Processes: Approaches in Design of Laboratory-Scale UASB Reactors

processes
Review
Approaches in Design of Laboratory-Scale
UASB Reactors
Yehor Pererva , Charles D. Miller and Ronald C. Sims *
Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA;
[email protected] (Y.P.); [email protected] (C.D.M.)
* Correspondence: [email protected]

Received: 6 June 2020; Accepted: 20 June 2020; Published: 24 June 2020
Abstract: Up-flow Anaerobic Sludge Blanket (UASB) reactors are popular tools in wastewater
treatment systems due to the ability to work with high feed rates and wastes with high concentration
of organic contaminants. While full-scale industrial applications of UASB reactors are developed and
described in the available literature, laboratory-scale designs utilized for treatability testing are not
well described. The majority of published studies do not describe the laboratory UASB construction
details or do use reactors that already had developed a trophic network in microbial consortia
under laboratory environment and therefore are more stable. The absence of defined guidelines for
geometry design, selection of materials, construction, operation rules, and, especially, the start-up
conditions, significantly hamper researchers who desire to conduct treatability testing using UASB
reactors in laboratory scale. In this article, we compiled and analyzed the information available in the
refereed literature concerning UASB reactors used in laboratory environment, where information on
geometry and/or operational conditions were provided in detail. We utilized the information available
in the literature and the experience gained in our laboratory (Sustainable Waste to Bioproducts
Engineering Center) to suggest a unified operation flowchart and for design, construction, operation,
and monitoring for a laboratory-scale UASB reactors.
Keywords: up-flow anaerobic sludge blanket reactors; anaerobic digestion; laboratory-

scale experiment
1. Introduction
Up-flow Anaerobic Sludge Blanket (UASB) reactor is an anaerobic digester for wastewater
treatment, and its operational concept can be described as a vertical up-flow pumping of liquid
substrate, including wastewater or growth media, through a layer of anaerobic sludge [1–6]. Microbial
consortia inside the sludge layer consume digestible components as substrate and decompose them
into smaller chemical compounds [7]. Within the scope of a wastewater treatment, the goal of anaerobic
digestion is a complete mineralization of organic compounds combined with the production of biogas
for the purpose of energy recovery.
A distinguishing feature of UASB reactors is the formation of microbial conglomerates, where the
metabolic product of one microbial group is a consumable substrate for another microbial group [8].
Such microbial conglomerates grow into spherical or bean-shaped granules over time [9–13]. The sizes
of granules may vary, but typically are reported in the range 0.5 to 6 mm, where longer operation leads
to larger sizes [14–16]. Granulation of sludge is promoted by the presence of microorganisms that are
able to produce and secrete Exocellular Polymeric Substances (EPS) [17]. The term “EPS” includes
multiple types of compounds, which serve as a glue to agglomerate microorganisms together and to
add some mechanical strength to a granule [18].
Processes 2020, 8, 734; doi:10.3390/pr8060734 www.mdpi.com/journal/processes

Processes 2020, 8, 734 2 of 26
A combination of developing trophic microbial connections and mechanical cementation with

EPS, results in higher resilience of larger granules to sudden changes of operation conditions including
a change in pH, temperature mode failure, substrate switch or inconsistency of a substrate strength
and content, feeding rate fluctuations, etc. [19,20]. In some cases, granules can be disrupted due to
hydrodynamic forces or inner gas pressure into several smaller fragments [12,21,22] which become
cores for the formation of new granules [23].
The traditional concept of a UASB reactor, as suggested by Lettinga [10,11,24–26], is represented
in Figure 1a. The substrate is pumped to a reactor through the distribution system into a bottom
layer of anaerobic sludge. Equally distributed in normal cross-section of the reactor, the substrate is
pushed through the sludge layer (called a “digestion zone”) creating a vertical up-flow. This process is
concurrent with the decomposition of organic compounds of substrate and a formation of gaseous
products. Besides feeding the reactor, the continuous vertical up-flow of substrate prevents the sludge
layer from clogging, keeping it afloat. However, the up-flow does wash out the unattached biomass
(microorganisms, that did not start to form flocs) and small flocs/granules. The liquid part above the
sludge layer (called “settling zone”) serves as a vertical settler and/or coagulation column to initiate
the biomass and solids retention process before the actual separation. The separation process occurs in
the compartment called Gas–Liquid–Solids separator (GLSS, a.k.a. three-phase separator). GLSS is
traditionally located on top of the reactor column and it starts with a baffle-shaped structure in its
bottom part, which serves the purpose of collecting and re-directing the gas bubbles to the main gas
collection part and preventing gas bubbles from escaping with effluent.
Figure 1. Operational concept of traditional Up-flow Anaerobic Sludge Blanket (UASB) reactors:
(a) traditional; (b) with modified gas collector; and (c) Y-shaped.
The construction concept of the GLSS is shown in Figure 1a, where it’s implemented via narrowing
the outlet of the reaction tube with baffles. Such baffles are typically referred to as “deflectors” or
“collar”. The side effect of narrowing the reaction tube outlet is a creation of local velocity gradient
(velocity shear), which slightly enhances the formation of granulated particles, their separation from
liquid and settling back to the bottom of the reactor. Above the baffles, the GLSS contains the
gas collecting structure, where the cross-section looks like a flipped upside-down funnel. In some
studies, this funnel is replaced by a tubular structure with diameter larger than the distance between
baffles [27,28]. The liquid is forced to flow through the space in between the lower edge of the gas
collector and the baffles, to go around the funnel and leave the reactor at the effluent port.
Processes 2020, 8, 734 3 of 26
Other existing modifications of GLSS in laboratory-scale reactors can improve the higher solids
retention time, such as installing a high rate settler in headspace [29] or modification of three-phase
separators [30].
In addition to the operational concept of the UASB reactor shown in Figure 1a, the same authors [11]
also describe UASB reactor with a modified gas collector, which is demonstrated in Figure 1b. However,
some studies [31] call such a modification of the Up-flow Anaerobic Sludge Baffled Reactor (UASBR).
It may also contain the inner mechanical agitation device to prevent foam formation in the gas collecting
area [32]. Recently, the Y-shaped variation of UASB reactor also became popular and is depictured
in Figure 1c. In the case of the Y-shaped reactor, the GLSS is split into two individual separators:
one separator is used to separate gas from the liquid and collect it directly at the top of a main tube,
whereas a second collector is a sidearm tube that serves as an inclined settler for separating solids from
liquid (similar to a Lamella clarifier). Use of a funnel-shaped gas-collecting element becomes optional
in such case, since it serves only the purpose of preventing gas flow to an effluent side-arm.
Considering the concepts described, the optimization goal of a laboratory scale UASB reactor
operation is to achieve better performance, where optimization targets for UASB performance include
the following:
• Higher removal of contaminants;

• Higher biogas production rate;
• Shortening of adaptation period; and
• Resilience (robustness) of sludge.
To achieve some of those optimizations, the classical UASB concept can be combined with other
types of reactors, resulting in a range of composite reactors. Some modifications are found in the
literature and are presented in Table 1. This table represents options, where another reactor type is
incorporated into the UASB itself, but not a sequence of two consecutive reactors.
Table 1. Existing hybrid versions of UASB reactors.
Unit to Incorporate into UASB Resulting Reactor Name Purpose of Incorporation Reference
Up-flow Anaerobic Increase the methane production
Electrolysis cell BioElectroChemical via partial capture of dissolved [33–36]
reactor (UABE) carbon dioxide
UASB-AF Increase retention of solids inside [37]
Anaerobic Filter Anaerobic hybrid of a reactor and prevent washout
of active biomass [14,31,38,39]
reactor (AHR)
Increase solids retention
Lamella settler No Name [40]
time (SRT)
In a holistic view, the purpose of UASB reactor optimization is to keep the microorganisms
in a stage of maximum substrate consumption and active growth. However, from an operational
perspective, the optimization of UASB functioning is achievable via adjusting operational parameters,
including, but not limited to:
• Organic Loading Rate (OLR) and Hydraulic Retention Time (HRT);

