by Clostridium acetobutylicum. The results 531 demonstrated the capacity of these microorganisms to hydrolyse the pretreated agave 532 biomass and improve hydrogen gas production, highlighting the potential of 533 bioaugmentation in biohydrogen generation. it was difficult to assign this to a clear phase 534 because H2 production relates to acidification but then it was applied in regard to hydrolysis. 535 Collectively, these findings underscore the potential of bioaugmentation strategies in 536 optimising anaerobic digestion processes, promoting higher yields of biogas and hydrogen, 537 and shedding light on the microbial dynamics responsible for enhanced degradation and 538 energy recovery from various organic waste substrates. Such insights can contribute to the 539 development of sustainable and efficient bioenergy production techniques with significant 540 implications for renewable energy applications. 541 Comparing all 33 articles, species from the following families have been successfully applied 542 in order to improve hydrolysis with the following substrates: Clostridiaceae (brewery spent 543 grain), Thermoanaerobacteraceae (corn stover), Thermotogaceae (sewage sludge), 544 Flavobacteriaceae (swine wastewater), Chlorellaceae (microalgal biomass), 545 Fibrobacteraceae (brewery spent grain) and Dictyoglomaceae (cattle manure). As the found 546 strains are mostly related to a better degradation of plant derived material, the importance 547 of lignocellulose needs to be highlighted. It is well known that lignocellulose is the most 548 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 34 abundant renewable material on the planet. It is easily accessible and also cost-effective. 549 However, its hydrolysis process is often difficult to complete due to its complex structure. 550 To move on to the next chapters, the case of Wang et al. (2024) will be described at this 551 point. They evaluated improved thermophilic anaerobic digestion (TAD) of food waste due 552 to four thermophilic strains: Ureibacillus suwonensis E11, Clostridium thermopalmarium 553 HK1, Bacillus thermoamylovorans Y25, and Caldibacillus thermoamylovorans QK5. Results 554 showed that cumulative methane production improved by 2.05% (E11), 14.54% (HK1), 555 19.79% (Y25), and 9.17% (QK5) compared to the control. Analysis of microbial community 556 composition revealed increased relative abundance of key hydrolytic bacteria, but also 557 methanogenic archaea. This highlights that the impact on the microbiome cannot just be 558 attributed to the functionality of the strains added. In the particular case of Wang et al., the 559 addition of hydrolytic bacteria was also affecting methanogenic archaea. 560 561 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 35 562 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 36 Figure 4. Pie chart indicating the diversity and frequency of microbial strains used for bioaugmentation 563 in anaerobic digestion systems. In the figure and throughout the study, the affiliations Methanothrix 564 and Methanosaeta have both been used. It must be noted that more recent works use Methanothrix 565 instead of Methanosaeta, as Methanosaetaceae were renamed to Methanotrichaceae. 566 3.5 Manipulation of acidogenesis 567 Previous research studies have investigated the role of bioaugmentation in acidogenesis. 568 With the chosen search terms, several articles were found, which were not only addressing 569 anaerobic methane production, but also dark fermentation related articles. Although the 570 present article is primarily not focussed on dark fermentation, these articles were not 571 excluded from the present set of literature. Acidogenesis is a crucial stage responsible for 572 converting complex organic compounds into valuable products such as volatile fatty acids 573 (VFAs) and hydrogen gas. In recent years, microbial bioaugmentation has emerged as a 574 promising approach to enhance acidogenesis efficiency by introducing specific microbial 575 species or mixed cultures into anaerobic systems. This section summarizes and analyses 576 several studies that explore the impact of different microbial bioaugmentation strategies on 577 acidogenesis performance. It needs to be highlighted again that it is difficult to separate 578 between hydrolytic and acidogenic bacteria, because many organisms are able to do both. 579 To cope with this conflict, the authors have shifted articles into the acidogenesis section, if 580 they were discussing the impact on volatile fatty acid (VFA) formation specifically. Articles 581 were also shifted into the acidogenesis section, if an improved hydrogen formation was 582 addressed, but without linking this specifically to syntrophic acetogenesis. Doing this, 583 remaining studies were exclusively related to dark fermentation. In this regard, many of the 584 found articles focus on the production of butyric acid and hydrogen gas (dark fermentation). 