• Recycle ratio of effluent;
• Regulation of pH;
• Retention of biomass; and
• Granulation enhancement.
Despite the long history since the invention and description of the UASB concept by
Lettinga et al. [41] and increasing its application in industry, UASB laboratory scale reactors used for
treatability studies are highly variable with regard to terminology, design, construction, and operation
Processes 2020, 8, 734 4 of 26
processes. This lack of uniformity leads to different results regarding water quality indicators,
for example, Chemical Oxygen Demand (COD), as well as bioenergy production, for example for
biomethane and biohydrogen. There is a lack of uniformity with regard to the guidelines for operation
of laboratory scale conditions, which is highlighted in this manuscript and recommendation are
provided for making UASB laboratory studies and results more uniform with results more transferrable
among laboratories and more useful for scale up activities. These lack of uniformity with laboratory
scale UASB reactors is addressed in this study and guidelines are provided for increasing the uniformity
so that results are comparable across different laboratories and are also more meaningful for scale up
applications of the UASB reactor process.
2. Review of Existing Solutions across Various Published Works

Despite a large number of available publications on wastewater treatment involving UASB reactors,
a majority of the studies only briefly mentions constructional concepts of the reactor, and dimensional
parameters are mentioned even more rarely. We collected available information on physical dimensions
among existing studies in Table 2, while Table 3 shows the geometry of either hybrid reactors or where
UASB reactors are installed in series with any other reactor. While building those tables we focused
on the geometry of the reactor and operational conditions including substrate strength expressed as
COD, Biological Oxygen Demand (BOD), etc.; loading rates; volume of reactor; and effluent recycling
rates. The type of the substrate used in reported studies is provided for reference purpose only.
Where Table 2 does not contain the geometric or operational parameter means that such value was
not specified in the reference. Also, Tables 2 and 3 do not provide calculations based on available
geometry. All information provided there is information stated in referenced publications, nothing
was added. The only modifications were made to units (for COD, BOD, etc.) where they were unified
across all publications.
As we can see from Tables 2 and 3, there is no uniformity in parameters of operating the UASB
reactor and, and what is in our opinion even more important, information on the start-up of a laboratory
UASB reactor. Such inconsistency may complicate the interpretation of results as well as the accuracy
and successfulness of an experiment in general. On the larger scale, it also complicates the comparison
of results obtained by different laboratories, which creates problems for feasibility studies, if the
literature is the primary source of information. To be more precise, in case of a failure, incomplete
information does not allow an interpretation of the data and to trace-back the reason for failure, such as
unadopted inoculum or its insufficient amount, problem of biomass washout due to the geometry,
problematic substrate properties, or wrong OLR or recycle ratio. Inconsistent reporting units (like OLR
calculated as per total volume of reactor or per volume of digestion zone) may also harm the attempt
to reproduce results of one laboratory in another one, or wrong implementation of a procedure.
Among the inconsistencies found across the studies, we also see terminology problem in the use
of terms ‘sludge blanket’ and ‘sludge bed’. The controversy of the terms is in that fact that they are:
• used interchangeably (equally)

• ‘sludge bed’ refers to a layer of sludge at the bottom, where it is concentrated and visually seems to
be a packed layer, while ‘sludge blanket’ refers to a part of the reactor where sludge is swimming
as flocs above the ‘sludge bed’
• ‘sludge bed’ refers to a bottom layer of sludge, and uses ‘transition zone’ instead of a ‘sludge blanket’
Below, in Table 4, we attempted to systematize all parameters we were able to identify in the
publications reviewed. Information in Table 4 does not intend criticize, but instead the intent is to
generalize and categorize information from publications referenced above.
Processes 2020, 8, 734 5 of 26
Table 2. Overview of available information of UASB reactors used in laboratory studies.
Used Type of Reactor, Material, Inoculum,

# Substrate Operating Conditions Constructional Geometry Reference
and Seeding
1. Hydrous ethanol vinasse HRT: 6 . . . 15 days Type: Figure 1b with added extra high rate Cylindrical part diameter: 11 cm [29]
kgCOD settler above gas collecting part Cylindrical part height: 35 cm
mg OLR: 7.27 . . . 22.16 m3 ·day
COD: 121,000 L Material: Acrylic Settler basement square side: 17 cm
Start-up OLR:
pH: 4 kgCOD Inoculum: taken from already functioning Settler height: 21 cm
Ethanol: 21,007 L
mg Days 1–6: 0.34 m3 ·day with UASB reactor treating vinasse of Settler is installed on top of cylindrical part.
mg
Acetic acid: 2237 L synthetic wastewater banana waster. Settler plates incline: 45◦
mg kgCOD Operational Volume: 3 L
Propionic acid: 4304 L Days 7–8: 5.9 m3 ·day
with substrate
No sampling ports
2. Distillery Spentwash HRT: 10 days Type: Figure 1a Digestion zone: 10 × 10 × 98 cm [42,43]
pH: 3.8 . . . 4.2 OLR: 11.75
kgCOD Material: Acrylic Transition zone: 10 × 10 × 6 cm
mg m3 ·day
COD: 122,000 L Inoculum: Laboratory enriched sludge from GLS zone: 19.2 × 19.2 × 25 cm
mg ongoing reactor by cow dung slurry. Digester volume: 16.75 L
TS: 121,020 L
Seeding: Seeding by filling the 50% of volume Settler volume: 7.15 L
with sludge mixture and multiple dilution by GLS opening angle: 53 ◦ C
wastewater sample. 8 sampling ports with 10 cm spacing
3. Spent wash of distillery plant pH is adjusted to 6.5 . . . 7.5 with Type: Figure 1a Operational Volume: 10 L [44]
lime (Ca(OH)2 ) Material: Acrylic Tube I: 11.7 cm
mg
COD: 90,000 . . . 100,000 L Dilution of substrate applied Inoculum: active sludge from anaerobic reactor Full height: 97 cm
mg kgCOD
BOD: 30,000 . . . 50,000 L OLR: 5.63 . . . 9.5 m3 ·day Inoculation: 3 L of sludge per reactor Digestion Zone: 78 cm
pH: 3.5 . . . 4.5 Temperature: 36 . . . 40 ◦ C
Several sampling ports as 5, 19, and 57 cm levels
Extra Sludge washing port
Suggests to adjust the ratio
Deflectors angle: 55 ◦ C
COD:N:P as 300:5:1 with urea and
GLS opening angle: 55 ◦ C
diammonium phosphate
4. Municipal sewage Temperature: 9 . . . 32 ◦ C Type: Figure 1a Full Volume: 1148 L [45]
HRT: 6 h Pre-existing functioning UASB reactor Height: 4 m
kgCOD
OLR: 2.4 m3 ·day
5. Synthetic wastewater based on Temperature: 30 ± 1◦ C Type: Figure 1b Total volume: 15 L [46]

unbleached pulp mill HRT: 30 h Inoculum: granulated sludge from UASB Digestion compartment:
mg
COD: 1400 L reactor treating poultry ID: 15 cm
pH: 6.3 . . . 8.3 slaughterhouse effluent Height: 52 cm
Settler cylindrical and conical compartment:
ID: 15 cm
Height: 30 cm
Processes 2020, 8, 734 6 of 26
Table 2. Cont.

and Seeding
6. Vanderbilt mineral Temperature: 35 ± 2 ◦ C Type: Figure 1a Total Volume: 2 Liters [47]
medium with gCOD Material: Stainless steel ID: 9 cm
OLR: 10.5 m3 ·day
tetrachloroethylene Inoculant: Flocculent anaerobic biomass from Total Height: 100 cm
mg HRT: 0.4 day
COD: 3500 L anaerobic Continuous Stirring-Tank Reactor GLS height: 15 cm
(CSTR) Seeding: 350 mL of sludge, equal to Equipped with 5 sampling ports and
8 g/L of TSS inner heater
7. Municipal sewage Ambient temperature: 24 . . . 28 ◦ C Type: Figure 1a Total volume: 62 L [48]
mg
COD: 97 . . . 196 L Material: Acrylic Sheets Total height: 270 cm
pH: 6.8 . . . 7.2 Non-cylindrical form
Sludge bed: height: 80 cm
Square cross-section: 16 cm
Gas collector slope: 60◦
8. Municipal landfill leachate Ambient temperature: 13–23 ◦ C Type: Figure 1a Height: 295 cm [49]
g
COD: 1.5 . . . 3.2 L kgCOD
OLR: 1.2 . . . 4 m3 ·day Material: Stainless steel with PVC tubing and Diameter: 13.5
pH: 6.5 . . . 7.0 HRT: 35 . . . 15 h
insulated with poly-urethane sheets GLS height: 50 cm
Inoculum: Mesophilic anaerobic sludge from Total Volume: 40 L
Added NaHCO3 as 0.5 g/L for
sewage treatment plant Recycle ratio: 3.5:1 (feed to recycle)
neutralization purpose and no
2 sampling ports
extra pH adjustment was done
Contained the heater
Recycle ratio: 3.5:1
Reports escape of methane
with effluent
9. Grey water from sewer pipe HRT: 8 . . . 20 h Type: Figure 1a Full volume: 7 L [50]
mg
COD: 647 . . . 681 L Ambient temperature: Material: not specified Diameter: 7 cm
14 . . . 24.5 ◦ C Inoculum: sludge from anaerobic digester Total height: 200 cm
treating primary and secondary sludge GLS height: 50 cm
Reactor sludge filling: 100 cm
10. Municipal wastewater HRT: 2.4 . . . 4 h Type: Figure 1c Full volume: 25 L [51]
mg
COD: 672 . . . 698 L Temperature: set of reactors Material: PVC Height: 1.35 m
operating in range 12 . . . 25 ◦ C as Inoculum: not specified ID: 15 cm
water bath made of PVC pipe Inclined arm angle: 45◦
Ø30 cm 4 sampling ports
11. Sanitary waste + aerated HRT: 6 h Type: Figure 1a Cylindrical (tubular) shape [52]
filter effluent Experiment duration of 120 days Material: not specified, but either PVC or Full volume: 7.8 L
mg
COD: 351 ± 166 L PMMA, based on provided images Total height: 60 cm
Inoculum: not specified Diameter: 14.8 cm
2 sampling ports
GLSS opening angle: ~60◦
Height from top to baffles: 15 cm
Processes 2020, 8, 734 7 of 26
Table 2. Cont.

and Seeding
12. Municipal wastewater Temperature: 20 . . . 28 ◦ C Type: Figure 1a Total Height: 3.85 m [15]
mg
COD: 176 . . . 224 L HRT: 3 h Material: Not specified Total volume: 2.5 m3
mgCOD mgVS
OLR: 0.014 L·day or 0.009 L·day Already existing and functioning reactors 3 sampling ports
Separate preheater of substrate before inlet point
13. Sugar cane vinasse Temperature: 22 ± 3 ◦ C Type: Figure 1c Total volume: 120 L [53]
mg kgCOD
COD: 19,220 L OLR: 0.5 . . . 32.4 Material: PVC Reaction zone volume: 60 L
mg m3 ·day
sCOD: 15,300 L Inoculum: Granular sludge from UASB treating Total height: 4 m
Up-flow velocities:
poultry slaughterhouse Reaction Zone Height: 2 m
pH: 5.2 0.008 . . . 0.292 mh
Seeding: 60 L of granular sludge of VVS Diameter: 19.5 mm
HRT: 33.33 . . . 0.86 days
content 37 g/L 8 sampling ports
Recycling ratio: 1:3
Added 0.3 g NaHCO3 per 1 g of
COD to adjust the pH
and alkalinity.
kgCOD
14. Mix of domestic waste OLR: 6 m3 ·day (1.5 start-up) Type: Figure 1a Diameter: 25 cm [54]
with molasses Temperature: 15 . . . 25 ◦ C Material: UPVC Height: 2 m
(0.5:785 mix ratio) HRT: 10–12 h Pre-existing reactors 4 sampling ports
mg
COD: 6597 L Vertical velocity: 0.5–0.7 m/h Volume: 98 L
mg
BOD: 3197 L
mg
TSS: 4500 L
15. Pre-digested chicken manure Feed rate; 500 mL/day/reactor + Type: Figure 1a Digestion zone height: 1 m [55]
pH: ~8.0 dilution with tap water Material: plexiglass Diameter: 90 mm
mg
COD: 807 ± 215 L HRT: 13 days Inoculum: sludge for internal circulation Volume: 6.5 L
mg Semi-continuous operation reactor treating paper/cardboard Extra sampling ports
sCOD: 295 ± 46 L
industry waste
Seeding: 1.3 L sludge per reactor (20% of
working volume)
16. Synthetic wastewater with Temperature: 37 ◦ C Type: Figure 1a Digestion zone height: 50 cm [27]
butyrate as a main substrate kgCOD Inoculum: flocculant sludge from anaerobic Digestion zone diameter: 8.4 cm
OLR: 4–83 m3 ·day
pH: 6.0–6.5 sludge digester, partially granulated in Digestion zone volume = 2.8 L
mg HRT: 12.5–4.5
COD: 2100–15,500 L pilot-scale reactor for 2 month growing Settler zone height: 25 cm
With water-jacket
on sucrose Settler zone diameter: 11.4 cm
pH: 7.1–7.9 by addition of NaHCO3
Seeding: 1.5 L of adapted sludge per reactor Settler zone volume: 2.0 L
5 sampling ports
Processes 2020, 8, 734 8 of 26
Table 2. Cont.