585 It needs to be highlighted that production and extraction of butyric acid and/or hydrogen is 586 something, which is usually not wanted in biogas plants, as they are supposed to enrich 587 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 37 methane and not hydrogen or organic acids. Therefore, it is crucial to distinguish clearly 588 which of these scenarios (dark fermentation or methane production) is addressed when 589 investigating the impact of bioaugmentation on acidogenesis. To distinguish between 590 hydrolysis and acidogenesis is not always simple. In this regard, Yang et al. (2016) used L. 591 hydrogenispora ethanolica LX-B in bioaugmentation experiments, which significantly 592 improved hydrogen production from complex substrates. The bioaugmentation with LX-B 593 resulted in hydrogen yields more than twice that of the control group in batch cultivation. 594 Regarding dark fermentation, the improved formation of hydrogen could be subjected to the 595 acidogenesis chapter. However, since improved degradation of complex substrates is 596 addressed, it could also be possible to discuss this article in the hydrolysis section. Another 597 example which fits clearer into the acidification section, has been published by Atasoy & 598 Cetecioglu, (2020). They investigated the enhancement of butyric acid production through 599 bioaugmentation with Clostridium butyricum in mixed cultures. Anaerobic sequencing batch 600 reactors were operated under alkaline conditions and fed with dairy industry wastewater as 601 the substrate. Bioaugmentation with Clostridium butyricum significantly increased butyric 602 acid production, indicating a positive influence of this specific microbial species on 603 acidogenesis in respect to dark fermentation. 604 605 In addition to this, Dams et al. (2016) investigated the potential of bioaugmentation with 606 Clostridium acetobutylicum ATCC 824 for hydrogen, organic acid, and alcohol production 607 using residual glycerol as the carbon source. Similarily to Clostridium butyricum, Clostridium 608 acetobutylicum ATCC 824 allows for the enrichment of hydrogen. Batch experiments were 609 conducted in pure and mixed cultures, with three different sources of inocula and the 610 experiments were conducted with mixed cultures. The work from Dams et al. is interesting, 611 as it shows the possibility to enrich other metabolites than just hydrogen and butyric acid. 612 Significant yields of hydrogen, but also 1,3-propanediol were achieved when Clostridium 613 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 38 acetobutylicum ATCC 824 was bioaugmented into the sludge from municipal wastewater 614 with 5 g/L of glycerol. One highlight of this work was further the application of glycerol, which 615 is regarded as a recalcitrant substrate. According to Dams et al. and with Clostridium 616 acetobutylicum ATCC 824 as a microbial additive, glycerol could be a promising substrate 617 for the generation of valuable products like hydrogen and 1,3-propanediol during dark 618 fermentation in mixed culture approaches. Similar as Atasoy & Cetecioglu (2020) or Dams 619 et al. (2016), Goud et al. (2014) evaluated the possibility of bioaugmentation for the 620 improvement of dark fermentation as well. However, they focused their work on indigenous 621 microorganisms, which are naturally present in the environment of dark fermentation 622 processes. They applied three acidogenic bacterial isolates belonging to the phyla 623 Firmicutes and Proteobacteria, in order to increase hydrogen formation, but also to cope 624 with elevated organic: for this they used the species Bacillus subtilis, Pseudomonas stutzeri, 625 and Lysinibacillus fusiformis. In addition to the work from Dams et al. (2016), Wang et al. 626 (2008) also demonstrated the potential of Clostridium acetobutylicum ATCC 824 in 627 biohydrogen production through dark fermentation but focused on using microcrystalline 628 cellulose as the carbon source. 629 Some studies on dark fermentation focus more on the production of organics acids. In this 630 regard, the work by Zheng et al. (2022) can be highlighted. They investigated a biochemical 631 strategy to enhance propionic acid production from kitchen waste acidification through 632 bioaugmentation with Propionibacterium acidipropionici. Their results showed that when the 633 inoculum of Propionibacterium acidipropionici comprised 30% (w/w) of the seeding sludge, 634 propionic acid production increased by 79.57%. 