and Seeding
17. Synthetic wastewater HRT at beginning: 12–1.8 h Type: Figure 1a Volume: 8.5 L Digestion (+ 5 L of GLS) [56]
mg kgCOD
COD: 6000–20,000 L OLR: 18–260 m3 ·day Inoculum: anaerobic digester treating Digestion zone ID: 104 mm
pH: 7.1–7.8 (caused by buffers municipal wastewater Digestion zone H: 1000 mm
Increasing OLR by 50% after each
in WW) Seeding: 6.5 L of inoculum (1.% VSS and GLS: ID 144 mm
achieving of removal rate of 80%
1.3 TSS) GLS: H 300 mm
Preheating of substrate: 37 ◦ C
7 evenly distributed sampling ports
Alkalinity spiked with NaHCO3
Volumetric loads calculated per
digestion zone volume only
18. Municipal sewage Temperature: 25 . . . 35 (ambient) Type: Figure 1a Working volume: 14 L [57]
pH: 4.4 Feed rate: 28 L/day Material: Glass sheets Length to height ration: 1:14
mg
COD: 531 L Up-flow velocity: 0.116 m/h Inoculum: adjusted cow dung manure Height: 140 cm (it is not cylindrical)
BOD: 359 L
mg kgCOD
OLR: 1.062 m3 ·day Inoculum adaptation: 9 L of inoculum + 1 L of Length of base: 10 cm
nutrients, grow for 120 days growing on Area of reactor: 100 cm2
pH adjusted with NaOH up to
sucrose with (NH4 )2 HPO4 . Remove
6.7 ± 0.1
undigested residuals via filtering through the
Reported granulation on 20th day
3 mm mesh.
for main experiment.
Seeding: 4 L of filtered sludge from adaptation
per reactor
19. Distillery effluent from 35 ± 2 ◦ C (constant temperature Type: Figure 1a Active volume: 2.3 m3 [58]
fermentation-based vitamin C room) Material: stainless steel Height: 5.90 m
production plant kgCOD Inoculum: sludge (VSS 31.0 g/L) from anaerobic Inner diameter: 0.8 m
OLR: 6 . . . 11.8 m3 ·day
mg
COD: 6000 . . . 38,000 L Upflow velocity: 0.52 m/h digester treating the municipal wastewater Conical shape of bottom
mg 5 sampling ports
BOD5 : 2000 . . . 14,000 L HRT < 10 h
pH: 4.5–6.2 252 days of total experiment Recycle line active
100 days of start-up
Adjusted COD:N:P as 300–600:5:1
with urea and KH2 PO4
pH adjusted with NaOH up to 7.2
20. High-strength distillery HRT: 4 . . . 2d Type: Figure 1a Inner diameter: 92.1 mm [59]
wastewater Temperature: 37 ◦ C with Material: borosilicate glass Total Height: 79.6 cm
pH: 3.42–5.84 water jacket Inoculum: the sludge from UASBR treating Digestion zone: 59.97 cm
mg kgCOD
TS: 31,520–126,240 L OLR: 15.34 m3 ·day distillery wastewater Digestion volume: 5 L
mg Seeding: ~30% of reactor volume Water jacket ID: 132.10 mm
TSS: 1040–26,640 L Experiment duration: 635 days
mg Sampling ports: 6
COD: 68,000–100,000 L Start-up: 65 days with HRT: 47.11 h
mg GLSS opening angle: 70◦ (flipped funnel)
BOD: 21,600–35,000 L
Processes 2020, 8, 734 9 of 26
Table 2. Cont.

and Seeding
21. Distillery wastewater Temperature: 37 ◦ C Type: Figure 1a Total Height: 1.35 m [28]
mg
COD: 107,000 L Flow rate: 2.2 . . . 3.3 L/d Seeding: 500 mL of inoculum per reactor, + 3 L Fluidization part volume: 3 L
mg Recycle: 50% of influent flow of glucose-based synthetic wastewater and Fluidization part H: 1.05 m
TOC: 39,200 L
NaHCO3 added as 3 g/L to adjust nutrients, including Ca2+ and PO4 3− to Fluidization part ID: 5.9 cm
pH to 7 stimulate granulation Settling part volume: 3 L
kgTOC
OLR: up to 3 m3 ·day
22. Cane molasses vinasse Temperature: 55 ◦ C with Type: Figure 1b with extra settler above Volume: 140 L (126 L digestion + extra for GLSS) [60]
g
COD: 10 L water jacket gas collector Digestion par: 20 cm ID × 4 m height
pH: 4.1 kgCOD
OLR: up to 28 m3 ·day Material: Stainless steel Solids separator was made of inclined plates: 60◦
g
COD: 120 L Inoculum: sludge (12 g VS/L) from suspended
g
Experiment duration: 430 days
BOD: 30 L growth type digester treating
Added 5 g/L of NaHCO3 to
g distillery wastewater
TS: 100 L maintain 7.3 pH
g Seeding: 87 L of sludge per reactor
TS: 50 L all-over experiment.
23. Wastewater with high Temperature: 37 ◦ C with pre-heater Type: Figure 1a Volume: 8.5 L of digestion zone + 5.0 L GLSS [61]
corn-starch content HRT: 24–12 h and Inoculum: Sludge from anaerobic digester Digestion ID: 104 mm
kgCOD
pH: 6.8–7.9 OLR: 3 . . . 150 m3 ·day treating sewage wastewater Digestion Height: 1000 mm
mg
COD: 3000–75,000 L Experiment duration: 510 days Seeding: 6.5 L of sludge per reactor GLS ID: 144 mm
OLR is calculated on the volume of GLS Height: 300 mm
digestion zone only.
pH adjusted with NaHCO3 equal
to COD, but < 8 g/L to prevent
toxicity of Na+ .
Reports pH of effluent as 6.8 at the
highest OLR
24. Recycled paper mill wastewater Temperature: 37 ± 2 ◦ C with helix Type:Figure 1a Volume: 70 L (digestion zone: 53 L) [62]
pH: 7.4 heat exchanger Inoculum: sludge from full-scale UASB Height: 1 m (30 cm of which is GLS)
mg
COD: 5330.5 L Feed: 0.5–4.5 l/h, increment by Seeding: 25 L of sludge per reactor Diameter: 30 cm
g 0.5 l/d
TS: 32.99 L
g kgCOD
VS: 27.28 L OLR: 1–10 m3 ·day
Load calculations per
digestion zone!
Processes 2020, 8, 734 10 of 26
Table 2. Cont.

and Seeding
25. Distillery wastewater Flowrate: 18 L/d Type: Figure 1a ID: 10.16 cm (4in) [63]
g
COD: 40.389 L Vertical up-flow velocity: 0.0925 mh Material: PVC Height: 142.24 cm (56 in) + 14.2 cm of GLSS
pH: 3.2 . . . 3.8 HRT: 15.6 h Inoculum: 18 L of cow dung and 2 L of Effective volume: 15.4 L
kgCOD
ORL: 53.75 m3 ·day (digestion zone) substrate and aged for 3 weeks and filtered D: H ratio: 1:14
pH: 6.7 ± 0.1 with NaOH through 3 mm mesh. 5 sample ports
Extra mixing pump inside Gas collection funnel opening angle: 55◦
of reactor
26. Vinasse cane Temperature: 35 ± 2 ◦ C Type: Figure 1a Digestion part: 53 cm H × 7.5 cm ID [64]
alcohol wastewater kgCOD Material: Glass Digestion part volume: 2.3 L
OLR: varied 1 . . . 6 m3 ·day
pH 4.03 . . . 4.44 Inoculum: sludge from wastewater treatment 6 sampling ports
g HRT: 109 . . . 25 days
COD: 57.59 . . . 128.63 L Up-flow velocity: 2 . . . 3 mh
plant treating mix of urban and
g industrial wastewater
TS: 17.85 . . . 113.98 L Biogas cleaned with 3N
g Seeding: 600 mL of inoculum resulting in
VS: 11.81 . . . 58.11 L NaOH solution
10.63 g VS/L in reactor
27. Distiller’s grains wastewater pH: ~7 with NaHCO3 Type: Figure 1a with second level of gas 6 sampling ports with spacing of 20 cm [65]
mg kgCOD
COD 16,500–22,520 L OLR: 3.2 . . . 48.3 m3 ·day collectors as on Figure 1b in between.
pH 3.3–4.3 Material: acrylic Inner diameter of Tube: 8.2 cm
mg (33.3 was optimal)
VFA: 3000–3600 L Inoculum: Sludge from mesophilic anaerobic Height: 190 cm (total), 155 cm (reaction zone)
No reactor heater, the substrate was
mg digester in sewage treatment plant Total volume: 8.18 L
VSS 190–640 L preheated to 37 ◦ C before entering
Seeding: Seeded with 5.2 L of sludge with VSS Inner diameter of GLS: 14 cm
the reactor
content of 12.3 g/L, degassed by OD of gas harvesting funnel: 10 cm
Start-up OLR:
auto-incubation at 37 ◦ C for three weeks. Funnel opening angle (60◦ )
Linear increase from 0.42 to 5.6
kgCOD Duration of experiment: 420 days
m3 ·day
for 27 days
Processes 2020, 8, 734 11 of 26
Table 3. Information on UASB modifications of multi-step reactors involving UASB.
# Substrate Operating Conditions Type, Material, Inoculum, Seeding Geometry Reference

1. Distillery spent wash Temperature: 20 . . . 40 ◦ C (ambient) Type: Figure 1a with packing materials. Operational liquid volume—5 L (45 cm of [14]
pH: 4 . . . 4.5 pH: ~7 with NaHCO3 So called Hybrid UASB reactor total height)
mg
COD: 80,000 . . . 12,000 L Substrate COD:N:P as 100:5:1 with Material: PMMA Diameter: 10 cm
mg NaH2 PO4 and Urea Inoculum: flocculent sludge from anaerobic Overall height: 77 cm
TS: 60,000 . . . 85,000 L
kgCOD
mg
BOD5 : 35,000 . . . 45,000 L OLR: 1.0 . . . 8.0 m3 ·day (start-up), digester of sewage treatment plant GLS separator was replaced with packing,
kgCOD Seeding: sieved through 1 mm mesh, loaded taking 19 cm of total height (volume 1.5 L)
36 m3 ·day as 15 g VSS/L (2.5 L per reactor)
HRT: 6 . . . 48 h
Observed granulation at day 50.
2. Tannery wastewater Temperature: 17 . . . 38 ◦ C (ambient) Type: two reactors as Figure 1a in line Volume: 94 L [66]
mg
COD: 8600 . . . 14,100 L Substrate was diluted to COD Material: UPVC Total height: 325 cm
mg
pH: 2.8 . . . 3.7 value of 5400 . . . 9400 L Digestion zone height: 240 cm
Experiment duration of 52 weeks Tube ID: 20 cm
Equalization tank (600 L) prior to 5 sampling ports every 55 cm
1st stage UASB Funnel overlap on baffles: 1.5 cm per side
Start-p OLR: 24 h
HRT: 5 . . . 24 h
3. Molasses-based ethanol HRT: 70 h, treating as 2nd stage Type: Figure 1a Digestion Volume: 10 L [67]
distillery wastewater after CSTR Digestion ID: 0.08 m
Feed flow: 3.4 L/d Digestion Height: 1.5 m
Temperature: ambient
Processes 2020, 8, 734 12 of 26
Table 4. Summary of the geometry and operational parameters for existing UASB reactor designs.
Criteria Options/Area of Application/Observations

Height No constraints on height. The smallest found reactor was 30 cm tall,
the largest as above 4 m. Perhaps, limited only by the available space in
a laboratory.
Volume Small volumes are 0.5, 0.75, 1, and 2–2.4 L. Larger volumes of 14 and 55 L
were also found. Usually, reactors with volume greater than 1 m3 are
referred as pilot-scale.
Height: Diameter (H:D) ratio Since the substrate has an up-flow velocity, the reaction part of UASB
reactor in some degree functions as a sedimentation or coagulation
column, where ratio H:D should help preventing the biomass
washout [19].
This parameter is very rarely reported, and reporting of it can be
confusing due to not clear geometry reference. There are reports of H:D
ratio as a diameter of a reactor to either a total height of a reactor or to the
height of a reactor without GLS. We see reasonable to calculate it as a
diameter of reactor to the height without GLSS, since the goal of GLSS is
to create a chamber for gas capture above the reaction tube of a reactor.
From review studies, such ratio for majority of cases is in range from 8 to
14. However, there are also extreme cases as 3.5 or 23.
Construction material For small volumes (up to couple liters): Borosilicate glass
For small and medium sizes: PVC and PMMA
For pilot scale: Stainless steel.
Gas–Solid–Liquid Separator The particular design varies with the concept of the reactor itself, and
(GLSS, Three-Phase Separator) options can be split into:
Implementation of baffles
Gas collection
For tubular reactor designs, the deflectors are typically made as an
O-rings with a triangular cross-section. For rectangular reactors, a series
of inclined baffles are installed to narrow a main liquid flow.
For smaller reactors, baffles are sometimes omitted, probably, because it’s
difficult to implement those in smaller volumes. Another case when
deflectors were noticed to be omitted is when GLSS is represented by a
separate part (either tube or funnel), wider than the major reaction tube,
and a diameter of a gas collector is close to a diameter of a reaction tube.
Gas collector is usually represented by a flipped upside-down funnel for
smaller reactors. For larger ones, it can be a separate compartment.
Y-shaped reactors do not have any specific structure inside.
Heating Among the reviewed designs the following heating systems were noticed:
Heating pads or tapes
Water jacket
No heating
Inner heaters (helix shaped)
Water jacket is the most common option for smaller designs but it
complicates the placement/insertion of sensors (like pH, ORP,
temperature, etc.) into a reactor. Larger reactors usually use heating pads
or a combination of heating pads with thermal insulation material.
Temperature ranges Mesophilic: 35 ± 2 ◦ C or 37 ± 1 ◦ C
Thermophilic: 55 ± 1 ◦ C
Ambient temperature
Ambient temperature with thermostat to prevent overcooling
Inoculum material No constraints:
Granular or flocculated sludge from another UASB
Non-granulated anaerobic or active sludge
Adjusted inoculum from non-sludge sources, like animal manure
Seeding (inoculating) Across the reviewed studies, this was the most inconsistent parameter,
which was not even always reported. The process was reported as:
(a) filling reactor with raw sludge up to a certain percentage of height;
(b) volumetric load of sludge per reactor, sometimes mentioning its VSS
and/or TSS equivalent; and (c) final concentration of sludge in reactor as
TSS or VSS. Also, few studies suggested to sieve the sludge through
1–3 mm mesh to remove any undigested particulate or residuals
before seeding.
Processes 2020, 8, 734 13 of 26
Table 4. Cont.
Criteria Options/Area of Application/Observations

Substrate preparation, feeding and Few studies considered the adjustment of substrate based on ratio
pH management COD:N:P. However, the final ratio does not match across publications and
varies for COD parameter 300–600:5:1. Surprisingly, no-one mentioned
adjusting the C:N ratio, which is recommended for anaerobic treatment in
general. Only one publication mentioned the addition of compounds to
stimulate granulation.
Substrate pH management Researchers use either pH adjustment in substrate directly or pumping
pH adjusting solution to the reactor. Used adjusting compounds are either
hydroxides or bicarbonates. Interesting fact: addition of 0.5–3 g of
NaHCO3 per 1 L of substrate was sufficient to maintain a stable effluent
pH around 7. In some extreme cases 8 g per 1 L of substrate were
kgCOD
sufficient to work with OLR 150 m3 ·day .
OLR and HRT HRT and OLR are interdependent values and both are optimization points
in research. Researchers aim to increase OLR and decrease HRT.
These parameters are points of inconsistent reporting:
Some sources report OLR and HRT as referred to the total volume of the
reactor (both reaction tube and GLSS)
Some sources report OLR and HRT as referred to the volume of the reactor
without the volume of GLSS
Higher limit for OLR is not specified, since it depends on chemical
composition of influent wastewater and its strength.
Substrate distribution system Typically is not reported, but where it is mentioned it’s either:
a circular tube with evenly distributed outlet holes and an inlet from the
side through the wall of reactor
an inlet into conical-shaped bottom of reactor
a side inlet through the wall into bottom compartment with
inclined bottom
3. Discussion
Studies, involving the UASB trials, are usually purposed for: (a) treatability testing and energy
recovery estimation; (b) microbiology studies on changes in microbial consortia during adaptation to
new substrates or long-term operation for further modeling of trophic network; or (c) toxicity and
granulation process studies. In the scope of this manuscript, we would like to identify the common
needs of such research and point out the differences, where it is important. Here, we would like to
focus on experimental aspects, which are needed to pay attention to, while designing the reactors and
its infrastructure.
3.1. Volume of Reactor