635 Most articles that addressed dark fermentation worked with single stages process. In this 636 regard, Liu et al. (2023a) stands out who addressed bioaugmentation in a more holistic 637 approach. They investigated the impact of bioaugmentation technology on anaerobic 638 digestion processes by directly manipulating microbial structure through bioaugmentation in 639 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 39 a two-stage co-digestion system. In their study, different doses of Thermoanaerobacterium 640 thermosaccharolyticum were introduced into the hydrogen producing pretreatment stage. 641 The system was operating at 55°C. The findings revealed that the addition of 642 Thermoanaerobacterium thermosaccharolyticum at 1.12 g had the most significant impact, 643 resulting in cumulative hydrogen and methane yields of 81.54 mL/g VS and 550.98 mL/g 644 VS, respectively. These values were 68.72% and 84.45% higher than those of the control 645 group. Microbial analysis indicated notable changes in microbial community structure, with 646 an increase in the relative abundance of Thermoanaerobacterium thermosaccharolyticum 647 during the hydrogen production stage. This increase led to higher levels of volatile fatty acids 648 (VFAs) and hydrogen content, suggesting a potential influence on the acidogenesis step of 649 anaerobic digestion. Like the other acidogenesis related studies, the work from Liu et al. is 650 addressing dark fermentation. However, and unlike the other articles, Liu et al. were the only 651 ones, who implemented this into a system with a subsequent methanation stage. 652 It needs to be highlighted that bioaugmentation in acidogenesis is not always about 653 improving biomass degradation. It can also be about the elongation of fatty acids. In this 654 regard, a study by Zagrodnik et al. (2020) can be highlighted. They used Clostridium kluyveri 655 (AS + CK) in chain elongation processes, where it produced medium-chain fatty acids, such 656 as caproic acid, from a mixed substrate. 657 658 3.6 Manipulation of acetogenesis 659 The acetogenesis section explores various strategies to enhance acetate production within 660 AD. Research has focused on utilizing specific microbial strains, such as Clostridium and 661 Thermoanaerobacterium (Kuribayashi et al., 2017) to improve acetate yields. These strains 662 have shown promise in optimizing the acetogenesis phase by facilitating more efficient 663 conversion of intermediates into acetate. As Bacillus species, including Brevibacillus sp. KH3 664 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 40 (Li et al., 2009), Bacillus subtilis (Xu et al., 2018), and B. licheniformis (He et al., 2017), have 665 also been studied for their role in stimulating hydrolytic enzymes and enhancing AD 666 performance, their contributions are more pertinent to the hydrolysis stage and are thus 667 discussed in the corresponding section. In the context of acetogenesis, interactions between 668 acetogens and methanogenic archaea or hydrogen-producing bacteria have been linked to 669 improved biogas production. In this regard, Wang et al. (2018) can be highlighted. They 670 developed a microbial consortium entitled D83. For this consortium they highlighted the 671 occurance of Syntrophospora bryantii, Sedimentibacter sp., and Thermosyntropha bryantii, 672 Methanosarcina sp. and Methanobacterium ferruginis. They further explained that D83 was 673 dominated by hydrogen-producing acetogens, which helped to enhance methane 674 production. Bioaugmentation with D83 doubled methane yield and rate from glucose 675 fermentation and improved COD removal in molasses wastewater treatment. This study 676 highlighted hydrogen-producing acetogenesis as a key step in methanogenesis, improving 677 both acidogenesis and methanogenesis. There are cases, where such an improvement of 678 methanogenesis is further interwoven with syntrophic relations. Syntrophic bacteria can be 679 involved due to their role in syntrophic acetate oxidation and hydrogen turnover (Zhang et 680 al., 2019). However, the specific role of methanogenic archaea in bioaugmentation is 681 beyond the scope of acetogenesis and is elaborated further in the methanogenesis section. 682 By focusing on acetogenic strains that directly contribute to acetate formation, researchers 683 aim to optimize this critical intermediate step in AD. In a study by Huang et al. (2020), the 684 factors influencing the growth and acetate production efficiency of the Clostridium sp. 685 NJUST19 strain were investigated under different environmental conditions. The 686 experimental results of digesting waste activated sludge (WAS) with the addition of 687 Clostridium sp. NJUST19 showed enhanced Total Suspended Solids (TSS) degradation and 688 increased concentrations of VFAs. The TSS degradation rate increased to 35.3%, which 689 was 13.4% higher than the control group. Additionally, the maximum VFAs concentration 690 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 41 reached 4200 mg/L, indicating a significant increase of 45.8% compared to the control 691 group. This is another example, which shows how intertwined the different phases of 692 anaerobic digestion are. Although the title from Huang et al. refer to acetogenesis, it remains 693 difficult to differentiate between hydrolysis, acidogenesis and acetogenesis in this specific 694 case. It stands out that in total just one article was fitting into the “acetogenesis” chapter. On 695 one hand, this might indicate a research gap. Amongst the detected articles, there were 696 almost no articles addressing syntrophic butyrateand propionate-degrading bacteria 697 (SBOBs and SPOBs) and it might be interesting to test the suitability of such organisms for 698 bioaugmentation. On the other hand, missing articles on bioaugmentation with a specific 699 focus on syntrophic, acetogenic bacteria might also be explained by difficulties in culturing 700 such bacteria. It might well be that it is just impractical to use syntrophic, acetogenic bacteria 701 for bioaugmentation. One study that was found, has been published by Shao et al. (2020). 702 They studied bioaugmentation to accelerate recovery in an anaerobic sequencing batch 703 reactor, which was exposed to an organic shock load. The bioaugmented reactor, with a 704 butyric acid-utilizing culture containing Methanobacteriales and Syntrophomonas, recovered 705 faster (40 days) than the non-bioaugmented reactor (110 days), by relieving feedback 706 inhibition and boosting propionic acid degradation. Another interesting article in the regard 707 comes from Tale et al. (2011), who utilized a propionate-degrading enrichment culture 708 dominated by Methanospirillum hungatei and Methanobacterium beijingense to bioaugment 709 anaerobic digesters. This approach enhanced recovery after organic overload by reducing 710 acid accumulation and shortening recovery time by approximately 25 days, demonstrating 711 the effectiveness of bioaugmentation in improving process stability. Both studies, the one by 712 Shao et al. and the one by Tale et al., include methanogens, which shows once again that 713 it is not always possible to clearly distinguish published cases regarding the different phases 714 of anaerobic digestion. Yet another case is the study by Akila et al. (2010). They isolated a 715 psychrotrophic xylanolytic acetogenic strain, Clostridium sp. PXYL1, and the 716 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 48 systems. Four reactors were subjected to ammonia shocks: one with biogel, one with 872 biochar, one with both, and a control. Results showed that reactors receiving both 873 supplements achieved 100% methane production recovery, while the other configurations 874 showed methane production losses. They described further that reaction with biochar, or the 875 combination of biochar and biogel facilitated the adaptation to higher ammonia levels. It 876 attracts attention that multiple studies tackled ammonia inhibition successful by applying 877 Methanoculleus or consortia containing Methanoculleus (Fotidis et al., 2013; Fotidis et al., 878 2017; Yan et al., 2021). In yet another study, Fotidis et al. (2014a) revealed that an increase 879 in methane levels in ammonia-rich environments could be directly linked to the presence of 880 Methanoculleus. They introduced a fast-growing hydrogenotrophic methanogen, 881 Methanoculleus bourgensis MS2T, into a reactor with high ammonia levels, achieving an 882 31.3% increase in methane production. High-throughput gene sequencing showed a 5-fold 883 rise in Methanoculleus spp. abundance after bioaugmentation. Although these results on 884 Methanoculleus appear quite promising, there are other hydrogenotrophic methanogens, 885 which could also help to overcome ammonia inhibition. In this regard, Wang et al. (2015) 886 worked with four different hydrogenotrophic methanogens, namely Methanoculleus 887 bourgensis, Methanobacterium congolense, Methanoculleus thermophilus, and 888 Methanothermobacter thermautotrophicus. These hydrogenotrophic methanogens were 889 applied together with two SAOBs, namely Tepidanaerobacter acetatoxydans and 890 Thermacetogenium phaeum. Under different ammonia concentrations (0.26, 3, 5, and 7 g 891 NH4+-N L-1), all strains showed the potential to improve the process performance. Yet in 892 another study, Gállego-Bravo et al. (2023) studied enhanced methane production from 893 municipal waste by bioaugmenting a thermophilic anaerobic digestion process with a 894 hydrogenotrophic methanogenic community. Interestingly, they did not work on ammonia 895 inhibition. Instead, the bioaugmentation improved methane yield from the organic fraction of 896 municipal solid waste by 4%. This indicates that bioaugmenting anaerobic digesters with 897 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 49 hydrogenotrophic methanogens is useful for more scenarios than just ammonia inhibition. 