The first thing that affects the final volume of a designed reactor is the available amount of
sample/substrate. Some samples of substrates are available in very limited quantities due to the policies
of supplier companies or may be a subject of special regulations preventing the dumping of effluent to
a sewer (Ex. industrial wastewater). Depending on the complexity of substrate and potential inhibitory
effects, the reactor start-up period might occupy a substantial period up to 120 days [14,57,59,65,68],
thus the volume of a reactor should allow to utilize the available sample volume for both start-up
period and experiment duration.
3.2. Material of the Reactor

Due to the specifics of laboratory studies, the reactor needs to be constructed with the feature to
visually inspect the content. It allows one to: (a) confirm the fact of granulation and (b) inspect the
foam or scum formation, etc. This significantly narrows the selection choice of materials, limiting it to
(a) polymethyl methacrylate (a.k.a., PMMA, Plexiglas, Perspex, acrylic glass), (b) borosilicate glass,
and (c) clear polyvinyl chloride. Each of the mentioned materials can be used, and in our opinion it is
more of a question of budget and available stock parts. We compare pros and cons of each material in
Table 5.
Processes 2020, 8, 734 14 of 26
Table 5. Comparison of materials used for UASB reactors in various studies.
Material Pros Cons

PMMA Less expensive than glass Needs machining equipment
Almost no film formation (unless scratched) If sterilization is needed: consider chemical
Optically clear, may have some UV-protective coating sterilization
Stronger that glass Easily scratchable
Machinable with mechanical tools
(CNC/lathe/mill/drill)
Cracks can be fixed with either solvent treatment,
epoxy of UV-curable resin in short time frame
Glass Optically clear Expensive
Non-UV degradable, chemically inert (under In case of cracks becomes sensitive to vibrations and
conditions of AD) not usable
Washable Requires specialist (glass blower) to build or repair/fix
Autoclavable Fragile
PVC Clear, but not optically. Has blueish color Degrades over time, becomes fragile
Machinable, but melts easily Non-UV-stable, becomes yellowish over long-term
Relatively cheap and available on the market, has a expose to light containing UV spectrum (sunlight)
wide set of existing fittings for quick assembly Microorganisms form biofilm on its surface
Borosilicate glass is an excellent option if used for studies with sterile cultures, since it can be
autoclaved. However, in the author’s opinion, the ideal reactor must be manufactured of stainless
steel and be featured with an inspection window, a water jacket and multiple sampling ports. Such a
design would be chemically resistant under conditions of anaerobic digestion, autoclavable, and meet
multiple research needs. However, such construction complicates the customization and should be
done for optimized and fully tested design after confirmation of its efficiency. The authors currently
use PMMA due to machinability of this material, its transparency, and stability under conditions of
anaerobic digestion (AD).
3.3. Heating of Reactor

Heating of reactor under laboratory conditions is defined by: (a) actual need for heating and
(b) necessity of sampling the content of reactor and location of sampling. If no sampling of reactor
content is needed, the water jacket would be the most suitable option. Otherwise, sampling ports
complicate the construction of water jacket. Without a water jacket, consider: (a) use of heating
tapes or flexible heating pads or (b) preheating of substrate and thermal insulation of reactor to keep
the temperature.
Heating tape on the outer surface of PMMA or PVC reactor is not recommended, since it could
cause local damage, when the contact point of wall material and heating tape is locally overheated,
causing melting or other types of damage. Our laboratory experienced problems with heating tapes
even under mesophilic conditions. The reactor that got damaged, was controlled by thermostat with
an external submergible temperature sensor. The damage consisted of the tape melting through the
wall of the reactor causing leakage. Thus, we moved to a water jacket in our projects.
Perhaps, the use of heating pads would be more secure due to a larger area of contact and, hence,
more uniform heating. Extra uniformity may be added by use of heat-transfer pastes, but they will
decrease the observability of the process in reactors. However, it is still a viable option when there is a
need for the presence of sampling ports on various levels or there is no way to implement a water
jacket due to other reasons.
3.4. Inoculum: Preparation, Adaptation, and Seeding

While the granulated anaerobic sludge is the desirable inoculum, authors fully realize the
probability of a situation when researchers do not have a source for granulated sludge. In such a
case, the manure sample of animal origin could be a source of methanogenic microbial consortia,
and referenced studies [27,42,43,57,63,65] suggest self-digestion of such sample or mixing it with
a substrate and conditioning for up to 3 weeks. The presence of methanogenic microorganisms is
Processes 2020, 8, 734 15 of 26
required for the generation of methane, but not every manure contains methanogens. The most typical
confirmed cases of manure containing methanogens are cattle and swine manures. The presence of
methanogenic bacteria could be confirmed by conducting specific methanogenic activity test [69,70],
which is very close in technique to a popular Bio-Methane potential (BMP) test [71], but conducted on
a nutrient media containing acetate as the only source of carbon [72].
Some studies suggest the sieving of inoculum through a 1–3 mm mesh to remove undigested or
large inert material. It is reasonable, if the inoculum originated from manure, since manure samples
may contain some animal bedding, or sewage wastewater treatment facility, which may contain hair,
etc. However, if the sample originated from an industrial wastewater treating facility, such sieving
could be optional, especially if sludge is already granulated and granules are large. Also, the effect of
exposing sludge or granules to air during the sieving is not clear. Perhaps, the sieving process should
be done in anaerobic chamber.
The seeding of reactor must be calculated and expressed as Volatile Suspended Solids (VSS),
introduced with the inoculum, per working volume of reactor according to [73,74] and seeding should
kg kg
be in the range 10 to 20 mVSS VSS
3 , (however, it also could be up to 25 m3 ) [10]. Inoculum should be analyzed
for Total Solids, (TS), Volatile Solids (VS), Total Suspended Solids (TSS), and Volatile Suspended Solids
(VSS) since sludge is also characterized by VS:TS and VSS:TSS ratio, as criteria of alive biomass if
condition of sludge is tracked over time [75] and ratio VSS:TSS of sludge in range of 0.7 to 0.85 is
likely to cause granulation [59]. The recommended method for solids content analysis is specified in
Standard Methods 2540 [76].
3.5. Substrate Adjustment

Before any adjustments is done to a substrate, the treatability can be roughly characterized by
the ratio BOD5 :COD, which is referred to as a biodegradability index (BI) [77,78]. For municipal raw
wastewater BI is usually in the range 0.4 to 0.8 [79,80] and it is considered to indicate good treatability.
Greater index means better bio-treatability and pretreatment can increase the value of BI [81,82].
Estimation of sample degradability based on BOD5 :COD is inconsistent, but can be generalized as:
(a) highly bio-treatable if greater than 0.5; (b) bio-treatable if greater than 0.3; and (c) not bio-treatable
when lower than 0.2 [81–85].
One of the primary adjustments for substrate is the C:N ratio [86] by mass, with the optimum in the
range 25 to 30 [87–89] or 20 to 30 [90] and higher temperature ranges require higher C:N ratio. However,
it also could be a substrate-specific optimization parameter [91–93]. Some authors also consider C:P
ratio for methanogens between 16:1 and 75:1 [94,95] as optimal, while C:N:P ratio is considered to
be favorable in the range 400:5:1 to 100:28:6 [95,96]. Some inconsistence to in attempt to meet those
ranges may come from measurement techniques, where various authors use either: (a) elemental
analyzers [97] or (b) total carbon and Total Kjeldahl Nitrogen (TKN) [90]. Across referenced in this
manuscript studies, following compounds were used to correct ratio: KH2 PO4 , NaH2 PO4 , CO(NH2 )2
(urea or carbamide), and NH4 H2 PO4 .
Individual studies stated the need to account for sulphur [98], and report C:N:P:S ratio as
600:15:5:3 to be the optimal for methanization [99]. Perhaps, such increase of considered elements
is reasonable, since elemental composition of anaerobic biomass is reported as C5 H7 O2 NP0.06 S0.1
according to [100–102], but not yet widely used in anaerobic digestion studies.
The ratio of COD:N:P of 250:5:1 is generally suggested for anaerobic treatment [103–105], however,
some variation exist between 900:5:1.7 and 150:5:1 [104,106,107] and could be even 300:1:0.1 [108].
Other studies recommend 300:5:1 as start-up conditions specifically for UASB [75,96,109,110]. Important
to mention, that “N” in such proportion refers to the total nitrogen [108]
pH adjustment for methanogenic bacteria should bring the pH in the optimal range 6.8 to
7.5, while outside of the 5 to 8.5 range, methanogenesis is fully suppressed [111–113]. However,
for anaerobic digester the range of 6.8 to 7.2 is recommended, due to widely used in wastewater
Processes 2020, 8, 734 16 of 26
treatment lime as pH adjusting chemical [114]. Across referenced studies we noticed NaOH and
NaHCO3 as widely usable compounds to adjust pH, however, the choice is wider [115].
3.6. Granulation Stimulation