898 Key microbes involved were the archaeon Methanoculleus and bacterial order MBA08. 899 To overcome ammonia inhibition the application of oxygen might be interesting too. Although 900 not linked to ammonia inhibition, there is a work the combines the application of oxygen and 901 bioaugmentation. Hua et al. (2022) explored the use of micro-aerobic microbial communities 902 at elevated temperatures. By introducing the methanogens Methanosarcina acetivorans 903 C2A and Methanosaeta thermophila NBRC 101360, they achieved a significant increase in 904 biogas production of about 44.78%. 905 So far, most articles about methanogenesis addressed ammonia inhibition or improved 906 syntrophic relations. But there are other stressors that could impair methanogenic 907 communities and, in this regard, it could be interesting to apply methanogens, which can 908 better cope with acidosis. That this is possible was already mentioned earlier with the work 909 by Chen et al., who highlighted the fast recovery of Methanosarcina. Another interesting 910 work to cope with acidosis has been presented by Savant et al. (2004). They used the acid911 tolerant hydrogenotrophic methanogen Methanobrevibacter acididurans to enhance 912 methane production and reduce VFA accumulation in acidic anaerobic digesters. In another 913 study, Li et al. (2021) demonstrated that Methanosaeta dominated in anaerobic digestion in 914 oxytetracycline contaminated sludges under acidic conditions with a pH of 4.6 at the first 915 compartment of an anaerobic baffled reactor. In a further study by Town & Dumonceaux 916 (2016), they introduced an acetoclastic consortium into acidified batch digesters, which 917 significantly reduced acetate accumulation and increased methane production. PCR 918 analysis revealed a substantial increase in an acetoclastic methanogens related to 919 Methanosarcina sp, which highlights once again the high potential of the genus 920 Methanosarcina. 921 922 923 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 50 3.8 Extraordinary approaches in bioaugmentation research 924 In table 1, the use of controls has been assessed as described further above. Numbers 1 - 925 4 were used to indicate whether controls were applied. However, not in all experimental set926 ups it is possible or useful to have control experiments, where no cells, autoclaved or non927 autoclaved cells are added. Several experiments are not focused on biogas production. 928 Some of them have an unusual set-up, which is designed to evaluate selected strains rather 929 than a complex microbiome. In table 1, such extraordinary cases have been defined as 930 “other”. This concerns for example the case of Arkatkar et al. (2020). The authors assessed 931 coculture conditions for multiple strains based on redox activity, electron transfer rate, 932 columbic efficiency, and internal resistances in a microbial fuel cell, which is very different 933 from typical anaerobic digestion experiments. In such experiments, the aim is not to 934 implement a certain strain into a complex microbiome and therefore, no controls are needed. 935 At least not in the sense as it was analysed in table 1. Nevertheless, such experiments have 936 a certain importance for bioaugmentation. Mapping of microbial interactions can help to 937 define conditions, which might be relevant for bioaugmentation in practice. Another exotic 938 example is from Rinland & Gómez, (2015), who have searched for strains that allow better 939 degradation of onion waste. In this case, multiple strains were isolated from onion waste. 