If granulation enhancement is needed, the Ca2+ in concentration 100–200 mg/L of substrate can
be added [116], or even 150 . . . 300 mg/L at the start-up [117,118]. The role of calcium in granulation
process is not clear, but it is assumed to form precipitates with carbonate and phosphate [21,119]. Use of
Mg2+ is not recommended, since it causes disaggregation of granules [120], even though it is expected
to precipitate as MgNH4 PO4 [10]. Normally, granulation should be observed within 4–6 weeks after
the start of the experiment [73].
3.7. Start-up Feeding

kgCOD
The original research of [73] recommends the OLR as 0.05 . . . 0.10 kgsludge VSS ×day for the start-up
period and increasing of OLR after achieving the removal rate of at least 80%, however, the increment
values are not specified. The expression of COD load per VSS of sludge per day is called “sludge
kgCOD
load”, but in studies OLR is usually reported as m3 ×day
, which is called “space load”. Based on
kg
the previously suggested inoculum seeding as 10 . . . 20 mVSS
3 , the start-up space load should be in
kgCOD
range 0.5 . . . 2 m3 ×day
, however exact calculation based on the loaded VSS of inoculum must be done.
The vertical velocity of the substrate is suggested not to exceed 0.5 . . . 1 mh [10] in general, but minimal
values are not reported and no details were found for the start-up period.
3.8. Infrastructure of UASB Reactor

Based on our experience and referenced here studies, we want to suggest a unified operational
process flow diagram as in Figure 2, where we would like to emphasize several aspects.
Figure 2. Recommended flowchart of UASB infrastructure set-up.

Processes 2020, 8, 734 17 of 26
3.8.1. pH Adjusting and Alkalinity

As mentioned above, the composition of pH adjusting solution is a point of choice [115],
but, regardless, the solution should be pumped directly into the reactor feeding line (the mixing
manifold on schematic of Figure 2.
Otherwise there is a potential for growth of competitive microorganisms in the substrate feeding
tank, leading to chemical changes of substrate. The concentration of pH adjusting solution should
be balanced based on: (1) daily amount of solution needed to pump (pumps may have lower limit
of pumping speed) and (2) minimizing substrate dilution. The interim decision could be to use
concentrated solution that is pumped and dosed on a timer, if calculated flowrates are below the
limits of a pump. High concentrations of pH adjusting solutions can be chemically aggressive.
To prevent the contact between the substrate and parts of pumping mechanism, the peristaltic pumps
are recommended for use.
Referenced here studies dissolve or add 0.5–3 g of NaHCO3 per 1 L of substrate and achieve the
stable pH in favorable methanogenic range. However, there are more general suggestions to maintain
g CaCO
the ratio between alkalinity of substrate (expressed as CaCO3 ) and its COD as 1.2–1.6 g COD3 [121].
This value can be used as a reference to calculate the dosage portions and intensity for pH adjusting
solution, but should be optimized later [122] downwards. Methanogenesis could occur until ratio of
g CaCO g CaCO
0.8 g COD3 with some extreme cases of 0.3 g COD3 , but lower values should inhibit methanogenesis
and stimulate the formation of hydrogen [123,124].
Another reference value for regulation of alkalinity is ratio of Volatile Fatty Acids (VFA) to
Total Alkalinity (TA). Industrial guidelines [114] recommends the ratio VFA:TA to be below 0.35 and
consider the value below 0.25 as best for anaerobic digesters, and below 0.15 as safe against pH
mg
changes in substrate. The VFA should be expressed as equivalent concentration of acetic acid in L
mg
and TA as equivalent of CaCO3 in L . The determination of alkalinity by titration is described in
Standard methods 2320 [76], as well as appropriate methods for VFA with gas chromatography is
covered by Standard methods 5560. However, there were attempts to substitute VFA determination by
titration [125–127], to avoid using of gas chromatograph.
3.8.2. Feeding and Recycling

The feeding of reactor, based on OLR and HRT is the point of optimization targeting the maximum
achievable loads, however, the [10] recommends to limit the vertical velocity of liquid depending on
the type of sludge and type of waste:
• Granular sludge + soluble wastewater: 3 mh continuous, up to 6 mh peak for a couple of hours

per day;
• Granular sludge + partially insoluble water: 1–1.25 mh , up to 2 mh peak for a couple of hours per
day; and
• Flocculent sludge: 0.5 mh continuous, up to 2 mh peak.
After sludge matured and granulated, the flow could be increased by 50%. In the case of
insufficient vertical velocity and to prevent clogging, the recycle line can be used to manage it and (a) to
dilute substrate with treated wastewater, (b) to reuse of alkalinity [28], or (c) enhance the granulation
by increasing the vertical up-flow [128].
Important remark: effluent recycle port must be separate and located below the effluent discharge
port. It is made to prevent back-pumping of air from the effluent discharge line. In our laboratory
set-up, we used the flow splitters on effluent port to obtain a recycle line, and we noticed some gas
bubbles in it.
3.8.3. Manual Injection Port

A manual injection is strongly recommended and is purposed for:
Processes 2020, 8, 734 18 of 26
• urgent (emergency) injection of solution for managing pH, coagulation/flocculation, or granulation

agent problems;
• testing an enhancement of inoculant via injection of specific microbial culture(s); and
• sampling of substrate which is supplied to a reactor after all mixing procedures.
3.8.4. Biogas Collection and Counting

Notice the installed one-way valves in the gas line in Figure 2.
Check valves are important to prevent the back flow of gas and there are several reasons for that
particular phenomenon:
• drop of the ambient temperature and, consequently, gas compression in gas lines according to the
combined gas law;
• at the beginning of UASB operation, when substrate gradually fills the reactor and gas tubes have
residual air. The oxygen from residual air is consumed and thus the volume shrinks.
Any of those reasons can lead to one of the two undesirable consequences:
• ingress of liquid from reactor to a gas line, which potentially grabs the foam and clogs the pipeline.
• if water displacement gas counter is used: backflow of liquid from counter back to a reactor.
Important clarification is to use check valves with low cracking pressure. ‘Cracking pressure’
is a pressure value when check valve starts opening (passing gas through itself) and this pressure
(converted into inches of water column) must be taken into consideration when designing the gas
separator, specifically, the height and the level difference between gas collecting part and the effluent
release port. Usage of valve with high cracking pressure result in need to build tall GLSS, increasing
the material needed to build reactor and its dead volume.
We also want to stress that the gas counter working on the water displacement principle is the
only option for raw biogas. There are gas counters working on heat transfer principle ( similar to
thermal conductivity detectors of gas chromatographs), which seem to be cheaper options, but they
should not be used. Those counters can be calibrated for gas flow with constant content only, which is
not a case for biogas. However, they can be theoretically applied if biogas was stripped with alkaline
solution to remove acidic gasses (carbon dioxide, hydrogen sulfide, etc.) and assumed to be upgraded
to bio-methane. We do not recommend the use of that.
3.9. Tracking Operational Parameters