940 However, Rinland and Gómez did not work with complete methanogenic communities. They 941 selected strains that showed good degradation capabilities, and in this case, biogas 942 formation was not a suited criterium to evaluate the experimental success. The authors 943 isolated strains from onion waste at different degradation stages and locations. Growth 944 patterns and carbon source utilisation of the isolates were analysed to identify promising 945 candidates. Among the selected strains, Bacillus subtilis sp.MB2-62 and Pseudomonas 946 poae VE-74 demonstrated characteristics making them potential candidates for 947 bioaugmentation or pretreatment in anaerobic digestion processes. As control, they always 948 used a sterilised tube without any active microbes. The work from Rinland & Gómez was 949 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 51 taken into account as they were screening for microbes with potential for bioaugmentation, 950 although these strains were not applied in bioaugmentation experiments yet. 951 Amongst the works, which compared different strains in regard to their biogas formation 952 potential, Jones et al. (2010) was also found as an extraordinary example. They focused on 953 methane generation from nonproductive coal. Coal is a rather unusual substrate for biogas 954 formation, which usually is not degraded. However, the respective production sites contain 955 degradable coal intermediates (geopolymers), for which Jones et al. were highlighting their 956 potential in respect to biogas formation as a potential fuel source. The researchers 957 stimulated methane production using two approaches: biostimulation with nutrient 958 supplementation and bioaugmentation with a consortium of bacteria and methanogens 959 enriched from wetland sediment. The approach differs strongly from typical anaerobic 960 digestion approaches, as they were not starting the experiments with manure from animals 961 or water treatment, which is usually the case in the biogas industry. Apparently, the coal had 962 some intrinsic methanogenic activity, which can be stimulated with nutrients. The biogas 963 formation was even better, if they used a mixed culture from wet-lands. However they did 964 not describe any control, where autoclaved cells were added. So it is difficult to say, which 965 amount of biogas could be attributed to the amount of COD, which was present in the cell 966 mixture added. 967 Although the focus of the present work was not on microbial fuels cells (MFCs), the used 968 search terms also related to some articles, which used this technology. One work that can 969 be highlighted here is from Arkatkar et al. (2020). They worked with pure cultures, which is 970 usually not regarded as bioaugmentation. However, Arkatkar et al. analysed the coculture 971 behaviour of multiple species. A deeper understanding on how different strains interact and 972 how they might be combined is indeed of interest for better understanding of 973 bioaugmentation. Arkatkar et al. performed coculturing experiments in the anodic chamber 974 of multiple strains, namely Pseudomonas aeruginosa BR, Alcaligenes faecalis SW and 975 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 52 Escherichia coli EC. Arkatkar highlighted that coculturing Pseudomonas aeruginosa BR with 976 Alcaligenes faecalis SW or Escherichia coli EC improved the energy generation in both 977 cases. Although the primary goal for Arkatkar et al. was to improve the performance of MFCs 978 and not typical digesters, such experiments can help to define synergies between 979 microorganisms. Although not found with the search terms applied in the systematic search 980 for this study, similar works can be found, when specifically searching for this. For example, 981 a recent work has demonstrated a light driven carbon dioxide reduction to methane by 982 Methanosarcina barkeri in an electric syntrophic coculture (Huang et al., 2022b). Bagchi & 983 Behera (2021) investigated the impact of bioaugmentation on microbial fuel cells (MFCs) by 984 introducing Pseudomonas aeruginosa into anaerobic sludge (MFCP) and comparing its 985 performance to a control MFC seeded with mixed anaerobic sludge (MFCC). They also 986 tested an additional MFC with intermittent aeration and bioaugmentation (MFCP+A). The 987 results showed that MFCP+A produced significantly more electricity than MFCP alone, with a 988 4% increase compared to MFCP and a 31% increase compared to MFCC. This improvement 989 was due to better organic degradation and more efficient electron transfer in the 990 bioaugmented MFCs. The MFCP+A configuration achieved a coulombic efficiency of 10.4%, 991 which was higher than both MFCP (9.7%) and MFCC (4.41%). These findings are relevant to 992 anaerobic digestion because they demonstrate that bioaugmentation with specific 993 microorganisms, along with intermittent aeration, can enhance the efficiency of electron 994 transfer and electricity generation. This approach can potentially be applied to improve 995 anaerobic digestion processes by optimizing microbial activity and enhancing overall system 996 performance. 997 Finally and in regard to extraordinary approaches, wastewater treatment should be 998 highlighted. Lin et al. (2018) used Pseudomonas aeruginosa for denitrification treatment 999 processes. This process is difficult to relate to any of the four phases of anaerobic digestion. 1000 1001 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 53 4. Conclusions 1002 In conclusion, the literature underscores the significant potential of augmenting 1003 microorganism populations to amplify biomethane production within anaerobic digestion 1004 (AD) systems. Bioaugmentation offers the potential to improve yield, speed and robustness 1005 through increased biomass conversion, faster digestion rates and / or enhanced process 1006 stability. While much of the research has been confined to laboratory settings, the prospect 1007 of scaling-up these strategies appears promising. The focus of bioaugmentation primarily 1008 on the hydrolysis/acidogenesis phase of anaerobic digestion (AD) is logical, as this stage 1009 plays a critical role in enhancing the degradation rate and yield of lignocellulosic compounds. 1010 This is particularly important given the abundance of lignocellulosic waste, which serves as 1011 the primary feedstock for AD and is often subjected to various stressors. The studies 1012 reviewed include a variety of microbial populations, ranging from single species to simple 1013 and complex consortia. Notably, multiple articles have shown that the augmentation of just 1014 one or a few species can significantly impact the composition of the entire microbiome, 1015 underscoring the importance of a metataxonomic approach to study the dynamics of 1016 bioaugmentation. Best practice recommended by the authors is to not only map the 1017 bioaugmentation culture itself and the microbiome of the anaerobic digester inoculum before 1018 bioaugmentation, but also monitor the anaerobic digester after bioaugmentation. This 1019 approach helps in understanding how bioaugmentation influences the microbial community 1020 structure and functionality over time, which is crucial for optimizing AD processes. Alongside 1021 taxonomic screening we recommend putting more attention on conducting the research at 1022 a recommended ISR to ensure practical relevance of the results. Moreover, research has 1023 consistently demonstrated that the addition of co-cultures or small consortia often produces 1024 more significant effects compared to the augmentation of single species. This highlights the 1025 potential of mixed-culture bioaugmentation as a promising field for further study, as these 1026 consortia can better mimic natural microbial communities, leading to more robust and 1027 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 24, 2025. ; https://doi.org/10.1101/2025.01.22.634285doi: bioRxiv preprint 54 efficient degradation processes. Among the microorganisms studied, numerous efficient 1028 species, such as those from the Clostridiaceae family, have shown particular promise in 1029 enhancing the hydrolysis/acidogenesis phase. Hydrogenotrophic methanogenic archaea 1030 have a great potential in improving digester robustness, it stands out that Methanoculleus 1031 was used most often. Additionally, despite the few articles, Methanosarcina methanogens 1032 exhibit remarkable resilience and versatility in biomethane production, making them prime 1033 candidates for enhancing the methanogenesis phase and avoiding the accumulation of 1034 acetate or hydrogen in digester systems. This improved speed and robustness is opening 1035 up the possibility of increasing digester OLR. Despite their potential, research into their 1036 utilization in bioaugmentation remains limited. One reason for this might be the difficulties in 1037 culturing them in pure culture. Leveraging these archaea could yield substantial benefits, 1038 particularly in addressing volatile fatty acid accumulation during the hydrolysis/acidogenesis 1039 phase. Thus, further exploration and implementation of bioaugmentation strategies, 1040 especially involving mixed cultures and key species like Methanosarcina, hold great promise 1041 for optimizing AD processes and advancing sustainable biogas production. 1042 1043 Acknowledgements 1044 The authors gratefully acknowledge financial support from the European Union under the 1045 MICRO4BIOGAS project (reference ID 101000470), funded by the European Union's 1046 Horizon 2020 research and innovation programme. 1047 1048 Author contributions 1049 MA: Conceptualization, Methodology, Software, Formal Analysis, Investigation, Resources, 1050 Data Curation, Writing – Original Draft, Visualization; JT: Conceptualization, Methodology, 1051 Writing – Review & Editing; ML: Methodology, Software, Writing – Original Draft, 1052 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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