The exact set of trackable parameters depend on the purpose of a particular study, but for general
cases, we listed those parameters in Table 6.
Table 6. Minimal list of parameters for tracking during UASB experiments.
Parameter Measurement For Used For

COD Influent Calculate the degradability rate of substrate
Effluent Calculate corrected OLR
Reference for energy yield calculation and substrate utilization rate
pH Influent Estimation how favorable are conditions for methanogenesis
Effluent Tracking the changes of substrate
Reactor entrance : The actual pH value in a sludge layer if recycle line is used
Gas Yield volume Estimation of yield per unit of substrate
Content Calculation of energy recovery
Balancing COD on biomass growth
Flowrate Feed rate Calculation of OLR, HRT, up-flow velocity
Recycle rate
These parameters are already enough to calculate the main operational parameters specified in
Table 7 [10,91,129,130]:
Processes 2020, 8, 734 19 of 26
Table 7. Main operational parameters of UASB.
Parameter Equation
CODin f luent −CODe f f luent
Substrate utilization rate U = HRT×VSSsludge in reactor (1)
CODin f luent −CODe f f luent
Removal efficiency E= CODin f luent × 100% (2)
working volume o f reactor
Hydraulic retention time (HRT) θ = volumetric f lowrate o f in f luent (3)
volumetric f lowrate o f in f luent×CODin f luent
Organic Loading rate (space load) OLRspace = working volume o f reactor
(4)
volumetric f lowrate o f in f luent×CODin f luent
Organic loading rate (sludge load) OLRsludge = volatile suspended solids o f sludge in reactor (5)
in f luent f lowrate+recycle f lowrate+ad justing f lowrate
Up-flow velocity ν= area o f horizontal crosssection o f reactor
(6)
In addition, the track of biogas composition during the UASB experiments, the total gas yield and
methane yield should be logged. Mentioned above parameters for logging and calculations on their
basis do provide a basic understanding of ongoing process inside of UASB reactors, while interpretation
of calculations result are not the scope of this manuscript to avoid swelling of it. However authors feel
also a need to mention, that if some deeper understanding of chemical process or COD balancing is
needed, other researchers [59,131–133] suggest calculation of what part of metabolism is presented by
certain process according to the equations, collected in Table 8:
Table 8. Equations for metabolic ratios estimation.
Parameter Equation
CODCH4 +sCODe f f luent −sCODin f luent
Hydrolysis H = CODin f luent −sCODin f luent × 100% (7)
CODCH4 +CODVFA−e f f luent −CODVFA−in f luent
Acidification A= CODin f luent −CODVFA−in f luent × 100% (8)
CODCH4
Methanogenesis M = CODin f luent × 100% (9)
COD mass balance CODin f luent = CODaccumulated + CODbiogas + CODe f f luent (10)
Other parameters, not included here, belong to some partial cases of UASB experiments and are
subjects of individual consideration. Examples for a category of such specialized studies could be effects
of salinity or metal ions on the process inside of UASB, which would require extra electrical conductivity,
ion-selective electrodes, or other quantitative measurements for both influent and effluent [134,135].
If the study is dedicated to toxicity or biodegradation of particular compound, the appropriate assay
tests for that compound or its metabolites should be added [136,137], etc.
4. Conclusions
With this article we would like to draw the researcher’s focus towards the need to report in their
publications more information on materials and methods, including specifically sketches/operational
flowcharts, seeding conditions, inoculum sources and pre-treatments, and all adjustments to the
substrate and feeding equipment. The consideration and addition of these details will help to facilitate
a strong scientific and engineering community with comparable research results and conditions.
Such detailed data and methods reporting will also significantly propel modeling studies that aim to
realistically predict bioreactor behavior in various process conditions.
Author Contributions: Y.P. conceived the study, collected and analyzed information, wrote the manuscript under
supervision and support from R.C.S. and C.D.M. All authors have read and agreed to the published version of
the manuscript.
Funding: The authors acknowledge the financial sponsorship of WesTech-Inc., Salt Lake City (A-43875) and the
Huntsman Environmental Research Center (A-17526).
Conflicts of Interest: The authors declare no conflict of interest.
Processes 2020, 8, 734 20 of 26
Abbreviations
AD Anaerobic Digestion sCOD Soluble COD
BOD Biological Oxygen Demand SRT Solids Retention Time
COD Chemical Oxygen Demand TA Total Alkalinity
CSTR Continuous Stirred-Tank Reactor TKN Total Kjeldahl Nitrogen
EPS Exocellular Polymeric Substances TOC Total Organic Carbon
GLSS Gas–Liquid–Solids Separator TS Total Solids
HRT Hydraulic Retention Time TSS Total Suspended Solids
ID Inner diameter UASB Up-flow Anaerobic Sludge Blanket
OD Outer diameter UPVC Unplasticized Polyvinyl Chloride
OLR Organic Loading Rate VFA Volatile Fatty Acids
PMMA Polymethyl methacrylate VS Volatile Solids
PVC PolyVinyl Chloride VSS Volatile Suspended Solids
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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processes

Keywords: up-flow anaerobic sludge blanket reactors; anaerobic digestion; laboratory-

Processes 2020, 8, 734; doi:10.3390/pr8060734 www.mdpi.com/journal/processes

A combination of developing trophic microbial connections and mechanical cementation with

• Higher removal of contaminants;

Table 1. Existing hybrid versions of UASB reactors.

• Organic Loading Rate (OLR) and Hydraulic Retention Time (HRT);

2. Review of Existing Solutions across Various Published Works

• used interchangeably (equally)

Table 2. Overview of available information of UASB reactors used in laboratory studies.

Used Type of Reactor, Material, Inoculum,

5. Synthetic wastewater based on Temperature: 30 ± 1◦ C Type: Figure 1b Total volume: 15 L [46]

Used Type of Reactor, Material, Inoculum,

Used Type of Reactor, Material, Inoculum,

Used Type of Reactor, Material, Inoculum,

Used Type of Reactor, Material, Inoculum,

Used Type of Reactor, Material, Inoculum,

Table 3. Information on UASB modifications of multi-step reactors involving UASB.

# Substrate Operating Conditions Type, Material, Inoculum, Seeding Geometry Reference

Criteria Options/Area of Application/Observations

Criteria Options/Area of Application/Observations

3.1. Volume of Reactor

3.2. Material of the Reactor

Table 5. Comparison of materials used for UASB reactors in various studies.

Material Pros Cons

3.3. Heating of Reactor

3.4. Inoculum: Preparation, Adaptation, and Seeding

3.5. Substrate Adjustment

3.6. Granulation Stimulation

3.7. Start-up Feeding

3.8. Infrastructure of UASB Reactor

Figure 2. Recommended flowchart of UASB infrastructure set-up.

3.8.1. pH Adjusting and Alkalinity

3.8.2. Feeding and Recycling

• Granular sludge + soluble wastewater: 3 mh continuous, up to 6 mh peak for a couple of hours

3.8.3. Manual Injection Port

• urgent (emergency) injection of solution for managing pH, coagulation/flocculation, or granulation

3.8.4. Biogas Collection and Counting

3.9. Tracking Operational Parameters

Table 6. Minimal list of parameters for tracking during UASB experiments.

Parameter Measurement For Used For

Table 7. Main operational parameters of UASB.

Table 8. Equations for metabolic ratios estimation.

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